Patent ID: 12209106

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail.

[Alkoxyphenyl Compounds and Derivatives Thereof]

The alkoxyphenyl compounds of the present invention and the derivatives thereof are the compound represented by the general formula (1) or a derivative thereof.

(In the formula,R each independently represents an optionally substituted alkyl group having 10 to 40 carbons.m represents an integer between 1 and 5. When m is 2 or more, a plurality of ROs may be the same or different.X represents O, S, NH, or NRN.n represents an integer from 1 to 4.RNrepresents an optionally substituted alkyl group having 1 to 6 carbons.)

In the general formula (1), R each independently represents an optionally substituted alkyl group having 10 to 40 carbons. The R may be an alkyl group having 13 to 30 carbons, or may be an alkyl group having 15 to 20 carbons, and among them, an alkyl group having 18 carbons may be preferred. In addition, when a plurality of Rs are present, they each independently may be a linear alkyl group or a branched alkyl group, but are preferably linear alkyl groups. Further, when the R is an alkyl group having 10 to 40 carbons, synthesis of the Tagged protected nucleoside and the Tagged protected nucleotide of the present invention becomes easy, and also an effect of improving solubility and separation ability can be obtained.

In the general formula (1), m represents an integer of 1 to 5. When m is 2 or more, a plurality of ROs may be the same or different. Preferably m is an integer from 2 to 4, more preferably m is 3. In addition, when the carbonyl group (C═O group) directly bonded to the phenyl group is set to the 1-position, the RO group is preferably bonded to the 3-position, the 4-position, or the 5-position, and when m=3, it is preferably bonded to the 3-position, the 4-position, or the 5-position. Further, by adjusting the value of m within the range, it is possible to adjust the solubility of the Tagged protected nucleoside and the Tagged protected nucleotide of the present invention in a nonpolar solvent.

In the general formula (1), X represents O, S, NH, or NRN. Among them, X is preferably O.

In the general formula (1), n represents an integer of 1 to 4. Among them, n is preferably 1 or 2.

In the general formula (1), RNrepresents an optionally substituted alkyl group having 1 to 6 carbons. Examples of the RNinclude methyl group, ethyl group, 1-propyl group, 1-methyl-1-ethyl group, 1-butyl group, 1-methyl-1-propyl group, 1,1-dimethyl-1-ethyl group, 2-methyl-1-propyl group, 1-pentyl group, and 2-pentyl group.

In addition, in the present invention, it is preferable that the alkoxyphenyl compound and a derivative thereof are a compound represented by the general formula (2) or a derivative thereof.

(In the formula,R1, R2, and R3each independently represent an optionally substituted alkyl group having 10 to 40 carbons.X represents O, S, NH, or NRN.n represents an integer from 1 to 4.RNrepresents an optionally substituted alkyl group having 1 to 6 carbons.)

In Formula (2), R1, R2, and R3each independently represent a n optionally substituted alkyl group having 10 to 40 carbons. The R1, R2and R3may be alkyl groups having 13 to 30 carbon, alkyl groups having 15 to 20 carbons, and among them, alkyl groups having 18 carbons are preferable. The R1, R2, and R3are independent of each of her, and may be straight-chain or branched-class groups, but all are preferably straight-chain groups.

In the general formula (2), X, n, and RNare the same as those of the general formula (1).

In addition, although the alkoxyphenyl compound of the present invention and a derivative thereof have been exemplified by the synthesis method as an example, they may be synthesized using a known method using a commercially available raw material.

[Tagged Protected Nucleosides and Tagged Protected Nucleotides, Etc.]

The Tagged protected nucleosides and the Tagged protected nucleotides in the present invention are a compound represented by the general formula (3) or a derivative thereof.

(In the formula,R each independently represents an optionally substituted alkyl group having 10 to 40 carbons.m represents an integer between 1 and 5. When m is 2 or more, a plurality of ROs present may be the same or different.X represents O, S, NH, or NRN.n represents an integer from 1 to 4.RNrepresents an optionally substituted alkyl group having 1 to 6 carbons.Y represents the following general formula (4) or general formula (5).)

(In the formula,Base represents a nucleobase which may be modified. When 2 or more Bases are present, a plurality of Bases present may be the same or different.B represents a hydrogen atom, an optionally protected hydroxyl group, or a halogen. When 2 or more Bs are present, a plurality of Bs present may be the same or different.W represents a hydrogen atom or protecting group.R4represents an alkyl group having 1 to 4 carbons and having an electron-withdrawing group. If 2 or more R4s are present, a plurality of R4s present may be the same or different.Z represents O or S. When 2 or more Zs are present, a plurality of Zs present may be the same or different.y1 represents any integer greater than or equal to 0.* represents a bonding position in the general formula (3).)

(In the formula,Base represents a nucleobase which may be modified. When 2 or more Bases are present, a plurality of Bases present may be the same or different.Q represents a hydrogen atom or protecting group.V represents an alkoxy group having 1 to 6 carbons, a di(C1-6alkyl)amino group, or a piperazino group in which the nitrogen atom at the 4-position is protected with a protecting group and may be further substituted. When 2 or more Vs are present, a plurality of Vs present may be the same or different.y2 represents any integer greater than or equal to 0.* represents a bonding position in the general formula (3).)

In the general formula (3), R, m, X, n, and RNare the same as those of the general formula (1).

In the general formula (3), Y represents the following general formula (4) or the general formula (5).

In the general formula (4), Base represents a nucleobase which may be modified. When 2 or more Bases are present, a plurality of Bases present may be the same or different.

Examples of the Base include adenyl groups, guanyl groups, sitosyl groups, thiminyl groups, or uracil groups, and derivatives thereof, as well as modifications thereof. As the above-mentioned modifications, for example, a polar group or the like may be protected by a known protecting group or the like. In addition, a plurality of Bases present in the oligonucleotide may be the same or different.

In the general formula (4), B represents a hydrogen atom, an optionally protected hydroxyl group, or a halogen. When 2 or more Bs are present, a plurality of Bs present may be the same or different. In addition, in the case of a protected hydroxyl group, a known method can be appropriately used as a protecting group and a protective method.

In the general formula (4), W represents a hydrogen atom or protecting group. When W is a protecting group, a known method can be appropriately used as a protecting group and a protecting method.

It is also preferred that W is a hydrogen atom, a trityl group, a di(C1-6alkoxy)trityl group, a mono(C1-18alkoxy)trityl group, a 9-(9-phenyl)xanthenyl group, or a 9-(9-phenyl)thioxanthenyl group. When W is any of the above, the synthesis of oligonucleotides can be more conveniently performed.

In the general formula (4), R4represents an alkyl group having 1 to 4 carbons and having an electron-withdrawing group. If 2 or more R4s are present, a plurality of R4s present may be the same or different. It is also preferred that R4is an ethylenic group having an electron-withdrawing group at the 2-position. Further, as the electron-withdrawing group, for example, a cyano group can be mentioned as a preferable example.

In the general formula (4), Z represents O or S. When 2 or more Zs are present, a plurality of Zs present may be the same or different.

In the general formula (4), y1 represents an arbitrary integer of 0 or more. y1 may be 1 to 100, 1 to 50, 1 to 30, 1 to 20, 1 to 10, or 0.

In the general formula (4), * represents a bonding position in the general formula (3).

In the general formula (5), Base is the same as in the general formula (4).

In the general formula (5), Q represents a hydrogen atom or protecting group. When Q is a protecting group, a known method can be appropriately used as a protecting group and a protecting method.

It is also preferred that Q is a hydrogen atom, a trityl group, a di(C1-6alkoxy)trityl group, a mono(C1-18alkoxy)trityl group, a 9-(9-phenyl)xanthenyl group, or a 9-(9-phenyl)thioxanthenyl group. When Q is any of the above, synthesis of oligonucleotides can be more conveniently performed.

In the general formula (5), V represents an alkoxy group having 1 to 6 carbons, a di(C1-6alkyl)amino group, or a piperazino group in which a nitrogen atom at the 4-position is protected with a protecting group and may be further substituted. When 2 or more Vs are present, a plurality of Vs present may be the same or different.

In the general formula (5), y2 represents an arbitrary integer of 0 or more. y2 may be 1 to 100, 1 to 50, 1 to 30, 1 to 20, 1 to 10, or 0.

In the general formula (5), * represents a bonding position in the general formula (3).

Further, it is preferable that the Tagged protected nucleoside and the Tagged protected nucleotide of the present invention be the compound represented by the general formula (6) or a derivative thereof.

(In the formula,R1, R2, and R3each independently represent an optionally substitute d, 10 to 40 carbons.X represents O, S, NH, or NRN.n represents an integer from 1 to 4.RNrepresents an optionally substituted alkyl group having 1 to 6 carbons.Y represents the following general formula (4) or general formula (5).)

(In the formula,Base represents a nucleobase which may be modified. When 2 or more Bases are present, a plurality of Bases present may be the same or different.B represents a hydrogen atom, an optionally protected hydroxyl group, or a halogen. When 2 or more Bs are present, a plurality of Bs present may be the same or different.W represents a hydrogen atom or protecting group.R4represents an alkyl group having 1 to 4 carbons and having an electron-withdrawing group. If 2 or more R4s are present, a plurality of R4s present may be the same or different.Z represents O or S. When 2 or more Zs are present, a plurality of Zs present may be the same or different.y1 represents any integer greater than or equal to 0.* represents a bonding position in the general formula (6).)

(In the formula,Base represents a nucleobase which may be modified. When 2 or more Bases are present, a plurality of Bases present may be the same or different.Q represents a hydrogen atom or protecting group.V represents an alkoxy group having 1 to 6 carbons, a di(C1-6alkyl) amino group, or a piperazino group in which the nitrogen atom at the 4-position is protected with a protecting group and may be further substituted. When 2 or more Vs are present, a plurality of Vs present may be the same or different.y2 represents any integer greater than or equal to 0.* represents a bonding position in the general formula (6).)

In the general formula (6), R1, R2, R3, X, n, and RNare the same as in the general formula (2).

In the general formula (6), Y is the same as in the general formula (3).

In addition, although the Tagged protected nucleoside and the Tagged protected nucleotide of the present invention have been exemplified by the synthesis method in the examples, they may be synthesized using a known method using a commercially available raw material.

As a method (synthesis method) for producing a Tagged protected nucleoside and a Tagged protected nucleotide of the present invention, for example, it can be obtained by a method of binding a nucleoside or a nucleotide with an alkoxyphenyl compound or a derivative thereof described above. As a binding method, for example, a method of subjecting the alkoxyphenyl compound or a derivative thereof to a condensation reaction with a nucleoside or a nucleotide can be mentioned. When using the condensation reaction above, for example, the method of combining the alkoxyphenyl compound represented by a general formula (1) or (2) and a nucleoside or a nucleotide using a condensation agent, or the method of binding the derivative (for example, ester derivative, acid chloride derivative, etc.) of the alkoxyphenyl compound represented by the general formula (1) or (2) whose reactivity has been enhanced by a substituent or the like to a nucleoside or a nucleotide, or the method of binding the alkoxyphenyl compo and represented by the general formula (1) or (2) with a nucleoside or a nucleotide whose reactivity has been enhanced by a substituent or the like, can be appropriately used.

[Method for Producing Protected Oligonucleotide and Method for Removing Tag Moiety, Etc.]

The method for producing a protected oligonucleotide of the present invention (synthesis method) and method for removing the Tag moiety of the present invention (substituent removing method) include a step (1) of subjecting a compound represented by the general formula (3) or a derivative thereof to reduction treatment.

(In the formula,R each independently represents an optionally substituted alkyl group having 10 to 40 carbons.m represents an integer between 1 and 5. When m is 2 or more, a plurality of ROs present may be the same or different.X represents O, S, NH, or NRN.n represents an integer from 1 to 4.RNrepresents an optionally substituted alkyl group having 1 to 6 carbons.Y represents the following general formula (4) or general formula (5).)

(In the formula,Base represents a nucleobase which may be modified. When 2 or more Bases are present, a plurality of Bases present may be the same or different.B represents a hydrogen atom, an optionally protected hydroxyl group, or a halogen. When 2 or more Bs are present, a plurality of Bs present may be the same or different.W represents a hydrogen atom or protecting group.R4represents an alkyl group having 1 to 4 carbons and having an electron-withdrawing group. If 2 or more R4s are present, a plurality of R4s present may be the same or different.Z represents O or S. When 2 or more Zs are present, a plurality of Zs present may be the same or different.y1 represents any integer greater than or equal to 0.* represents a bonding position in the general formula (3).)

(In the formula,Base represents a nucleobase which may be modified. When 2 or more Bases are present, a plurality of Bases present may be the same or different.Q represents a hydrogen atom or protecting group.V represents an alkoxy group having 1 to 6 carbon atoms, a di(C1-6alkyl)amino group, or a piperazino group in which the nitrogen atom at the 4-position is protected with a protecting group and may be further substituted. When V are present in an amount of 2 or more, a plurality of V present may be the same or different.y2 represents any integer greater than or equal to 0.* represents a bonding position in the general formula (3).)

(In the formula,Base represents a nucleobase which may be modified. When 2 or more Bases are present, a plurality of Bases present may be the same or different.B represents a hydrogen atom, an optionally protected hydroxyl group, or a halogen. When 2 or more Bs are present, a plurality of Bs present may be the same or different.W represents a hydrogen atom or protecting group.R4represents an alkyl group having 1 to 4 carbons and having an electron-withdrawing group. If 2 or more R4s are present, a plurality of R4s present may be the same or different.Z represents O or S. When 2 or more Zs are present, a plurality of Zs present may be the same or different.y1 represents any integer greater than or equal to 0.)

(In the formula,Base represents a nucleobase which may be modified. When 2 or more Bases are present, a plurality of Bases present may be the same or different.Q represents a hydrogen atom or protecting group.V represents an alkoxy group having 1 to 6 carbons, a di(C1-6alkyl)amino group, or a piperazino group in which the nitrogen atom at the 4-position is protected with a protecting group and may be further substituted. When 2 or more Vs are present, a plurality of V presents may be the same or different.y2 represents any integer greater than or equal to 0.)

In the method for producing a protected oligonucleotide described above, the general formulas (3), (4), and (5) are the same as described above.

In the general formula (7) in the method for producing a protected oligonucleotide above, Base, B, W, R4, Z, and y1 are the same as those of the general formula (4).

In the general formula (8) in the method for producing a protected oligonucleotide, Base, Q, V, and y2 are the same as those of the general formula (5).

In the method for producing a protected oligonucleotide of the present invention and method for removing the Tag moiety of the present invention, it is preferable to include a step (1) of subjecting a compound represented by the general formula (6) or a derivative thereof to reduction treatment.

(In the formula,R1, R2, and R3each independently represent an optionally substituted alkyl group having 10 to 40 carbons.X represents O, S, NH, or NRN.n represents an integer from 1 to 4.RNrepresents an optionally substituted alkyl group having 1 to 6 carbons.Y represents the following general formula (4) or general formula (5).)

(In the formula,Base represents a nucleobase which may be modified. When 2 or more Bases are present, a plurality of Bases present may be the same or different.B represents a hydrogen atom, an optionally protected hydroxyl group, or a halogen. When 2 or more Bs are present, a plurality of Bs present may be the same or different.W represents a hydrogen atom or protecting group.R4represents an alkyl group having 1 to 4 carbons and having an electron-withdrawing group. If 2 or more R4s are present, a plurality of R4s present may be the same or different.Z represents O or S. When 2 or more Zs are present, a plurality of Zs present may be the same or different.y1 represents any integer greater than or equal to 0.* represents a bonding position in the general formula (6).)

(In the formula,Base represents a nucleobase which may be modified. When 2 or more Bases are present, a plurality of Bases present may be the same or different.Q represents a hydrogen atom or protecting group.V represents an alkoxy group having 1 to 6 carbons, a di(C1-6alkyl)amino group, or a piperazino group in which the nitrogen atom at the 4-position is protected with a protecting group and may be further substituted. When 2 or more Vs are present, a plurality of Vs present may be the same or different.y2 represents any integer greater than or equal to 0.* represents a bonding position in the general formula (6).)

(In the formula,Base represents a nucleobase which may be modified. When 2 or more Bases are present, a plurality of Bases present may be the same or different.B represents a hydrogen atom, an optionally protected hydroxyl group, or a halogen. When 2 or more Bs are present, a plurality of Bs present may be the same or different.W represents a hydrogen atom or protecting group.R4represents an alkyl group having 1 to 4 carbons and having an electron-withdrawing group. If 2 or more R4s are present, a plurality of R4s present may be the same or different.Z represents O or S. When 2 or more Zs are present, a plurality of Zs present may be the same or different.y1 represents any integer greater than or equal to 0.)

(In the formula,Base represents a nucleobase which may be modified. When 2 or more Bases are present, a plurality of Bases present may be the same or different.Q represents a hydrogen atom or protecting group.V represents an alkoxy group having 1 to 6 carbons, a di(C1-6alkyl)amino group, or a piperazino group in which the nitrogen atom at the 4-position is protected with a protecting group and may be further substituted. When 2 or more Vs are present, a plurality of V presents may be the same or different.y2 represents any integer greater than or equal to 0.)

In the method for producing the protected oligonucleotide and method for removing the Tag moiety of the present invention, the general formulas (3), (4), and (5) are the same as described above.

In the method for producing the protected oligonucleotide and the inventive method for removing the Tag moiety, in the general formula (7), Base, B, W, R4, Z, and y1 are the same as those of the general formula (4).

In the method for producing the protected oligonucleotide and method for removing the Tag moiety of the present invention, in the general formula (8), Base, Q, V, and y2 are the same as those of the general formula (5).

In the method for producing the protected oligonucleotide and method for removing the Tag moiety of the present invention, it is preferable that the reduction treatment is one using a boron-containing reducing agent or using a boron-containing reducing agent and an a mine. By having the configuration, a protected oligonucleotide can be more conveniently synthesized.

As the boron-containing reducing agent, a known one can be used, but among them, lithium borohydride, sodium borohydride, lithium triethylborohydride, tetrabutylammonium borohydride, and the like are preferred. These compounds may be used alone, or 2 or more of them may be used in combination. By having the configuration, a protected oligonucleotide can be conveniently synthesized in better yield.

When the boron-containing reducing agent and an amine are used, a known one can be used as the amine, but among them, diisopropylethylamine, triethylamine, pyridine, and the like are preferred. These compounds may be used alone, or 2 or more of them may be used in combination. By having the configuration, a protected oligonucleotide can be conveniently synthesized in better yield.

In the step (1), a known method may be used using a commercially available raw material, unless the chemical structure of the compound or a derivative thereof where the synthesis method or the re action method is represented by the general formula (6) or the like are damaged.

The step (1) may be, for example, a step of dissolving or mixing a compound represented by the general formula (6) or the like or a derivative thereof with a boron-containing reducing agent or a boron-containing reducing agent and an amine in an organic solvent, and stirring and reacting the mixture at −10° C. or at room temperature.

In addition, a method for producing a protected oligonucleotide of the present invention and a method for removing a Tag moiety of the present invention illustrate a synthesis method and a reaction method thereof in Examples, but other known methods may be used using a commercially available raw material.

As a method for producing a Tagged protected oligonucleotide of the present invention, for example, a 2 mer is taken as an example, and a Tagged protected nucleoside or a Tagged protected nucleotide described is subjected to a coupling reaction with an amidited nucleoside or nucleotide (Step A), and the obtained 2 mer is subjected to phosphoric esterification in an oxidation step (Step B), and deprotection of the protecting group W is performed (Step C), and filtration (Step D) is performed, whereby a 2 mer of a Tagged protected nucleotide can be obtained. Further, examples of the higher order (n+1) mer include a method in which the method is similarly obtained n times, and a method in which a nucleotide derivative having a certain length is used to bind them to each other.

EXAMPLES

Hereinafter, examples and the like specifically showing the configuration and effects of the present invention will be described. Hereinafter, the concentration operation in the examples was carried out under reduced pressure unless otherwise specified. The evaluation items in Examples and the like were measured as follows.

<Measurement of1H-NMR Spectrum>

1H-NMR spectrum was measured using a nuclear magnetic resonance apparatus (manufactured by Nippon Electronics Co., Ltd., product name AL400, and product name ECS-600) and tetramethylsilane as an internal standard.

<Determination of13C-NMR Spectrum>

13C-NMR spectrum was measured using nuclear magnetic resonators (manufactured by Japan Electronics Co., Ltd., under the product name AL400, and by Japan Electronics Co., Ltd., under the product name ECS-600) and using tetramethylsilane as an internal standard.

<Measure TOF/MS Spectrum>

TOF/MS spectra were measured using time-of-flight mass spectrometry (Waters, product name LCT-PremierXE and Japanese Electronic, product name JMS T-100LP).

1. Synthesis of 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetic Acid

(A) Process Via Tert-Butyl Ester

Example 1-1

(1) Synthesis of 2-(tert-butoxy)-2-oxoethyl 3,4,5-Tris(octadecyloxy)benzoate

Triethylamine (0.39 g, 3.87 mmol) and 2-butyl tert-bromoacetate (0.76 g, 3.87 mmol) was added to a suspension of 3,4,5-tris(octadecyloxy)benzoic acid (1.80 g, 1.94 mmol) in THF (10 mL), and then stirred at 60° C. for 2 hours. After cooling the reaction solution to room temperature, the solid precipitated by dropping methanol (54 mL) was filtered. The solid was washed with methanol, and then dried in vacuo at 50° C. to give the title compound (1.95 g, 97%) as a white solid.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.16-1.41 (m, 84H), 1.41-1.57 (m, 6H), 1.49 (s, 9H), 1.66-1.88 (m, 6H), 3.95-4.11 (m, 6H), 4.70 (s, 2H), 7.31 (s, 2H).

13C-NMR (100 MHz, CDCl3): δ 14.02, 22.48, 22.66, 22.83, 26.08, 26.11, 27.88, 28.08, 29.35, 29.40, 29.56, 29.64, 29.65, 29.70, 29.97, 30.22, 30.36, 31.76, 31.93, 32.10, 61.66, 69.35, 73.51, 82.32, 108.69, 108.90, 123.82, 123.93, 143.11, 143.38, 152.93, 165.83, 166.90.

TOF/MS (ESI): calcd for C67H124O7Na [M+Na]+1063.9245. found 1063.9254.

Example 1-2

(2) Synthesis of 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetic Acid)

Trifluoroacetic acid (11.40 g, 100.0 mmol) was added to a solution of 2-(tert-butoxy)-2-oxoethyl 3,4,5-tris(octadecyloxy)benzoate (5.21 g, 5.0 mmol) in chloroform (20 ml), and then stirred at 50° C. for 17 hours. After cooling the reaction solution to room temperature, the solid precipitated by dropping acetonitrile (50 mL) was filtered. The solid was washed with acetonitrile-chloroform mixture solvent, and then dried in vacuo at 50° C. to give the title compound (4.89 g, 99%) as a white solid.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=7.0 Hz), 1.16-1.54 (m, 90H), 1.69-1.88 (m, 6H), 3.95-4.11 (m, 6H), 4.87 (s, 2H), 7.30 (s, 2H).

13C-NMR (100 MHz, CDCl3): δ 14.08, 22.68, 26.06, 26.10, 29.36, 29.41, 29.57, 29.65, 29.66, 29.72, 30.35, 31.93, 60.49, 69.30, 73.57, 108.53, 123.31, 143.17, 152.96, 165.74.

TOF/MS (ESI): calcd for C63H116O7Na [M+Na]+1007.8619. found 1007.8639.

(B) Process Via Benzyl Esters

Example 1-3

(1) Synthesis of 2-Benzyloxy-2-oxoethyl 3,4,5-Tris(octadecyloxy)benzoate

Triethylamine (0.20 g, 1.94 mmol) and benzyl 2-bromoacetate (0.45 g, 1.94 mmol) was added to a suspension of 3,4,5-tris(octadec yloxy)benzoic acid (0.90 g, 0.97 mmol) in chloroform (5 mL), and then stirred at 50° C. for 14 hours. After cooling the reaction solution to room temperature, the solid precipitated by dropping methanol (18 mL) was filtered. The solid was washed with methanol, and then dried in vacuo at 50° C. to give the title compound (1.02 g, 97%) as a white solid.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.16-1.41 (m, 84H), 1.41-1.54 (m, 6H), 1.66-1.91 (m, 6H), 3.95-4.08 (m, 6H), 4.86 (s, 2H), 5.23 (s, 2H), 7.24-7.41 (m, 7H).

13C-NMR (100 MHz, CDCl3): δ 14.08, 22.71, 26.13, 26.16, 29.40, 29.43, 29.46, 29.62, 29.69, 29.71, 29.76, 30.00, 30.43, 31.98, 61.24, 67.07, 69.40, 73.59, 108.76, 123.69, 128.32, 128.50, 128.65, 135.35, 143.28, 152.96, 153.01, 165.84, 167.77.

Example 1-4

(2) Synthesis of 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetic Acid

10% Pd—C (0.09 g) was added to a solution of 2-benzyloxy-2-oxoethyl 3,4,5-tris(octadecyloxy)benzoate (0.91 g, 0.85 mmol) in THF (4.3 mL), and then stirred under a hydrogen balloon at 40° C. for 19 hours. The reaction solution was cooled to room temperature, and then 10% Pd—C was removed by Celite filtration. After concentrating the filtrate, the solid precipitated by dropping acetonitrile (18 mL) to the residue was filtered. The solid was washed with acetonitrile, and then dried in vacuo at 50° C. to give the title compound (0.84 g, 100%) as a white solid.

2. Synthesis of thymidinyl-[3′→5′]-deoxycytidinyl-[3′→5′]-deoxycytidinyl-[3′→5′]-deoxycytidinyl-[3′→5′]-deoxyguanidyl-[3′→5′]-deoxycytidinyl-[3′→5′]-deoxycytidinyl-[3′→5′]-thymidinyl-[3′→5′]-deoxyguanidyl-[3′→5′]-thymidinyl-[3′→5′]-deoxyguanidyl-[3′→5′]-deoxyadenylyl-[3′→5′]-deoxycytidinyl-[3′→5′]-deoxyadenylyl-[3′→5′]-thymidinyl-[3′→5′]-deoxyguanidyl-[3′→5′]-deoxycytidinyl-[3′→5′]-deoxyadenylyl-[3′→5′]-thymidinyl-[3′→5′]-thymidine (20 Mer Oligonucleic Acid)

Example 2-1

(1) Synthesis of 5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl 2-((3, 4,5-tris(octadecyloxy)benzoyl)oxy)acetate

COMU (4.73 g, 11.05 mmol) was added to a suspension of 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetic acid (4.95 g, 5.02 mmol), 5′-O-(4,4′-dimethoxytrityl)thymidine (3.28 g, 6.03 mmol), and 1-methylimidazole (2.27 g, 27.62 mmol) in THF (50 mL), and then stirred at room temperature for 3.5 hours. The solid precipitated by dropping acetonitrile (248 mL) to the reaction solution was filtered. The solid was washed with acetonitrile-THF mixture solvent, and then dried in vacuo at 50° C. to give the title compound (7.16 g, 95%).

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.16-1.54 (m, 93H), 1.69-1.88 (m, 6H), 2.42-2.58 (m, 2H), 3.42-3.55 (m, 2H), 3.78 (s, 6H), 3.95-4.05 (m, 6H), 4.18 (s, 1H), 4.82 (s, 2H), 5.56 (d, 1H, J=5.6 Hz), 6.40-6.50 (m, 1H), 6.80-6.90 (m, 4H), 7.20-7.40 (m, 9H), 7.61 (s, 1H), 8.03 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ 11.62, 14.10, 22.71, 26.11, 26.15, 29.38, 29.45, 29.61, 29.68, 29.75, 30.40, 31.96, 37.88, 55.28, 61.12, 63.68, 69.35, 73.60, 83.93, 84.40, 87.36, 108.59, 111.66, 113.43, 123.26, 127.29, 128.08, 128.19, 130.10, 130.15, 135.20, 135.25, 135.32, 144.22, 150.11, 153.02, 158.92, 163.16, 165.85, 167.50.

TOF/MS (ESI): calcd for C94H146N2O13Na [M+Na]+1534.0723. found 1534.0759.

Example 2-2

(2) Synthesis of thymidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

Pyrrole (1.54 g, 22.98 mmol) and trifluoroacetic acid (0.66 g, 5.75 mmol) were added to a solution of 5′-O-(4,4′-dimethoxytrityl)thyuridine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (6.95 g, 4.60 mmol) in dichloromethane (174 mL), and then stirred at room temperature for 2.5 hours. the solid precipitated by dropping acetonitrile (52 mL) and acetonitrile (156 mL) to the reaction solution was filtered. The solid was washed with acetonitrile-acetone-dichloromethane mixture solvent and acetonitrile, and then dried in vacuo at 50° C. to give the title compound (5.04 g, 91%).

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.11-1.39 (m, 84H), 1.43-1.51 (m, 6H), 1.66 (s, 1H), 1.71-1.85 (m, 6H), 1.95 (s, 3H), 2.36-2.58 (m, 3H), 3.89-3.97 (m, 2H), 3.97-4.08 (m, 6H), 4.15 (dd, 1H, J=2.0, 2.4 Hz), 4.84 (s, 2H), 5.45-5.53 (m, 1H), 6.20 (dd, 1H, J=6.0, 8.8 Hz), 7.29 (s, 2H), 7.45 (d, 1H, J=1.2 Hz), 8.59 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ 12.58, 14.12, 22.70, 26.07, 26.11, 29.33, 29.38, 29.43, 29.59, 29.67, 29.68, 29.73, 30.35, 31.94, 36.99, 61.12, 62.57, 69.24, 73.58, 75.97, 84.91, 86.46, 108.36, 111.48, 123.17, 136.41, 143.09, 150.29, 152.96, 163.40, 165.91, 167.71.

TOF/MS (ESI): calcd for C73H128N2O11Na [M+Na]+1231.9416. found 1231.9432.

Example 2-3

(3) Synthesis of 5′-O-((cyanoethoxy)(thymidine-3′-yl)phosphoryl)thymidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

Under argon atmosphere, 5-benzylthio-1H-tetrazole (0.32 g, 1.65 mmol) was added to a suspension of thymidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (1.00 g, 0.83 mmol) and 5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl-phosphoroamidite (1.23 g, 1.65 mmol) in dichloromethane (15 mL), and then stirred at room temperature for 1.5 hours. Next, cumene hydroperoxide (content: 82%) (0.23 g, 1.24 mmol) was added, and then stirred at room temperature for 2 h ours. Pyrrole (0.56 g, 8.27 mmol) and trifluoroacetic acid (0.28 g, 2.48 mmol) was added to the reaction solution, and then stirred at room temperature for 2 hours. The solid precipitated by dropping acetone (15 mL) and acetonitrile (45 mL) to the reaction solution was filter ed. The solid was washed with acetonitrile-acetone-dichloromethane mixture solvent and acetonitrile, and then dried in vacuo at 50° C. to give the title compound (1.20 g, 92%) as a white solid.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.11-1.55 (m, 90H), 1.65-2.05 (m, 12H), 2.40-2.60 (m, 4H), 2.75-2.90 (m, 2H), 3.84 (s, 2H), 3.95-4.10 (m, 6H), 4.15-4.50 (m, 6H), 4.85 (s, 2H), 5.15-5.25 (m, 1H), 5.35-5.55 (m, 1H), 6.10-6.35 (m, 2H), 7.29 (s, 2H), 7.35 (d, 1H, J=1.2 Hz), 7.47 (s, 1H), 9.60-9.95 (m, 2H).

13C-NMR (100 MHz, CDCl3): δ 12.42, 12.46, 14.10, 19.72, 19.79, 22.71, 26.12, 26.19, 29.38, 29.43, 29.49, 29.63, 29.70, 29.75, 30.43, 31.96, 36.47, 36.56, 38.46, 61.14, 61.91, 62.00, 62.63, 62.68, 62.75, 62.80, 67.53, 69.42, 73.64, 74.66, 74.77, 78.94, 79.12, 82.33, 82.40, 82.48, 85.60, 85.66, 85.79, 86.02, 86.21, 86.35, 108.61, 111.27, 111.33, 111.73, 111.82, 116.58, 116.66, 123.15, 135.87, 135.94, 136.53, 136.62, 143.41, 150.49, 150.59, 153.07, 163.94, 164.02, 165.96, 167.79, 167.83.

TOF/MS (ESI): calcd for C86H144N5O18PNa [M+Na]+1589.0142. found 1589.0104.

Example 2-4

(4) 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyano ethoxy)(5′-O-((2′-cyanoethoxy)(5′-O-((2′-cyanoethoxy)(5′-O-((2′-cyano ethoxy)(5′-O-((2′-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy) (5′-O-((2′-cyanoethoxy)(5′-O-((2′-cyanoethoxy)(5′-O-((2′-cyanoethoxy)(5′-O-((2′-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(thymidine-3′-yl)phosphoryl)-N4-Benzoyldeoxycytidine-3′-yl)phosphoryl)-N4-benzoyldeoxycytidine-3′-yl)phosphoryl)-N4-benzoyldeoxycytidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)-N4-benzoyldeoxycytidine-3′-yl)phosphoryl)-N4-benzoyl deoxycytidine-3′-yl)phosphoryl)-thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)-thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)-N6-benzoyldeoxyadenosine-3′-yl)phosphoryl)-N4-benzoyldeoxycytidine-3′-yl)-phosphoryl)-N6-benzoyldeoxyadenosine-3′-yl)phosphoryl)-thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)-N2-benzoyldeoxycytidine-3′-yl)phosphoryl)-N6-benzoyldeoxyadenosine-3′-yl)phosphoryl)-thymidine-3′-yl)phosphoryl)-thymidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The same procedure as in Example 2-3 was repeated 18 times to give the title compound (1.10 g)

TOF/MS (ESI): m/z 3114.3 [M−3H]3−, 2335.6 [M−4H]4−, 1868.9 [M−5H]5−, 1557.6 [M−6H]6−.

Example 2-5

(5) Deprotection and Purification Steps

28% ammonia water (2.1 mL) was added to the compound synthesized in Example 2-4 (50 mg, 5.4 μmol), and then heated and stirred at 80° C. for 1.5 hours. After concentrating the reaction solution under reduced pressure,

the insoluble compound precipitated by dropping acetonitrile (50 mL) adding 0.1 mol/L-triethylamine acetate buffer to the concentrated residue was filtered. The eluate obtained by purifying C-18 cartridges of the filtrate was dried, and then Thymidinyl-[3′→5′]deoxycytidinyl-[3′→5′]-deoxycytidinyl-[3′→5′]-deoxycytidinyl-[3′→5′]-deoxyguanidyl-[3′→5′]-deoxycytidinyl-[3′→5′]-deoxycytidinyl-[3′→5′]-thymidinyl-[3′→5′]-deoxyguanidyl-[3′→5′]-thymidinyl-[3′→5′]-deoxyguanidyl-[3′→5′]-deoxyadenylyl-[3′→5′]-deoxycyidinyl-[3′→5′]-deoxyadenylyl-[3′→5′]-thymidinyl-[3′→5′]-deoxyguanidyl-[3′→5′]-deoxycytidinyl-[3′→5′]-deoxyadenylyl-[3′→5′]-thymidinyl-[3′→5′]-thymidine was obtained.

HPLC (shodexODP (4.6 φ×150 mm), flow rate: 1.0 mL/min, transfer: 0.1 mol/L triethylamine-acetate buffer, acetonitrile gradient: 0-15 min (2 to 98% acetonitrile), Rt=3.9 min (98.6%)

TOF/MS (ESI): m/z 3020.9 [M−2H]2−, 2013.4 [M−3H]3−, 1509.8 [M−4H]4−.

(6) Synthesis of 5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

Example 2-6

(When EDCI—HCl is Used as the Condensing Agent)

4-dimethylaminopyridine (0.02 g, 0.18 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.34 g, 1.77 mmol) was added to a solution of 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetic acid (0.87 g, 0.89 mmol) and 5′-O-(4,4′-dimethoxytrityl)thymidine (0.97 g, 1.77 mmol) in THF (9 mL), and then stirred at room temperature for 14 hours. The solid precipitated by dropping acetonitrile (18 mL) to the reaction solution was filtered. The solid was washed with acetonitrile-THF mixture solvent, and then dried under reduced pressure at 50° C. to obtain a crude compound (1.28 g). The crude compound was purified by column chromatography (spherical neutral silica gel, eluent: chloroform-THF) to give the title compound (1.06 g, 79%) as a white solid.

Example 2-7

(When CDI is Used as the Condensing Agent)

1,1′-carbonyldiimidazole (2.43 g, 15.0 mmol) was added to a suspension of dichloromethane (210 mL) of 2-((3,4,5-tris(octadecyloxy) benzoyl)oxy)acetic acid (9.86 g, 10.0 mmol) and 4-dimethylaminopyridine (0.12 g, 1.0 mmol) and stirred at room temperature for 1 hour, and then stirred at 30 to 35° C. for 4 hours. Next, 5′-O-(4,4′-dimethoxytrityl)thymidine (10.89 g, 20.0 mmol) was added, and then stirred at room temperature for 17 hours. Further, after 5′-O-(4,4′-dimethoxy trityl)thymidine (1.09 g, 0.4 mmol) was added to the reaction solution, the solution was stirred at room temperature for 4.5 hours. After dichloromethane (10 mL) was added to the reaction solution, the solid precipitated by dropping acetonitrile (400 mL) was filtered. The solid was washed with acetonitrile-dichloromethane mixture solvent, and then dried under reduced pressure at 50° C. to obtain a crude compound (14.44 g). The crude compound was purified by column chromatography (spherical neutral silica gel, eluent: dichloromethane-methanol) to give the title compound (11.11 g, 74%) as a white solid.

3. Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)thymidine-3′-yl)phosphoryl)-N6-benzoyldeoxyadenosine-3′-yl 2-((3,4,5-Tris(octadecyloxy)benzoyl)oxy)acetate

Example 3-1

(1) Synthesis of 5′-O-(4,4′-dimethoxytrityl)-N6-benzoyldeoxyadenosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (3.11 g, 96%) was obtained from 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetic acid (1.97 g, 2.00 mmol) and 5′-O-(4,4′-dimethoxytrityl)-N6-benzoyldeoxyadenosine (1.58 g, 2.40 m mol) according to the procedure of Example 2-1.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.11-1.40 (m, 84H), 1.42-1.50 (m, 6H), 1.71-1.84 (m, 7H), 2.73 (dd, 1H, J=4.4, 12.8 Hz), 3.04-3.13 (m, 1H), 3.46 (d, 2H, J=4.0 Hz), 3.77 (s, 6H), 3.99-4.05 (m, 6H), 4.34-4.37 (m, 1H), 4.86 (s, 2H), 5.67 (d, 1H, J=5.6 Hz), 6.51 (dd, 1H, J=5.6, 8.4 Hz), 6.79 (d, 2H, J=9.4 Hz), 7.18-7.29 (m, 7H), 7.31 (s, 2H), 7.38 (d, 2H, J=7.2 Hz), 7.53 (dd, 2H, J=7.2, 7.6 Hz), 7.59-7.63 (m, 1H), 8.03 (d, 2H, J=7.2 Hz), 8.17 (s, 1H), 8.74 (s, 1H), 8.98 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ 14.13, 22.71, 26.08, 26.11, 29.33, 29.38, 29.43, 29.60, 29.67, 29.74, 30.36, 31.94, 38.00, 55.23, 61.16, 63.52, 69.23, 73.58, 84.35, 84.48, 86.82, 108.35, 113.25, 123.22, 123.32, 127.03, 127.85, 127.95, 128.08, 128.91, 129.99, 130.04, 132.82, 133.66, 135.38, 135.42, 141.21, 143.07, 144.33, 149.53, 151.56, 152.75, 152.97, 158.63, 164.48, 165.89, 167.38.

TOF/MS (ESI): calcd for C101H149N5O12Na [M+Na]+1647.1100. found 1647.1149.

Example 3-2

(2) Synthesis of N6-benzoyldeoxyadenosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (2.25 g, 92%) was obtained from 5′-O-(4,4′-dimethoxytrityl)-N6-benzoyldeoxyadenosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (3.00 g, 1.85 mmol) according to the procedure of Example 2-2.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.11-1.40 (m, 84H), 1.44-1.50 (m, 6H), 1.71-1.85 (m, 7H), 2.53 (dd, 1H, J=5.6, 14.0 Hz), 3.21-3.28 (m, 1H), 3.89-3.96 (m, 1H), 3.98-4.06 (m, 6H), 4.36 (s, 1H), 4.87 (s, 2H), 5.70 (d, 1H, J=5.2 Hz), 5.91 (brs, 1H), 6.32 (dd, 1H, J=5.6, 10.0 Hz), 7.32 (s, 2H), 7.54 (dd, 2H, J=7.2, 8.0 Hz), 7.61-7.65 (m, 1H), 8.03 (d, 2H, J=7.2 Hz), 8.07 (s, 1H), 8.79 (s, 1H), 9.07 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ 14.12, 22.70, 26.07, 26.11, 29.33, 29.37, 29.42, 29.59, 29.67, 29.73, 30.35, 31.94, 37.76, 61.20, 63.17, 69.28, 73.60, 87.18, 87.61, 108.39, 123.17, 124.66, 127.90, 128.96, 133.01, 133.39, 142.42, 143.17, 150.40, 150.68, 152.26, 153.00, 164.42, 165.97, 167.41.

TOF/MS (ESI): calcd for C80H131N5O10Na [M+Na]+1344.9794. found 1344.9828.

Example 3-3

(3) Synthesis of 5′-O-((2-cyanoethoxy)(thymidine-3′-yl)phosphoryl)-N6-benzoyldeoxyadenosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (2.50 g, 97%) was obtained from N6-benzoyldeoxyadenosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (2.00 g, 1.51 mmol) and DMTr-dT-CE-phosphoroamidite (2.25 g, 3.02 mmol) according to the methods of Examples 2-3.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.12-1.40 (m, 84H), 1.43-1.50 (m, 6H), 1.71-1.85 (m, 9H), 1.94 (s, 1H), 2.22-2.51 (m, 2H), 2.70-2.78 (m, 3H), 3.14-3.26 (m, 1H), 3.45-3.56 (m, 1H), 3.69-3.85 (m, 2H), 3.95-4.07 (m, 6H), 4.07-4.32 (m, 3H), 4.40-4.48 (m, 3H), 4.88 (s, 2H), 4.99-5.11 (m, 1H), 5.67-5.73 (m, 1H), 6.11-6.22 (m, 1H), 6.50-6.58 (m, 1H), 7.30 (s, 2H), 7.41-7.53 (m, 3H), 7.57-7.61 (m, 1H), 8.02-8.06 (m, 2H), 8.38 (m, 1H), 8.81 (m, 1H), 9.58-9.67 (m, 2H).

13C-NMR (100 MHz, CDCl3): δ 12.43, 14.14, 19.66, 19.73, 19.78, 22.71, 26.09, 26.15, 29.35, 29.38, 29.46, 29.61, 29.68, 29.74, 29.99, 3.38, 31.94, 36.57, 36.87, 38.35, 61.13, 61.84, 61.92, 62.45, 62.49, 62.58, 62.62, 67.39, 69.27, 73.61, 74.81, 75.06, 78.89, 79.19, 79.24, 82.98, 83.05, 83.12, 84.47, 84.54, 85.43, 85.48, 85.62, 85.67, 85.79, 85.93, 108.34, 111.22, 111.27, 116.52, 116.64, 123.01, 123.82, 124.08, 128.27, 128.31, 128.69, 128.72, 132.87, 132.94, 133.17, 133.41, 136.45, 141.68, 141.86, 143.18, 149.93, 150.00, 150.47, 151.84, 152.05, 152.67, 153.01, 163.81, 163.86, 165.43, 165.53, 165.99, 167.76.

TOF/MS (ESI): calcd for C93H147N8O17PNa [M+Na]+1702.0520. found 1702.0604.

Examples 3-4

(4) Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)thymidine-3′-yl)phosphoryl-N6-benzoyldeoxyadenosine-3′-yl 2-((3,4,5-Tris(octadecyloxy)benzoyl)oxy)acetate

5-Benzylthio 1H tetrazole (2.74 g, 14.25 mmol) was added to a suspension of 5′-O-((2-cyanoethoxy)(thymidine-3′-yl)phosphoryl)-N6-benzoyldeoxyadenosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)acetate (7.98 g, 4.75 mmol) and N2-isobutyryl-DMTr-dG-CE phosphoroamidite (11.97 g, 14.25 mmol) in dichloromethane (90 ml), and then stirred at room temperature for 3 hours. Next, cumene hydroperoxide (content: 82%) (2.64 g, 14.25 mmol) was added, and then stirred at room temperature for 1 hours. The solid precipitated by dropping Acetone (120 mL) and acetonitrile (359 mL) to the reaction solution was filtered. The solid was washed with a mixture solvent of acetonitrile-acetone-dichloromethane, acetonitrile, and methanol, and then dried in vacuo at 50° C. to give the title compound (10.86 g, 94%).

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.12-1.40 (m, 90H), 1.43-1.50 (m, 6H), 1.71-1.93 (m, 9H), 1.95-2.16 (m, 2H), 2.35-2.87 (m, 9H), 3.16-3.37 (m, 3H), 3.76 (s, 6H), 3.99-4.05 (m, 6H), 4.09-4.43 (m, 10H), 4.86 (s, 2H), 5.07-5.26 (m, 2H), 5.65-5.71 (m, 1H), 5.91-6.22 (m, 2H), 6.49-6.54 (m, 1H), 6.78 (d, 4H, J=8.4 Hz), 7.15-7.27 (m, 8H), 7.30 (s, 2H), 7.34-7.36 (m, 2H), 7.43-7.48 (m, 2H), 7.54-7.59 (m, 1H), 7.69-7.71 (m, 1H), 7.98-8.02 (m, 2H), 8.30-8.41 (m, 1H), 8.75-8.82 (m, 1H), 9.43-9.58 (m, 1H), 9.88-10.27 (m, 1H), 12.09-12.22 (m, 1H).

13C-NMR (100 MHz, CDCl3): δ 12.23, 12.36, 14.13, 18.84, 19.00, 19.05, 19.65, 19.73, 22.70, 26.09, 26.15, 29.35, 29.37, 29.46, 29.61, 29.68, 29.74, 29.77, 30.38, 31.94, 35.89, 36.47, 36.63, 55.26, 61.13, 62.47, 62.63, 62.68, 63.42, 67.55, 69.28, 73.61, 74.86, 75.05, 79.76, 81.43, 83.08, 83.59, 84.68, 86.85, 108.33, 111.11, 111.17, 113.15, 113.26, 116.48, 116.75, 116.88, 121.42, 121.59, 123.03, 124.05, 127.07, 127.12, 127.78, 127.85, 127.98, 128.06, 128.74, 129.14, 130.05, 132.88, 133.37, 135.23, 139.48, 141.92, 142.01, 143.18, 144.23, 147.35, 148.11, 148.27, 148.49, 149.98, 150.09, 151.80, 152.60, 153.01, 155.40, 155.47, 158.62, 158.68, 164.77, 165.20, 165.96, 167.71, 167.74, 179.81, 180.21.

TOF/MS (ESI): calcd for C131H186N14O26P2Na [M+Na]+2456.3036. found 2456.2988.

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)-N4-benzoyldeoxycytidine-3′-yl 2-((3,4,5-Tris(octadecyloxy)benzoyl)oxy)acetate

Example 3-5

(1) Synthesis of 5′-O-(4,4′-dimethoxytrityl)-N4-benzoyldeoxycytidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (3.10 g, 97%) was obtained from 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetic acid (1.97 g, 2.00 mmol) and 5′-O-(4,4′-dimethoxytrityl)-N4-benzoyldeoxycytidine (1.52 g, 2.40 mmol) according to the procedure of Example 2-1.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.12-1.53 (m, 90H), 1.71-1.85 (m, 6H), 2.36-2.43 (m, 1H), 2.83-2.95 (m, 1H), 3.49 (ddd, 2H, J=3.2, 10.4, 18.0 Hz), 3.78 (s, 3H), 3.79 (s, 3H), 3.95-4.07 (m, 6H), 4.30-4.35 (m, 1H), 4.83 (dd, 2H, J=16.0, 23.6 Hz), 5.51-5.58 (m, 1H), 6.33 (dd, 1H, J=6.0, 7.6 Hz), 6.86 (dd, 4H, J=2.4, 8.8 Hz), 7.20-7.38 (m, 11H), 7.50-7.54 (m, 2H), 7.60-7.64 (m, 1H), 7.88 (d, 2H, J=7.2 Hz), 8.14 (d, 1H, J=7.6 Hz), 8.40-8.60 (brs, 1H).

13C-NMR (100 MHz, CDCl3): δ 14.09, 22.70, 26.10, 26.15, 29.38, 29.46, 29.61, 29.68, 29.75, 30.40, 31.95, 39.47, 55.26, 61.10, 63.17, 69.33, 73.59, 75.77, 84.68, 87.32, 108.54, 113.46, 123.32, 127.22, 128.10, 128.13, 129.10, 130.04, 130.10, 133.20, 135.13, 135.30, 144.07, 153.01, 158.84, 165.81, 167.43.

TOF/MS (ESI): calcd for C100H149N3O13Na [M+Na]+1623.0988. found 1623.1069.

Examples 3-6

(2) Synthesis of N4-benzoyldeoxycytidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (2.39 g, 97%) was obtained from 5′-O-(4,4′-dimethoxytrityl)-N6-benzoyldeoxycytidine-3′-yl 2-((3,4,5-tris(octadec yloxy)benzoyl)oxy)acetate (3.04 g, 1.90 mmol) according to the procedure of Example 2-2.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.20-1.51 (m, 90H), 1.71-1.85 (m, 6H), 2.46-2.53 (m, 1H), 2.69-2.80 (m, 1H), 3.92-4.04 (m, 8H), 4.27 (d, 1H, J=2.4 Hz), 4.84 (dd, 2H, J=16.4, 20.4 Hz), 5.51-5.53 (m, 1H), 6.26 (dd, 1H, J=5.6, 7.6 Hz), 7.29 (s, 2H), 7.48-7.62 (m, 4H), 7.88 (d, 2H, J=7.6 Hz), 8.28 (d, 1H, J=7.2 Hz), 8.66-8.91 (brs, 1H).

13C-NMR (100 MHz, CDCl3): δ 14.10, 22.71, 26.10, 26.14, 29.38, 29.45, 29.61, 29.68, 29.75, 30.39, 31.95, 38.46, 61.15, 62.37, 69.34, 73.61, 75.80, 85.82, 88.44, 108.55, 123.25, 127.61, 129.07, 133.25, 143.25, 145.32, 153.01, 162.38, 165.89, 167.79.

TOF/MS (ESI): calcd for C79H131N3O11Na [M+Na]+1320.9681. found 1320.9723.

Examples 3-7

(3) Synthesis of 5′-O-((2-cyanoethoxy)(N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)-N4-benzoyldeoxycytidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (3.02 g, 99%) was obtained from N4-benzoyldeoxycytidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (2.27 g, 1.75 mmol) and N2-isobutyryl-DMTr-dG-CE-phosphoroamidite (2.94 g, 3.50 mmol) according to the methods of Examples 2-3.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.08-1.53 (m, 96H), 1.70-1.84 (m, 6H), 2.36-2.62 (m, 2H), 2.75-3.30 (m, 7H), 3.75-3.87 (m, 2H), 3.98-4.04 (m, 6H), 4.26-4.53 (m, 6H), 4.78-4.89 (m, 2H), 5.26-5.29 (m, 1H), 5.42-5.52 (m, 1H), 5.92-6.11 (m, 1H), 6.24-6.38 (m, 1H), 7.26-7.28 (m, 2H), 7.38-7.67 (m, 4H), 7.80-7.89 (m, 2H), 7.95-8.00 (m, 1H), 8.14-8.24 (m, 1H), 10.22-10.70 (m, 1H), 12.08-12.22 (m, 1H).

13C-NMR (100 MHz, CDCl3): δ 14.14, 18.96, 18.99, 19.05, 19.81, 19.87, 22.71, 26.08, 26.15, 29.34, 29.38, 29.46, 29.61, 29.68, 29.75, 30.37, 31.94, 36.01, 36.06, 38.37, 38.83, 39.11, 61.09, 62.01, 62.59, 62.64, 62.77, 62.82, 67.20, 67.51, 69.25, 73.60, 74.76, 75.03, 80.49, 83.50, 83.75, 84.57, 84.81, 86.32, 87.91, 88.04, 97.29, 97.50, 108.30, 116.55, 116.68, 120.72, 121.05, 123.01, 127.73, 127.80, 128.88, 128.93, 132.71, 132.81, 133.28, 138.19, 138.30, 143.13, 144.20, 144.42, 147.79, 147.99, 148.16, 148.33, 152.99, 155.25, 155.65, 162.86, 163.04, 165.92, 165.95, 167.77, 167.85, 179.77, 180.03.

TOF/MS (ESI): calcd for C96H152N9O18PNa [M+Na]+1773.0891. found 1773.0914.

Examples 3-8

(4) Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)-N4-benzoyldeoxycytidine-3′-yl 2-((3,4,5-Tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (3.44 g, 89%) was obtained from 5′-O-((2-cyanoethoxy)(N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)-N4-benzoyldeoxycytidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (2.80 g, 1.60 mmol) and DMTr-dT-CE phosphoroamidite (3.58 g, 4.80 mmol) according to the methods of Examples 3-4.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.13-1.55 (m, 99H), 1.71-1.84 (m, 6H), 1.95-2.14 (m, 2H), 2.27-2.95 (m, 9H), 3.00-3.55 (m, 3H), 3.73-3.81 (m, 6H), 3.99-4.04 (m, 6H), 4.14-4.48 (m, 11H), 4.82-4.85 (m, 2H), 5.05-5.53 (m, 3H), 6.00-6.46 (m, 3H), 6.76-6.87 (m, 4H), 7.20-7.36 (m, 11H), 7.44-7.64 (m, 4H), 7.72-7.88 (m, 3H), 8.11-8.22 (m, 1H), 9.00-9.15 (m, 1H), 10.29-10.49 (m, 1H), 12.14-12.17 (m, 1H).

13C-NMR (100 MHz, CDCl3): δ 11.67, 11.71, 14.13, 18.86, 18.90, 18.94, 18.98, 19.00, 19.59, 19.67, 19.81, 19.88, 22.70, 26.08, 26.15, 29.35, 29.37, 29.46, 29.61, 29.67, 29.74, 29.77, 30.37, 31.94, 35.88, 38.67, 55.29, 61.13, 62.66, 62.73, 62.87, 63.33, 69.25, 73.59, 84.20, 84.28, 85.31, 87.29, 108.31, 111.69, 111.73, 113.15, 113.35, 116.64, 116.79, 123.01, 127.31, 127.71, 127.77, 128.08, 128.99, 129.14, 130.09, 133.33, 134.93, 134.97, 135.01, 135.17, 143.14, 143.96, 144.04, 147.84, 147.93, 148.16, 150.40, 150.48, 152.99, 155.48, 158.83, 163.52, 165.96, 167.86, 179.65, 179.76.

TOF/MS (ESI): calcd for C130H186N12O27P2Na [M+Na]+2432.2923. found 2432.2937.

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl 2-((3,4,5-Tris(octadecyloxy)benzoyl)oxy)acetate

Examples 3-9

(1) Synthesis of 5′-O-(4,4′-dimethoxytrityl)-N2-isobutyryldeoxyguanosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (3.14 g, 98%) was obtained from 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetic acid (1.97 g, 2.00 mmol) and 5′-O-(4,4′-dimethoxytrityl)-N2-isobutyryldeoxyguanosine (1.54 g, 2.40 mmol) according to the procedure of Example 2-1.

1H-NMR (400 MHz, CDCl3): δ 0.86-0.89 (m, 12H), 1.03 (d, 3H, J=6.8 Hz), 1.11-1.40 (m, 84H), 1.42-1.50 (m, 6H), 1.70-1.83 (m, 6H), 1.95 (sept, 1H, J=6.8 Hz), 2.51 (dd, 1H, J=5.2, 12.4 Hz), 3.17-3.29 (m, 2H), 3.42 (dd, 1H, J=3.2, 10.4 Hz), 3.76 (s, 3H), 3.77 (s, 3H), 3.98-4.05 (m, 6H), 4.22 (s, 1H), 4.83 (s, 2H), 5.70 (d, 1H, J=6.0 Hz), 6.07 (dd, 1H, J=5.2, 9.2 Hz), 6.76-6.82 (m, 4H), 7.16-7.38 (m, 9H), 7.43-7.50 (m, 2H), 7.71 (s, 1H), 7.73 (s, 1H), 11.96 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ 14.13, 18.64, 18.77, 22.71, 26.05, 26.11, 29.32, 29.37, 29.42, 29.60, 29.66, 29.68, 29.74, 29.75, 29.77, 30.33, 31.94, 36.17, 37.21, 55.24, 61.24, 63.48, 69.30, 73.64, 76.16, 83.94, 84.44, 86.49, 108.33, 113.27, 113.29, 122.34, 123.24, 127.15, 128.02, 129.99, 135.47, 135.77, 137.83, 143.04, 144.70, 147.18, 148.02, 152.94, 155.38, 158.74, 166.00, 167.35, 178.30.

TOF/MS (ESI): calcd for C98H151N5O13Na [M+Na]+1629.1206. found 1629.1240.

Examples 3-10

(2) Synthesis of N2-isobutyryldeoxyguanosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (2.42 g, 98%) was obtained from 5′-O-(4,4′-dimethoxytrityl)-N2-isobutyryldeoxyguanosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (3.05 g, 1.90 mmol) according to the procedure of Example 2-2.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.20-1.50 (m, 96H), 1.71-1.85 (m, 6H), 2.48 (dd, 1H, J=5.6, 14.0 Hz), 2.72 (sept, 1H, J=6.8 Hz), 2.97-3.05 (m, 1H), 3.49 (brs, 2H), 3.86-4.05 (m, 8H), 4.26 (s, 1H), 4.86 (s, 2H), 5.59 (d, 1H, J=6.0), 6.13 (dd, 1H, J=5.6, 9.2 Hz), 7.30 (s, 2H), 7.86 (s, 2H), 8.82 (s, 1H), 12.16 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ 14.10, 18.94, 18.97, 22.71, 26.09, 26.15, 29.38, 29.45, 29.61, 29.69, 29.75, 30.39, 31.96, 36.50, 37.85, 61.26, 62.92, 69.43, 73.67, 86.27, 86.34, 108.59, 122.49, 123.26, 138.42, 143.34, 147.09, 147.73, 153.04, 155.01, 166.02, 167.52, 178.69.

TOF/MS (ESI): calcd for C77H133N5O11Na [M+Na]+1326.9899. found 1326.9879.

Example 3-11

(3) Synthesis of 5′-O-((2-cyanoethoxy)(thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (2.68 g, 94%) was obtained from N2-isobutyryldeoxyguanosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (2.25 g, 1.72 mmol) and DMTr-dT-CE-phosphoroamidite (2.57 g, 3.45 mmol) according to the methods of Examples 2-3.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.05-1.52 (m, 96H), 1.71-1.89 (m, 9H), 2.24-2.78 (m, 8H), 3.38-3.49 (m, 1H), 3.69-4.05 (m, 8H), 4.17-4.89 (m, 7H), 5.20-5.29 (m, 1H), 5.55-5.66 (m, 1H), 6.08-6.24 (m, 2H), 7.30 (s, 2H), 7.42-7.54 (m, 1H), 7.68-7.73 (m, 1H), 8.64-8.83 (m, 1H), 10.20-10.32 (m, 1H), 12.11-12.22 (m, 1H).

13C-NMR (100 MHz, CDCl3): δ 12.46, 14.14, 18.59, 18.93, 19.06, 19.42, 19.66, 19.71, 19.73, 19.78, 22.71, 26.07, 26.14, 29.33, 29.38, 29.45, 29.61, 29.68, 29.74, 30.36, 31.94, 35.57, 35.93, 38.42, 61.12, 61.24, 61.71, 61.89, 62.56, 62.64, 62.69, 69.26, 73.62, 75.69, 75.81, 79.29, 82.80, 85.73, 86.04, 86.41, 108.29, 111.23, 111.32, 116.46, 116.51, 122.39, 122.98, 123.03, 136.55, 136.78, 139.04, 143.11, 143.18, 147.93, 147.98, 150.30, 150.37, 152.98, 155.39, 155.59, 163.53, 163.62, 166.00, 166.12, 167.69, 167.86, 179.92, 180.04.

TOF/MS (ESI): calcd for C90H149N8O18PNa [M+Na]+1684.0625. found 1684.0648.

Example 3-12

(4) Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl 2-((3,4,5-Tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (3.11 g, 89%) was obtained from 5′-O-((2-cyanoethoxy)(thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (2.50 g, 1.50 mmol) and DMTr-dT-CE phosphoroamidite (3.36 g, 4.51 mmol) according to the methods of Examples 3-4.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.4 Hz), 1.05-1.53 (m, 96H), 1.71-1.88 (m, 13H), 2.24-2.87 (m, 10H), 3.25-3.53 (m, 3H), 3.72-3.81 (m, 7H), 3.95-4.05 (m, 6H), 4.05-4.72 (m, 10H), 4.81-4.91 (m, 2H), 5.03-5.24 (m, 2H), 5.55-5.61 (m, 1H), 5.95-6.43 (m, 3H), 6.82-6.85 (m, 4H), 7.18-7.37 (m, 12H), 7.53 (s, 1H), 7.73 (s, 1H), 9.10-9.31 (m, 2H), 10.05-10.09 (m, 1H), 12.18 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ 11.68, 11.72, 12.33, 12.37, 14.14, 18.78, 18.88, 19.13, 19.24, 19.58, 19.65, 19.70, 19.77, 22.70, 26.08, 26.16, 29.34, 29.38, 29.46, 29.62, 29.68, 29.74, 29.77, 30.36, 31.94, 35.84, 35.99, 37.65, 38.95, 55.29, 61.18, 62.62, 62.77, 62.83, 63.26, 67.10, 67.45, 69.27, 73.63, 75.55, 79.91, 82.62, 82.87, 84.27, 87.31, 108.29, 111.50, 111.80, 113.15, 113.35, 116.66, 116.74, 116.86, 122.51, 122.59, 123.07, 127.32, 128.08, 128.13, 129.13, 130.13, 134.95, 135.12, 136.32, 138.75, 143.10, 143.99, 147.93, 147.97, 148.04, 150.28, 150.37, 150.60, 150.69, 152.98, 155.49, 155.54, 158.81, 163.63, 163.68, 163.79, 166.02, 167.69, 167.71, 179.73, 179.77.

TOF/MS (ESI): calcd for C124H183N11O27P2Na [M+Na]+2343.2658. found 2343.2742.

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)thymidine-3′-yl 2-((3,4,5-Tris(octadecyloxy)benzoyl)oxy)acetate

Example 3-13

(1) Synthesis of 5′-O-((2-cyanoethoxy)(N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)thymidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (2.43 g, 98%) was obtained from thymidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (1.82 g, 1.50 mmol) and N2-isobutyryl-DMTr-dG-CE phosphoroamidite (2.52 g, 3.00 mmol) according to the methods of Examples 2-3.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=7.2 Hz), 1.00-1.41 (m, 90H), 1.41-1.54 (m, 6H), 1.71-1.85 (m, 6H), 1.92-2.00 (m, 3H), 2.24-2.97 (m, 6H), 3.68-4.08 (m, 8H), 4.14-4.54 (m, 6H), 4.78-4.92 (m, 2H), 5.14-5.78 (m, 2H), 5.95-6.24 (m, 2H), 7.27-7.43 (m, 3H), 7.87-7.95 (m, 1H), 10.19-10.49 (m, 2H), 12.24-12.38 (m, 1H).

13C-NMR (100 MHz, CDCl3): δ 12.35, 14.14, 19.01, 19.06, 19.10, 19.73, 19.80, 19.85, 22.70, 26.07, 26.14, 29.33, 29.38, 29.45, 29.61, 29.68, 29.74, 30.02, 30.36, 31.77, 31.94, 35.80, 35.92, 36.62, 38.19, 38.48, 61.05, 61.13, 62.26, 62.44, 62.49, 62.70, 62.75, 66.12, 67.37, 69.21, 73.58, 74.51, 80.47, 80.64, 82.33, 82.40, 85.33, 85.52, 86.12, 86.55, 86.84, 87.31, 108.24, 111.24, 112.18, 116.50, 116.83, 121.43, 121.59, 123.05, 123.08, 136.55, 137.73, 138.66, 138.99, 143.02, 143.06, 147.75, 147.78, 148.49, 150.42, 151.06, 152.96, 152.97, 155.23, 155.27, 164.76, 164.83, 165.92, 165.96, 167.73, 167.80, 180.26, 180.45.

TOF/MS (ESI): calcd for C90H149N8O18PNa [M+Na]+1684.0625. found 1684.0643.

Examples 3-14

(2) Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)thymidine-3′-yl 2-((3,4,5-Tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (2.76 g, 90%) was obtained from 5′-O-((2-cyanoethoxy)(N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)thymidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (2.20 g, 1.32 m mol) and DMTr-dT-CE-phosphoroamidite (2.96 g, 3.97 mmol) according to the methods of Examples 3-4.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.13-1.47 (m, 96H), 1.71-2.01 (m, 15H), 2.35-2.85 (m, 10H), 3.31-3.51 (m, 2H), 3.78 (s, 6H), 3.96-4.04 (m, 6H), 4.05-4.59 (m, 10H), 4.84 (s, 2H), 5.13-5.55 (m, 3H), 6.11-6.42 (m, 3H), 6.83 (d, 4H, J=8.8 Hz), 7.16-7.37 (m, 12H), 7.45-7.55 (m, 1H), 7.72-7.80 (m, 1H), 9.23-10.39 (m, 3H), 12.15-12.21 (m, 1H).

13C-NMR (100 MHz, CDCl3): δ 11.70, 12.39, 12.45, 14.14, 18.82, 18.86, 19.04, 19.08, 19.58, 19.65, 19.74, 19.80, 22.70, 26.08, 26.15, 29.34, 29.38, 29.46, 29.61, 29.68, 29.74, 29.77, 30.37, 31.94, 35.89, 36.13, 36.52, 38.68, 55.29, 61.12, 62.74, 62.89, 63.22, 63.32, 67.12, 69.23, 73.60, 74.76, 79.03, 79.98, 82.49, 84.28, 85.14, 87.28, 108.26, 111.37, 111.49, 111.69, 111.72, 113.14, 113.34, 116.74, 116.84, 116.92, 121.89, 121.96, 123.02, 127.08, 127.31, 127.76, 127.85, 128.08, 129.13, 130.11, 134.94, 134.99, 135.03, 135.20, 135.30, 136.93, 138.26, 139.46, 143.07, 143.96, 144.04, 147.98, 148.02, 148.14, 148.23, 150.46, 150.57, 150.62, 152.97, 155.42, 158.60, 158.79, 163.79, 164.43, 164.50, 165.96, 167.79, 179.87, 179.95.

TOF/MS (ESI): calcd for C124H183N11O27P2Na [M+Na]+2343.2658. found 2343.2693.

Examples 3-15

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-thymidine-3′-yl)phosphorothioyl)-thymidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

5-benzylthio-1H-tetrazole (0.58 g, 3.00 mmol) was added to a suspension of thymidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy) acetate (1.82 g, 1.50 mmol) and DMTr-dT-CE phosphoroamidite (2.23 g, 3.00 mmol) in dichloromethane (27 mL), and then stirred at room temperature for 1.5 hours. Next, ((N,N-dimethylaminomethylidene) amino)-3H-1,2,4-dithiazoline-3-thione (1.13 g, 4.50 mmol) and pyridine (0.87 g, 9.00 mmol) were added, and then stirred at room temperature for 1.5 hours. The solid precipitated by dropping acetonitrile (109 mL) to the reaction solution was filtered. The solid was washed with acetonitrile-dichloromethane mixture solvent, acetonitrile, and methanol, and then dried in vacuo at 50° C. to give the title compound (2.71 g, 96%).

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.11-1.53 (m, 93H), 1.72-1.84 (m, 6H), 1.92-1.95 (m, 3H), 2.28-2.49 (m, 3H), 2.57-2.66 (m, 2H), 2.77 (dd, 1H, J=6.0, 7.2 Hz), 3.39-3.50 (m, 2H), 3.79 (s, 6H), 3.97-4.04 (m, 6H), 4.09-4.40 (m, 6H), 4.83 (d, 2H, J=6.8 Hz), 5.31-5.43 (m, 2H), 6.27-6.32 (m, 1H), 6.35-6.41 (m, 1H), 6.82-6.86 (m, 4H), 7.22-7.32 (m, 10H), 7.37-7.39 (m, 2H), 7.56 (s, 1H), 8.69-8.91 (m, 2H).

13C-NMR (100 MHz, CDCl3): δ 11.79, 12.58, 14.14, 19.31, 19.40, 19.47, 19.55, 22.70, 26.07, 26.13, 29.32, 29.38, 29.44, 29.60, 29.68, 29.73, 30.35, 31.94, 36.78, 39.00, 55.29, 61.06, 62.65, 62.69, 62.90, 62.94, 63.21, 67.63, 69.19, 73.56, 75.01, 75.09, 80.18, 82.27, 82.36, 84.40, 84.53, 84.91, 85.39, 87.31, 108.25, 111.74, 111.90, 113.37, 116.15, 116.39, 123.06, 127.28, 128.05, 128.11, 130.09, 134.97, 135.03, 135.06, 135.30, 143.01, 144.04, 150.22, 150.27, 150.35, 152.94, 158.78, 163.36, 163.46, 165.86, 165.88, 167.71, 167.76.

TOF/MS (ESI): calcd for C107H162N5O19PSNa [M+Na]+1907.1220. found 1907.1223.

4. Synthesis of N4-benzoyl-5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-2′-O-methyluridine-3′-yl)phosphoryl)-2′-O-methylcytidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

Example 4-1

(1) Synthesis of N4-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methylcytidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (2.96 g, 91%) was obtained from 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetic acid (1.97 g, 2.00 mmol) and N4-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methylcytidine (1.59 g, 2.40 mmol) according to the procedure of Example 2-1.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.10-1.38 (m, 84H), 1.42-1.50 (m, 6H), 1.70-1.85 (m, 6H), 3.48 (d, 1H, J=11.6 Hz), 3.62 (s, 3H), 3.69 (d, 1H, J=11.2 Hz), 3.71 (s, 1H), 3.83 (s, 5H), 3.97-4.04 (m, 6H), 4.18 (d, 1H, J=4.8 Hz), 4.36 (d, 1H, J=8.4 Hz), 4.80-4.89 (m, 2H), 5.29 (dd, 1H, J=4.8 Hz, 8.4 Hz), 6.11 (d, 1H, J=1.6 Hz), 6.90 (dd, 4H, J=2.0, 8.8 Hz), 7.27-7.42 (m, 11H), 7.52 (t, 2H, J=7.6 Hz), 7.62 (t, 1H, J=7.6 Hz), 7.89 (d, 2H, J=7.6 Hz), 8.55 (d, 1H, J=6.4 Hz), 8.61-8.72 (br s, 1H).

13C-NMR (100 MHz, CDCl3): δ 14.14, 22.70, 26.07, 26.09, 26.11, 29.31, 29.38, 29.43, 29.59, 29.66, 29.68, 29.73, 30.34, 31.94, 52.31, 55.24, 59.15, 60.47, 60.72, 61.06, 69.14, 70.07, 73.53, 80.42, 82.21, 87.49, 88.91, 108.22, 113.43, 123.33, 123.50, 127.27, 127.49, 128.17, 129.06, 130.06, 130.08, 133.17, 135.07, 135.22, 142.85, 143.86, 152.87, 152.90, 158.75, 165.64, 165.84, 167.17, 168.42.

TOF/MS (ESI): calcd for C101H151N3O14Na [M+Na]+1653.1094. found 1653.1171.

Example 4-2

(2) Synthesis of N4-benzoyl-2′-O-methylcytidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (2.26 g, 99%) was obtained from N4-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methylcytidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (2.80 g, 1.72 mmol) according to the methods of Examples 2-2.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.10-1.55 (m, 90H), 1.70-1.85 (m, 6H), 3.51 (s, 3H), 3.79-3.83 (m, 2H), 3.95-4.08 (m, 7H), 4.34 (d, 1H, J=4.8 Hz), 4.50 (t, 1H, J=4.8H z), 4.83-4.90 (m, 2H), 5.44 (t, 1H, J=4.8 Hz), 5.73 (d, 1H, J=4.4 Hz), 7.29-7.30 (m, 2H), 7.47-7.66 (m, 3H), 7.90 (d, 2H, J=7.6 Hz), 8.16 (d, 1H, J=7.6 Hz), 8.75-9.00 (brs, 1H).

13C-NMR (100 MHz, CDCl3): δ 14.14, 22.71, 26.07, 26.11, 29.30, 29.38, 29.42, 29.59, 29.66, 29.68, 29.73, 30.34, 31.94, 52.32, 59.19, 60.97, 61.07, 61.24, 69.15, 69.20, 71.18, 73.56, 80.58, 83.43, 92.64, 108.28, 123.20, 123.50, 127.61, 129.09, 132.80, 133.36, 142.99, 147.07, 152.87, 152.93, 162.63, 165.85, 167.66, 168.43.

TOF/MS (ESI): calcd for C80H133N3O12Na [M+Na]+1350.9787. found 1350.9855.

Example 4-3

(3) Synthesis of N4-benzoyl-5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-2′-O-methyluridine-3′-yl)phosphoryl)-2′-O-methylcytidine-3′-yl 2-((3,4,5-Tris(octadecyloxy)benzoyl)oxy)acetate

A crude compound (2.80 g) was obtained from N4-benzoyl-2′-O-methylcytidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (2.10 g, 1.58 mmol) and 5′-O-DMTr-2′-O-methyluridine-CE phosphoro amidite (2.40 g, 3.16 mmol) according to the methods of Examples 3-4. The crude compound was purified by column chromatography (spherical neutral silica gel, eluent: dichloromethane-methanol) to give the title compound (2.21 g, 70%).

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.10-1.53 (m, 90H), 1.71-1.83 (m, 6H), 2.50-2.74 (m, 1H), 2.79 (t, 1H, J=6.4 Hz), 3.46-3.76 (m, 8H), 3.77 (s, 3H), 3.79 (s, 3H), 3.97-4.04 (m, 6H), 4.11-4.58 (m, 7H), 4.82-4.95 (m, 2H), 5.13-5.30 (m, 3H), 5.86-6.08 (m, 2H), 6.82-6.88 (m, 4H), 7.23-7.36 (m, 11H), 7.51 (t, 2H, 8.0 Hz), 7.61 (t, 1H, J=7.2 Hz), 7.82-8.05 (m, 4H), 8.69-8.96 (m, 2H).

13C-NMR (100 MHz, CDCl3): δ 14.14, 19.52, 19.58, 22.70, 26.07, 26.13, 29.33, 29.38, 29.45, 29.60, 29.68, 29.73, 30.35, 31.94, 55.25, 55.30, 58.84, 59.27, 60.82, 61.14, 62.35, 62.40, 62.68, 62.73, 65.55, 66.16, 69.19, 69.90, 70.16, 73.56, 74.28, 74.38, 79.38, 79.46, 79.52, 79.60, 81.32, 81.38, 81.52, 82.19, 86.71, 86.87, 87.46, 90.07, 90.64, 102.58, 102.62, 108.24, 113.34, 113.36, 116.36, 116.43, 123.19, 127.39, 127.44, 127.64, 128.10, 128.12, 128.26, 128.29, 129.04, 130.21, 130.27, 130.30, 133.28, 134.64, 134.73, 139.53, 139.75, 142.96, 143.82, 143.90, 150.02, 150.07, 152.93, 158.81, 158.84, 158.86, 162.70, 162.73, 165.77, 165.81, 167.22, 167.26.

TOF/MS (ESI): calcd for C114H167N6O22PNa [M+Na]+2026.1769. found 2026.1764.

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-2′-fluorodeoxyuridine-3′-yl)phosphoryl)-2′-fluoro-N2-isobutyryldeoxyguanosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate [Example 4-4]

(1) Synthesis of 5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-N2-isobutyryldeoxyguanosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (3.13 g, 96%) was obtained from 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetic acid (1.97 g, 2.00 mmol) and 5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-N2-isobutyryldeoxyguanosine (1.58 g, 2.40 mmol) according to the procedure of Example 2-1.

1H-NMR (400 MHz, CDCl3): δ 0.81 (d, 3H, J=6.8 Hz), 0.88 (t, 9H, J=6.8 Hz), 0.94 (d, 3H, J=7.2 Hz), 1.10-1.53 (m, 90H), 1.70-1.87 (m, 6H), 1.93 (sept, 1H, J=6.8 Hz), 3.17 (d, 1H, J=11.2H z), 3.59 (d, 1H, J=11.2 Hz), 3.77 (s, 6H), 3.97-4.05 (m, 6H), 4.32-4.34 (m, 1H), 4.85 (d, 1H, J=16.0 Hz), 4.92 (d, 1H, J=16.0 Hz), 5.92-6.07 (m, 2H), 6.18-6.24 (m, 1H), 6.78 (dd, 4H, J=5.2, 8.8 Hz), 7.16-7.29 (m, 7H), 7.30 (s, 2H), 7.36 (d, 2H, J=8.0 Hz), 7.79 (s, 1H), 8.01 (s, 1H), 11.92 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ 14.14, 18.39, 18.47, 18.88, 18.91, 22.70, 25.61, 26.06, 26.11, 29.30, 29.38, 29.42, 29.59, 29.66, 29.68, 29.73, 30.34, 31.94, 36.25, 36.43, 55.24, 55.25, 60.68, 60.84, 61.24, 67.98, 69.25, 70.85, 71.00, 73.61, 79.97, 81.44, 83.15, 86.36, 86.47, 86.67, 90.96, 108.26, 113.15, 113.22, 122.23, 123.17, 127.08, 127.13, 127.76, 127.85, 127.89, 127.98, 129.13, 129.85, 129.92, 135.20, 135.66, 138.43, 138.70, 139.43, 143.05, 144.47, 147.30, 147.34, 147.47, 147.90, 152.93, 152.96, 155.04, 155.31, 158.62, 158.69, 165.65, 165.89, 167.23, 167.60, 178.18, 178.76.

TOF/MS (ESI): calcd for C98H150N5O13F [M+H]+ 1623.1136. found 1623.1160.

Example 4-5

(2) Synthesis of 2′-fluoro-N2-isobutyryldeoxyguanosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (2.40 g, 98%) was obtained from 5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-N2-isobutyryldeoxyguanosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (3.00 g, 1.85 mmol) according to the procedure of Example 2-2.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.10-1.55 (m, 96H), 1.70-1.85 (m, 6H), 2.71 (sept, 1H, J=6.8 Hz), 3.37-3.72 (brs, 2H), 3.80 (d, 1H, J=11.6 Hz), 3.99-4.05 (m, 7H), 4.34 (d, 1H, J=4.0 Hz), 4.94 (s, 2H), 5.69 (dt, 1H, J=4.4, 52.0 Hz), 5.78-5.84 (m, 1H), 5.97 (dd, 1H, J=4.0, 16.0 Hz), 7.31 (s, 2H), 7.90 (s, 1H), 9.22 (s, 1H), 12.15 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ 14.14, 18.85, 18.95, 22.70, 26.06, 26.11, 29.30, 29.38, 29.43, 29.60, 29.68, 29.74, 30.33, 31.94, 36.32, 60.86, 69.24, 71.25, 71.39, 73.60, 82.89, 87.22, 87.55, 89.60, 91.55, 108.27, 121.56, 123.15, 138.45, 142.99, 147.29, 148.08, 152.91, 155.03, 165.91, 167.26, 179.15.

TOF/MS (ESI): calcd for C77H132N5O11FNa [M+Na]+1344.9805. found 1344.9834.

Examples 4-6

(3) Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-2′-fluorodeoxyuridine-3′-yl)phosphoryl)-2′-fluoro-N2-isobutyryldeoxyguanosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (3.07 g, 93%) was obtained from 2′-fluoro-N2-isobutyryldeoxyguanosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl) oxy)acetate (2.20 g, 1.66 mmol) and 5′-O-DMTr-2′-fluoro-deoxyuridine-CE phosphoroamidite (2.49 g, 3.33 mmol) according to the methods of Examples 3-4.

1H-NMR (400 MHz, CDCl3): 0.88 (t, 9H, J=7.2 Hz), 0.97-1.53 (m, 96H), 1.71-1.83 (m, 6H), 2.56-2.75 (m, 3H), 3.37-3.66 (m, 2H), 3.74-3.79 (m, 6H), 3.97-4.55 (m, 12H), 4.78-5.08 (m, 2H), 5.08-5.50 (m, 3H), 5.70-6.01 (m, 4H), 6.80-6.84 (m, 4H), 7.17-7.40 (m, 11H), 7.57-7.73 (m, 2H), 8.94-9.07 (m, 1H), 10.00 (s, 1H), 12.18-12.26 (m, 1H).

δ13C-NMR (100 MHz, CDCl3): δ 14.14, 18.51, 18.76, 18.92, 19.20, 19.55, 19.62, 22.70, 26.07, 26.14, 29.33, 29.38, 29.45, 29.60, 29.68, 29.74, 30.36, 31.94, 35.90, 35.99, 55.23, 55.30, 60.70, 60.88, 62.85, 69.26, 73.61, 79.54, 79.63, 87.17, 87.21, 102.93, 108.31, 113.31, 116.34, 116.52, 122.55, 122.61, 122.95, 122.98, 127.25, 127.31, 128.07, 128.10, 130.14, 134.79, 134.88, 134.92, 138.65, 140.55, 143.15, 143.19, 143.92, 144.02, 147.71, 147.74, 148.30, 148.43, 149.68, 149.98, 152.98, 152.99, 155.30, 155.48, 158.74, 158.79, 162.67, 165.89, 166.00, 167.20, 179.68.

TOF/MS (ESI): calcd for C110H163N8O20F2PNa [M+Na]+2008.1587. found 2008.1593.

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-2′-O-(tert-butyldimethylsilyl)uridine-3′-yl)phosphoryl)-2′-O-(tert-butyldimethylsilyl)uridine-3′-yl 2-((3,4,5-Tris(octadecyloxy)benzoyl)oxy)acetate [Examples 4-7]

(1) Synthesis of 5′-O-(4,4′-dimethoxytrityl)-2′-O-(tert-butyldimethylsilyl)uridine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

4-dimethylaminopyridine (0.03 g, 0.25 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.58 g, 3.00 mmol) was added to a suspension of 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy) acetic acid (2.46 g, 2.50 mmol) and 5′-O-(4,4′-dimethoxytrityl)-2′-O-(tert-butyldimethylsilyl)uridine (1.98 g, 3.00 mmol) in dichloromethane (25 mL), and then stirred at room temperature for 5 hours. The solid precipitated by dropping acetonitrile (148 mL) to the reaction solution was filtered. The solid was washed with acetonitrile-dichloromethane mixture solvent, and then dried under reduced pressure at 50° C. to obtain a crude compound (2.89 g). The crude compound was purified by column chromatography (spherical neutral silica gel, eluent: dichloromethane-THF) to give the title compound (2.76 g, 68%) as a white solid.

1H-NMR (400 MHz, CDCl3): δ 0.08 (s, 3H), 0.11 (s, 3H), 0.81-0.92 (m, 18H), 1.04-1.53 (m, 90H), 1.70-1.84 (m, 6H), 3.49 (dd, 2H, J=10.8, 27.6 Hz), 3.79 (s, 6H), 3.79-4.04 (m, 6H), 4.26-4.27 (m, 1H), 4.56 (t, 1H, J=5.2 Hz), 4.81 (d, 1H, J=16.0 Hz), 4.96 (d, 1H, 16.4 Hz), 5.30 (d, 1H, J=8.0 Hz), 5.47 (t, 1H, J=4.0 Hz), 6.02 (d, 1H, J=5.6 Hz), 6.85 (d, 4H, J=8.4 Hz), 7.23-7.37 (m, 11H), 7.85 (d, 1H, J=8.4 Hz), 8.37 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ −5.22, −5.09, 14.14, 17.87, 22.71, 25.51, 26.07, 26.11, 29.30, 29.38, 29.43, 29.59, 29.67, 29.74, 30.34, 31.94, 55.25, 60.75, 62.47, 69.14, 73.36, 73.54, 74.40, 81.20, 87.58, 88.03, 102.56, 108.25, 113.40, 113.43, 123.33, 127.33, 128.03, 128.14, 130.03, 130.17, 134.71, 134.83, 140.00, 142.87, 144.10, 150.14, 152.89, 158.78, 158.83, 162.59, 165.67, 167.10.

TOF/MS (ESI): calcd for C99H158N2O14SiNa [M+Na]+1650.1380. found 1650.1455.

Examples 4-8

(2) Synthesis of 2′-O-(tert-butyldimethylsilyl)uridine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (2.05 g, 97%) was obtained from 5′-O-(4,4′-dimethoxytrityl)-2′-O-(tert-butyldimethylsilyl)uridine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (2.60 g, 1.60 mmol) according to the procedure of Example 2-2.

1H-NMR (400 MHz, CDCl3): δ 0.04 (s, 3H), 0.07 (s, 3H), 0.81-0.92 (m, 18H), 1.11-1.53 (m, 90H), 1.70-1.85 (m, 6H), 2.95-3.10 (brs, 1H), 3.77-3.82 (m, 1H), 3.93-4.04 (m, 7H), 4.25-4.28 (m, 1H), 4.67 (t, 1H, J=5.2 Hz), 4.81 (d, 1H, 16.0 Hz), 4.96 (d, 1H, 16.0 Hz), 5.34 (dd, 1H, J=3.2, 4.8 Hz), 5.60 (d, 1H, J=5.6 Hz), 5.76 (d, 1H, J=8.0 Hz), 7.29 (s, 2H), 7.63 (d, 1H, J=8.0 Hz), 8.71 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ −5.22, −5.19, 14.14, 17.88, 22.71, 25.52, 26.07, 26.10, 29.30, 29.38, 29.42, 29.59, 29.66, 29.68, 29.73, 30.34, 31.94, 60.84, 61.84, 69.20, 72.78, 73.24, 73.57, 82.99, 92.46, 102.70, 108.32, 123.24, 141.88, 142.99, 150.17, 152.92, 162.72, 165.78, 167.38.

TOF/MS (ESI): calcd for C78H140N2O12SiNa [M+Na]+1348.0073. found 1348.0115.

Examples 4-9

(3) Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-2′-O-(tert-butyldimethylsilyl)uridine-3′-yl)phosphoryl)-2′-O-(tert-butyldimethylsilyl)uridine-3′-yl 2-((3,4,5-Tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (2.84 g, 92%) was obtained from 2′-O-(tert-butyldimethylsilyl)uridine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (1.95 g, 1.47 mmol) and 5′-O-DMTr-2′-O-(tert-butyldimethylsilyl)uridine-CE phosphoroamidite (2.53 g, 2.94 mmol) according to the methods of Examples 3-4.

1H-NMR (400 MHz, CDCl3): δ 0.03-0.13 (m, 12H), 0.86-0.90 (m, 27H), 1.02-1.53 (m, 90H), 1.68-1.84 (m, 6H), 2.45-2.69 (m, 1H), 2.73 (t, 1H, J=6.0 Hz), 3.48 (d, 1H, J=10.8 Hz), 3.60-3.68 (m, 1H), 3.76-3.84 (m, 6H), 3.97-4.04 (m, 6H), 4.10-4.58 (m, 8H), 4.73-4.84 (m, 1H), 4.90-5.03 (m, 2H), 5.20-5.25 (m, 2H), 5.67-5.78 (m, 2H), 5.95-6.03 (m, 1H), 6.80-6.89 (m, 4H), 7.21-7.44 (m, 12H), 7.84 (d, 1H, J=8.0 Hz), 8.72-8.83 (m, 2H)

13C-NMR (100 MHz, CDCl3): δ −5.27, −5.24, −5.07, −4.94, −4.92, 14.14, 17.86, 18.06, 19.50, 19.57, 19.65, 22.70, 25.50, 25.55, 25.60, 26.07, 26.13, 29.32, 29.38, 29.44, 29.60, 29.68, 29.74, 30.35, 31.94, 55.27, 55.30, 60.69, 61.96, 62.37, 62.42, 62.58, 62.63, 69.19, 71.39, 71.60, 72.86, 73.00, 73.57, 74.47, 79.43, 79.51, 81.75, 87.65, 87.68, 87.99, 88.18, 90.56, 91.22, 102.54, 102.64, 103.05, 103.13, 108.28, 113.39, 116.31, 116.40, 123.21, 127.43, 128.13, 128.21, 128.25, 130.25, 130.27, 130.32, 134.56, 134.65, 134.70, 134.73, 139.84, 140.05, 140.15, 142.97, 143.01, 143.92, 143.97, 149.95, 150.19, 150.40, 152.92, 158.83, 158.87, 162.53, 162.58, 162.69, 165.64, 165.68, 167.05, 167.12.

TOF/MS (ESI): calcd for C117H186N5O22Si2PNa [M+Na]+2123.2763. found 2123.2769.

5. Synthesis of 3-((3,4,5-tris(octadecyloxy)benzoyl)oxy)propionic Acid

Example 5-1

(1) Synthesis of tert-butyl 3-((3,4,5-tris(octadecyloxy)benzoyl)oxy)propionate

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.86 g, 4.5 mmol) was added to a suspension of 3,4,5-tris(octadecyloxy)benzoic acid (2.78 g, 3.0 mmol), 4-dimethylaminopyridine (0.04 g, 0.3 mmol), tert-butyl 3-hydroxypropionate (4.39 g, 30.0 mmol), and triethylamine (0.46 g, 4.5 mmol) in dichloromethane (60 mL), and then stirred at room temperature for 20 hours. The solid precipitated by dropping acetonitrile (180 mL) to the reaction solution was filtered. The solid was washed with acetonitrile-dichloromethane mixture sol vent, and then dried under reduced pressure at 50° C. to obtain a cru de compound (3.07 g). The crude compound was purified by column chromatography (spherical neutral silica gel, eluent: dichloromethane) to afford the title compound (1.90 g, 60%) as a white solid.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=7.0 Hz), 1.15-1.55 (m, 99H), 1.70-1.85 (m, 6H), 2.68 (t, 2H, J=6.4 Hz), 3.95-4.05 (m, 6H), 4.53 (t, 2H, J=6.4 Hz), 7.23 (s, 2H).

13C-NMR (100 MHz, CDCl3): δ 14.08, 22.68, 26.08, 28.09, 29.35, 29.40, 29.57, 29.65, 29.71, 30.35, 31.93, 35.42, 60.72, 69.25, 73.51, 80.97, 108.19, 124.57, 152.84, 166.14, 169.93.

TOF/MS (ESI): calcd for C68H126O7Na [M+Na]+1077.9401. found 1077.9427.

Example 5-2

(2) Synthesis of 3-((3,4,5-tris(octadecyloxy)benzoyl)oxy)propionic Acid

Methanesulfonic acid (3.75 g, 39.0 mmol) was added to a solution of tert-butyl 3-((3,4,5-tris(octadecyloxy)benzoyl)oxy)propionate (2.75 g, 2.6 mmol) in dichloromethane (52 mL), and then stirred at room temperature for 20 hours. The solid precipitated by dropping acetonitrile (156 mL) to the reaction solution was filtered. The solid was washed with acetonitrile-dichloromethane mixture solvent and acetonitrile, and then dried in vacuo at 50° C. to give the title compound (2.54 g, 98%) as a white solid.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=7.0 Hz), 1.15-1.55 (m, 90H), 1.70-1.85 (m, 6H), 2.85 (t, 2H, J=6.6 Hz), 3.95-4.10 (m, 6H), 4.57 (t, 2H, J=6.4 Hz), 7.23 (s, 2H).

13C-NMR (100 MHz, CDCl3): δ 14.10, 22.71, 26.10, 26.13, 29.38, 29.45, 29.61, 29.68, 29.75, 30.38, 31.96, 33.54, 59.99, 69.34, 73.57, 108.35, 124.32, 142.88, 152.91, 166.17.

TOF/MS (ESI): calcd for C64H118O7Na [M+Na]+1021.8775. found 1021.8817.

Synthesis of 2-(N-methyl-3,4,5-tris(octadecyloxy)benzamide)acetic Acid

Example 5-3

(1) Synthesis of tert-butyl 2-(N-methyl-3,4,5-tris(octadecyloxy) benzamido)acetate

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.86 g, 4.5 mmol) was added to a suspension of 3,4,5-tris(octadecyloxy)benzoic acid (2.78 g, 3.0 mmol), 4-dimethylaminopyridine (0.04 g, 0.3 mmol), tert-butyl sarcosinate hydrochloride (2.73 g, 15.0 mmol), and triethylamine (1.52 g, 15.0 mmol) in dichloromethane (60 mL), and then stirred at room temperature for 20 hours. The solid precipitated by dropping acetonitrile (180 mL) to the reaction solution was filtered. The solid was washed with acetonitrile-dichloromethane mixture solvent, and then dried under reduced pressure at 50° C. to obtain a crude compound (3.07 g). The crude compound was purified by column chromatography (spherical neutral silica gel, eluent: dichloromethane-THF) to give the title compound (2.66 g, 84%) as a white solid.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=7.0 Hz), 1.15-1.55 (m, 99H), 1.70-1.85 (m, 6H), 3.04 (s, 3H), 3.80-4.20 (m, 8H), 6.63 (s, 2H).

13C-NMR (100 MHz, CDCl3): δ 14.01, 22.66, 26.13, 26.15, 28.16, 29.35, 29.42, 29.48, 29.65, 29.71, 30.39, 31.93, 69.51, 73.52, 82.02, 106.19, 130.61, 139.93, 153.25, 168.33, 172.01.

TOF/MS (ESI): calcd for C68H127NO6Na [M+Na]+1076.9561. found 1076.9591.

Example 5-4

(2) Synthesis of 2-(N-methyl-3,4,5-tris(octadecyloxy)benzamide)acetic Acid

The title compound (2.38 g, 99%) was obtained from tert-butyl 2-(N-methyl-3,4,5-tris(octadecyloxy)benzamido)acetate (2.53 g, 2.4 mmol) as white solids according to the procedure of Examples 5-2.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=7.0 Hz), 1.15-1.55 (m, 90H), 1.70-1.85 (m, 6H), 3.11 (s, 3H), 3.85-4.35 (m, 8H), 6.15-6.30 (brs, 1H), 6.66 (s, 2H).

13C-NMR (100 MHz, CDCl3): δ 14.05, 22.67, 26.12, 29.36, 29.43, 29.61, 29.66, 29.72, 29.98, 30.36, 31.93, 39.20, 50.02, 69.49, 73.56, 106.47, 129.16, 140.27, 152.97, 153.24, 172.11, 172.92.

TOF/MS (ESI): calcd for C64H118NO6[M−H]−996.8959. found 996.8947.

Synthesis of 3-(3,4,5-Tris(octadecyloxy)benzamide)propionic Acid

Example 5-5

(1) Synthesis of tert-butyl 3-(3,4,5-Tris(octadecyloxy)benzamide)propionate

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.86 g, 4.5 mmol) was added to a suspension of 3,4,5-tris(octadecyloxy)benzoic acid (2.78 g, 3.0 mmol), 4-dimethylaminopyridine (0.04 g, 0.3 mmol), 3-alanine tert-butyl ester hydrochloride (2.73 g, 15.0 mmol), and triethylamine (1.52 g, 15.0 mmol) in dichloromethane (60 mL), and then stirred at room temperature for 19.5 hours. The solid precipitated by dropping acetonitrile (180 mL) to the reaction solution was filtered. The solid was washed with acetonitrile-dichloromethane mixture solvent, and then dried under reduced pressure at 50° C. to obtain a crude compound (3.12 g). The crude compound was purified by column chromatography (spherical neutral silica gel, eluent: dichloromethane-THF) to give the title compound (2.75 g, 87%) as a white solid.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=7.0 Hz), 1.15-1.55 (m, 99H), 1.70-1.85 (m, 6H), 2.55 (t, 2H, J=5.8 Hz), 3.66 (q, 2H, J=6.0 Hz), 3.95-4.05 (m, 6H), 6.72 (t, 1H, J=6.0 Hz), 6.94 (s, 2H).

13C-NMR (100 MHz, CDCl3): δ 14.09, 22.71, 26.11, 28.16, 29.38, 29.43, 29.61, 29.68, 29.74, 30.36, 31.95, 35.18, 35.58, 69.45, 73.53, 81.19, 105.86, 129.57, 141.32, 153.14, 167.20, 172.27.

TOF/MS (ESI): calcd for C68H127NO6Na [M+Na]+1076.9561. found 1076.9579.

Example 5-6

(2) Synthesis of 3-(3,4,5-Tris(octadecyloxy)benzamide)propionic Acid

The title compound (2.52 g, 101%) was obtained from tert-butyl 3-(3,4,5-tris(octadecyloxy)benzamido)propionate (2.64 g, 2.5 mmol) as white solids according to the procedure of Examples 5-2.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=7.0 Hz), 1.15-1.55 (m, 90H), 1.65-1.85 (m, 6H), 2.74 (t, 2H, J=5.8 Hz), 3.72 (t, 2H, J=5.8 Hz), 3.90-4.05 (m, 6H), 6.95 (s, 2H).

13C-NMR (100 MHz, CDCl3): δ 14.06, 22.71, 26.16, 26.19, 29.40, 29.50, 29.53, 29.65, 29.71, 29.77, 30.44, 31.98, 33.68, 35.85, 39.37, 69.77, 73.67, 106.51, 128.33, 142.29, 153.35, 168.43, 175.46.

TOF/MS (ESI): calcd for C64H118NO6[M−H]−996.8959. found 996.8912.

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl 3-((3,4,5-tris(octadecyloxy)benzoyl)oxy)propionate

Examples 5-7

(1) Synthesis of thymidine-3′-yl 3-((3,4,5-tris(octadecyloxy)benzoyl)oxy)propionate

COMU (2.06 g, 4.80 mmol) was added to a suspension of 3-((3,4,5-tris(octadecyloxy)benzoyl)oxy)propionic acid (2.40 g, 2.40 mmol), 5′-O-(4,4′-dimethoxytrityl)thymidine (1.57 g, 2.88 mmol), and 1-methylimidazole (0.99 g, 12.00 mmol) in THF (24 mL), and then stirred at room temperature for 6.5 hours. Further, COMU (0.21 g, 0.48 mmol) was added, and then stirred at room temperature for 17.5 hours. The solid precipitated by dropping acetonitrile (120 mL) to the re action solution was filtered. The solid was washed with acetonitrile-THF mixture solvent, and then dried under reduced pressure at 50° C. to obtain a crude compound (2.76 g). The crude compound was used for the next reaction without purification.

Pyrrole (0.57 g, 8.50 mmol) and trifluoroacetic acid (0.24 g, 2.13 mmol) were added to a dichloromethane solution (68 mL) of the c rude compound (2.60 g), and then stirred at room temperature for 3.5 hours. Further, trifluoroacetic acid (0.05 g, 0.43 mmol) was added, and then stirred at room temperature for 1.5 hours. The solid precipitated by dropping acetone (20 mL) and acetonitrile (61 mL) to the re action solution was filtered. The solid was washed with acetonitrile-acetone-dichloromethane mixture solvent and acetonitrile, and then dried under reduced pressure at 50° C. to obtain a crude compound (2.44 g). The crude compound was purified by column chromatography (spherical neutral silica gel, eluent: dichloromethane-THF) to give the title compound (0.52 g, 18%) as a white solid.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.15-1.55 (m, 90H), 1.65-1.85 (m, 7H), 1.92 (s, 3H), 2.30-2.40 (m, 1H), 2.45-2.55 (m, 1H), 2.60-2.70 (m, 1H), 2.80-2.90 (m, 2H), 3.85-3.95 (m, 2H), 3.95-4.05 (m, 6H), 4.05-4.10 (m, 1H), 4.55-0.65 (m, 2H), 5.40-5.55 (m, 1H), 6.10-6.20 (m, 1H), 7.22 (s, 2H), 7.41 (s, 1H), 8.58-8.64 (m, 1H).

13C-NMR (100 MHz, CDCl3): δ 12.57, 14.14, 22.71, 26.07, 26.11, 29.33, 29.38, 29.43, 29.59, 29.67, 29.74, 30.02, 30.34, 31.94, 34.16, 36.90, 60.05, 62.58, 69.24, 73.54, 75.25, 85.10, 86.67, 108.06, 111.42, 124.14, 136.56, 142.69, 150.26, 152.87, 163.40, 166.10, 170.43.

TOF/MS (ESI): calcd for C74H130N2O11Na [M+Na]+1245.9572. found 1245.9548.

Example 5-8

(2) Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl 3-((3,4,5-tris(octadecyloxy)benzoyl)oxy)propionate

The title compound (2.22 g, 96%) was obtained as a white solid from thymidine-3′-yl 3-((3,4,5-tris((octadecyloxy)benzoyl)oxy)propionate (1.50 g, 1.23 mmol) and DMTr-dT-CE phosphoroamidite (1.83 g, 2.45 mmol) according to the methods of Examples 3-4.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.10-1.38 (m, 84H), 1.41-1.50 (m, 9H), 1.70-1.83 (m, 7H), 1.91 (s, 3H), 2.30-2.46 (m, 3H), 2.62-2.68 (m, 2H), 2.75 (t, 1H, J=6.0 Hz), 2.84 (dd, 2H, J=4.8, 6.4 Hz), 3.37-3.42 (m, 1H), 3.50-3.55 (m, 1H), 3.79 (s, 6H), 3.98-4.02 (m, 6H), 4.12-4.38 (m, 6H), 4.56 (dd, 2H, J=4.8, 7.2 Hz), 5.16-5.20 (m, 1H), 5.28-5.35 (m, 1H), 6.21-6.26 (m, 1H), 6.38-6.44 (m, 1H), 6.84 (d, 4H, J=7.6 Hz), 7.21-7.32 (m, 10H), 7.34-7.37 (m, 2H), 7.50-7.59 (m, 1H), 8.91-9.06 (m, 2H).

13C-NMR (100 MHz, CDCl3): δ 11.72, 12.46, 14.13, 19.59, 19.66, 19.75, 22.70, 26.08, 26.15, 29.37, 29.46, 29.61, 29.67, 29.74, 30.36, 31.94, 33.94, 36.62, 38.95, 55.29, 59.89, 59.93, 62.39, 62.45, 62.54, 62.59, 63.23, 67.62, 69.27, 73.55, 74.09, 79.77, 79.82, 79.87, 79.92, 82.52, 84.29, 84.34, 84.46, 84.53, 85.36, 85.57, 87.34, 108.14, 111.70, 111.82, 113.36, 116.17, 116.36, 124.17, 127.34, 128.08, 128.14, 130.13, 134.94, 135.03, 135.07, 135.35, 135.49, 142.74, 143.97, 143.99, 150.23, 150.27, 150.38, 150.46, 152.89, 158.84, 163.40, 163.50, 166.14, 170.29, 170.34.

TOF/MS (ESI): calcd for C108H164N5O20PNa [M+Na]+1905.1605. found 1905.1636.

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl 2-(N-methyl-3,4,5-tris(octadecyloxy)benzamide)acetate

Examples 5-9

(1) Synthesis of 5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl 2-(N-methyl-3,4,5-tris(octadecyloxy)benzamide)acetate

COMU (1.80 g, 4.20 mmol) was added to a suspension of 2-(N-methyl-3,4,5-tris(octadecyloxy)benzamide)acetic acid (2.10 g, 2.10 m mol), 5′-O-(4,4′-dimethoxytrityl)thymidine (1.37 g, 2.52 mmol) and 1-methylimidazole (0.86 g, 10.50 mmol) in THF (32 mL), and stirred at room temperature for 3.5 hours. Further, COMU (0.27 g, 0.63 mmol) was added, and then stirred at room temperature for 20 hours. The solid precipitated by dropping acetonitrile (158 mL) to the reaction solution was filtered. The solid was washed with acetonitrile-THF mixture solvent, and then dried under reduced pressure at 50° C. to obtain a crude compound (2.96 g). The crude compound was purified by column chromatography (spherical neutral silica gel, eluent: dichloro methane-THF) to give the title compound (2.51 g, 78%) as a white solid.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.15-1.55 (m, 93H), 1.65-1.90 (m, 6H), 2.35-2.60 (m, 2H), 3.08 (s, 3H), 3.45-3.55 (m, 2H), 3.79 (s, 6H), 3.85-4.00 (m, 6H), 4.10-4.40 (m, 3H), 5.50-5.60 (m, 1H), 6.40-6.50 (m, 1H), 6.65 (s, 2H), 6.80-6.90 (m, 4H), 7.20-7.40 (m, 9H), 7.62 (s, 1H), 8.36 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ 11.64, 14.07, 22.71, 26.17, 29.38, 29.48, 29.65, 29.70, 29.75, 30.41, 31.97, 38.01, 55.30, 63.79, 69.54, 73.58, 76.72, 84.09, 84.54, 87.42, 106.36, 111.64, 113.49, 127.31, 128.09, 128.26, 129.90, 130.15, 135.30, 135.38, 140.18, 144.29, 150.20, 153.29, 159.01, 163.21, 168.70, 172.20.

TOF/MS (ESI): calcd for C95H149N3O12Na [M+Na]+1547.1039. found 1547.1042.

Example 5-10

(2) Synthesis of thymidine-3′-yl 2-(N-methyl-3,4,5-tris(octadecyloxy)benzamide)acetate

The title compound (1.86 g, 98%) was obtained as a white solid from 5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl 2-(N-methyl-3,4,5-tris(octadecyloxy)benzamide)acetate (2.36 g, 1.55 mmol) according to the method of Example 2-2.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.05-1.55 (m, 90H), 1.65-1.85 (m, 6H), 1.92 (s, 3H), 2.30-2.60 (m, 3H), 3.09 (s, 3H), 3.85-4.05 (m, 8H), 4.05-4.35 (m, 3H), 5.45 (s, 1H), 6.05-6.30 (brs, 1H), 6.64 (s, 2H), 7.44 (s, 1H), 8.28 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ 12.50, 14.08, 22.71, 26.17, 29.38, 29.47, 29.51, 29.65, 29.70, 29.75, 30.41, 31.97, 37.20, 39.06, 62.67, 69.59, 73.60, 75.74, 85.09, 86.62, 106.33, 111.45, 129.86, 136.38, 140.23, 150.26, 153.33, 163.22, 168.96, 172.29.

TOF/MS (ESI): calcd for C74H131N3O10Na [M+Na]+1244.9732. found 1244.9786.

Example 5-11

(3) Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl 2-(N-methyl-3,4,5-tris(octadecyloxy)benzamide)acetate

The title compound (2.54 g, 96%) was obtained from thymidine-3′-yl 2-(N-methyl-3,4,5-tris(octadecyloxy)benzamide) acetate (1.71 g, 1.40 mmol) and DMTr-dT-CE-phosphoroamidite (2.09 g, 2.80 mmol) as white solids according to the methods of Examples 3-4.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.6 Hz), 1.05-1.55 (m, 93H), 1.65-1.85 (m, 6H), 1.91 (s, 3H), 2.30-2.55 (m, 3H), 2.60-2.80 (m, 3H), 3.08 (s, 3H), 3.35-3.45 (m, 1H), 3.50-3.60 (m, 1H), 3.78 (s, 3H), 3.79 (s, 3H), 3.90-4.05 (m, 6H), 4.10-4.45 (m, 8H), 5.10-5.20 (m, 1H), 5.30-5.45 (m, 1H), 6.10-6.30 (brs, 1H), 6.35-6.45 (m, 1H), 6.64 (s, 2H), 6.75-6.90 (m, 4H), 7.15-7.40 (m, 10H), 7.49 (d, 1H, J=6.4 Hz), 8.50-8.75 (m, 2H).

13C-NMR (100 MHz, CDCl3): δ 11.76, 12.41, 12.46, 14.07, 19.63, 19.70, 19.78, 19.85, 22.70, 26.20, 29.37, 29.50, 29.52, 29.70, 29.75, 29.79, 30.43, 31.96, 36.86, 38.50, 39.03, 55.30, 55.34, 61.99, 62.10, 62.50, 62.55, 62.63, 63.33, 67.69, 69.61, 69.64, 73.60, 74.61, 79.78, 81.51, 82.43, 84.41, 84.52, 84.57, 85.65, 85.88, 86.36, 87.47, 106.43, 111.40, 111.78, 111.83, 111.89, 113.31, 113.50, 116.07, 116.42, 127.09, 127.38, 127.86, 128.11, 128.27, 129.21, 130.20, 135.07, 135.18, 135.45, 135.54, 136.37, 139.66, 140.30, 144.12, 147.51, 150.16, 150.21, 150.26, 150.38, 153.30, 158.81, 159.03, 163.33, 168.90, 172.25, 172.43.

TOF/MS (ESI): calcd for C108H165N6O19PNa [M+Na]+1904.1765. found 1904.1780.

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl 3-(3,4,5-tris(octadecyloxy)benzamide)propionate

Example 5-12

(1) Synthesis of 5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl 3-(3, 4,5-tris(octadecyloxy)benzamide)propionate

COMU (1.88 g, 4.40 mmol) was added to a suspension of 3-(3,4,5-tris(octadecyloxy)benzamide)propionic acid (2.20 g, 2.20 mmol), 5′-O-(4,4′-dimethoxytrityl)thymidine (1.44 g, 2.64 mmol), and 1-methylimidazole (0.90 g, 11.00 mmol) in THF (44 mL), and stirred at room temperature for 3.5 hours. Further, 1-methylimidazole (0.36 g, 4.4 mmol) and COMU (0.94 g, 2.20 mmol) were added, and the mixture was stirred at room temperature for 68 hours. The solid precipitated by dropping acetonitrile (220 mL) to the reaction solution was filtered. The solid was washed with acetonitrile-THF mixture solvent and methanol, and then dried under reduced pressure at 50° C. to obtain a crude compound (3.07 g). The crude compound was purified by column chromatography (spherical neutral silica gel, eluent: dichloromethane-THF) to give the title compound (2.01 g, 60%) as a white solid.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.15-1.55 (m, 93H), 1.60-1.85 (m, 6H), 2.40-2.55 (m, 2H), 2.65-2.75 (m, 2H), 3.40-3.55 (m, 2H), 3.65-3.75 (m, 2H), 3.78 (s, 6H), 3.95-4.05 (m, 6H), 4.14 (d, 1H, J=1.6 Hz), 5.47 (d, 1H, J=4.8 Hz), 6.42 (t, 1H, J=6.2 Hz), 6.59 (t, 1H, J=6.0 Hz), 6.80-6.90 (m, 4H), 6.94 (s, 2H), 7.20-7.45 (m, 9H), 7.60 (s, 1H), 8.43 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ 11.62, 14.14, 22.71, 26.11, 29.38, 29.45, 29.61, 29.68, 29.74, 30.33, 31.94, 34.05, 35.35, 37.92, 55.24, 63.65, 69.36, 73.51, 75.85, 83.94, 84.34, 87.24, 105.67, 111.65, 113.15, 113.32, 127.26, 127.76, 127.85, 128.06, 128.10, 129.12, 130.04, 130.08, 135.03, 135.24, 135.35, 141.26, 144.12, 150.23, 153.10, 158.78, 163.39, 167.36, 172.25.

TOF/MS (ESI): calcd for C95H149N3O12Na [M+Na]+1547.1039. found 1547.1014.

Example 5-13

(2) Synthesis of thymidine-3′-yl 3-(3,4,5-tris(octadecyloxy)benzamide)propionate

The title compound (1.51 g, 99%) was obtained as a white solid from 5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl 3-(3,4,5-tris(octadec yloxy)benzamide)propionate (1.91 g, 1.25 mmol) according to the method of Example 2-2.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.15-1.55 (m, 90H), 1.65-1.85 (m, 6H), 1.91 (s, 3H), 2.30-2.55 (m, 2H), 2.55-2.75 (m, 3H), 3.65-3.75 (m, 2H), 3.85-3.95 (m, 2H), 3.95-4.05 (m, 6H), 4.05-4.10 (m, 1H), 5.35-5.40 (m, 1H), 6.16 (dd, 1H, J=6.0, 8.4 Hz), 6.55-6.65 (m, 1H), 6.95 (s, 2H), 7.43 (s, 1H), 8.25-8.55 (m, 1H).

13C-NMR (100 MHz, CDCl3): δ 12.51, 14.09, 22.71, 26.13, 26.16, 29.38, 29.47, 29.62, 29.69, 29.75, 29.99, 30.38, 31.96, 34.26, 35.57, 37.12, 62.57, 69.57, 73.59, 75.09, 85.06, 86.65, 106.04, 111.41, 129.08, 136.51, 141.64, 150.33, 153.20, 163.40, 167.46, 172.39.

TOF/MS (ESI): calcd for C74H131N3O10Na [M+Na]+1244.9732. found 1244.9789.

Examples 5-14

(3) Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl 3-(3,4,5-tris(octadecyloxy)benzamide)propionate

The title compound (2.07 g, 95%) was obtained as white solids from thymidine-3′-yl 3-(3,4,5-tris(octadecyloxy)benzamide)propionate (1.41 g, 1.15 mmol) and DMTr-dT-CE-phosphoroamidite (1.71 g, 2.30 mmol) according to the methods of Examples 3-4.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.15-1.55 (m, 93H), 1.65-1.85 (m, 7H), 1.90 (d, 3H, J=1.2 Hz), 2.25-2.55 (m, 3H), 2.55-2.80 (m, 5H), 3.30-3.45 (m, 1H), 3.50-3.60 (m, 1H), 3.60-3.75 (m, 2H), 3.78 (s, 3H), 3.79 (s, 3H), 3.90-4.05 (m, 6H), 4.05-4.40 (m, 6H), 5.10-5.25 (m, 1H), 5.30-5.40 (m, 1H), 6.15-6.25 (m, 1H), 6.40-6.50 (m, 1H), 6.75-6.90 (m, 4H), 6.99 (d, 2H, J=3.6 Hz), 7.20-7.40 (m, 9H), 7.50-7.60 (m, 1H), 8.95-9.05 (m, 1H), 9.05-9.15 (m, 1H).

13C-NMR (100 MHz, CDCl3): δ 11.72, 12.49, 14.14, 19.59, 19.67, 19.75, 22.71, 26.11, 26.14, 29.38, 29.47, 29.63, 29.69, 29.75, 29.78, 30.35, 31.94, 34.03, 35.53, 35.58, 36.64, 38.95, 55.29, 62.36, 62.41, 62.52, 62.57, 63.24, 67.60, 69.38, 73.53, 73.84, 79.90, 82.35, 82.44, 84.25, 84.32, 84.39, 85.48, 85.67, 87.35, 105.86, 111.64, 111.74, 111.88, 113.14, 113.35, 116.25, 116.44, 127.34, 127.76, 127.85, 128.09, 129.03, 129.13, 130.13, 134.91, 134.95, 135.00, 135.07, 135.48, 135.61, 141.22, 143.93, 143.97, 150.21, 150.26, 150.46, 150.49, 153.03, 158.83, 163.48, 163.53, 167.47, 167.51, 172.19.

TOF/MS (ESI): calcd for C108H165N6O19PNa [M+Na]+1904.1765. found 1904.1807.

6. Synthesis of (4-(((6-(N6-benzoyladenine-9-yl)-4-tritylmorpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(thymine-1-yl)morpholin-2-yl)methyl 2-((3,4,5-Tris(octadecyloxy)benzoyl)oxy)acetate

Example 6-1

(1) Synthesis of (6-(thymine-1-yl)-4-tritylmorpholin-2-yl)methyl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (2.85 g, 98%) was obtained from 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetic acid (1.97 g, 2.00 mmol) and 6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-5-methylpyrimidine-2,4-(1H, 3H)-dione (1.16 g, 2.40 mmol) according to the procedure of Example 2-1.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.11-1.53 (m, 91H), 1.68-1.83 (m, 6H), 1.84 (s, 3H), 3.13 (d, 1H, J=12.0 Hz), 3.37 (d, 1H, J=11.2 Hz), 3.98-4.04 (m, 6H), 4.17 (d, 2H, J=5.2 Hz), 4.37-4.41 (m, 1H), 4.78 (s, 2H), 6.13 (dd, 1H, J=2.0, 9.6 Hz), 7.00 (s, 1H), 7.15-7.26 (m, 3H), 7.26-7.38 (m, 8H), 7.34-7.58 (m, 6H), 8.34 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ 12.45, 14.13, 22.70, 26.07, 26.11, 29.32, 29.38, 29.42, 29.59, 29.66, 29.68, 29.73, 30.35, 31.94, 49.01, 51.85, 60.89, 65.08, 69.19, 73.55, 74.58, 80.50, 108.31, 110.68, 123.33, 126.64, 127.97, 129.13, 135.33, 142.95, 149.63, 152.91, 163.23, 165.75, 167.64.

TOF/MS (ESI): calcd for C92H143N3O10Na [M+Na]+1473.0671. found 1473.0629.

Example 6-2

(2) Synthesis of (6-(thymine-1-yl)morpholin-2-yl)methyl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate trifluoroacetic Acid Salt

The title compound (2.44 g, 97%) was obtained from (6-(thy mine-1-yl)-4-tritylmorpholin-2-yl)methyl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (2.76 g, 1.90 mmol) according to the method of Ex ample 2-2.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.10-1.39 (m, 84H), 1.42-1.50 (m, 6H), 1.70-1.83 (m, 6H), 1.87 (s, 3H), 2.95-3.16 (m, 2H), 3.42 (d, 1H, J=11.2 Hz), 3.62-3.75 (m, 1H), 3.95-4.05 (m, 6H), 4.31 (d, 1H, J=8.4 Hz), 4.46 (d, 2H, J=10.0 Hz), 4.86 (s, 2H), 6.17 (d, 1H, J=9.6 Hz), 7.25 (s, 1H), 7.30 (s, 2H), 10.15-10.75 (brs, 1H).

13C-NMR (100 MHz, CDCl3): δ 12.48, 14.13, 22.71, 26.09, 26.15, 29.35, 29.39, 29.47, 29.63, 29.69, 29.75, 29.78, 30.38, 31.94, 43.24, 44.99, 60.87, 63.56, 69.21, 72.38, 73.57, 108.30, 112.18, 114.99, 117.90, 123.12, 134.33, 143.10, 150.40, 152.99, 162.55, 162.90, 163.78, 166.02, 167.45.

TOF/MS (ESI): calcd for C73H129N3O10—CF3COOH [M−H]−1320.9528. found 1320.9517.

Example 6-3

(3) Synthesis of (4-(((6-(N6-benzoyladenine-9-yl)-4-tritylmorpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(thymine-1-yl)morpholin-2-yl)methyl 2-((3,4,5-Tris(octadecyloxy)benzoyl)oxy)acetate

(6-(N6-benzoyladenine-9-yl)-4-tritylmorpholin-2-yl)methyldimethylphosphoramidechloridate (3.81 g, 5.27 mmol) was added to a solution of (6-(thymine-1-yl)morpholin-2-yl)methyl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate trifluoroacetic acid salt (2.32 g, 1.76 mmol) and N,N-diisopropylethylamine (4.16 g, 32.16 mmol) in 1,3-dimethyl-2-imidazolidinone (16 mL)-THF (18 mL), and then stirred at room temperature for 2.5 hours. The slurry liquid by adding methanol (93 m L) to the reaction solution was obtained, and then was concentrated under reduced pressure. The slurry liquid obtained by adding methanol to the concentrated residue was stirred at room temperature for 30 minutes, and then filtered. The solid was washed with methanol, and then dried under reduced pressure at 50° C. to obtain a crude compound (3.23 g). The crude compound was purified by column chromatography (spherical neutral silica gel, eluent: dichloromethane-methanol) to give the title compound (1.96 g, 59%) as a white solid.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.10-1.53 (m, 90H), 1.55-1.97 (m, 12H), 2.46 (d, 3H, J=10.4 Hz), 2.63 (d, 3H, J=9.6 Hz), 2.46-2.63 (m, 1H), 3.12-3.60 (m, 4H), 3.75-4.07 (m, 10H), 4.15-4.50 (m, 2H), 4.78-4.91 (m, 2H), 5.41-5.53 (m, 1H), 6.42 (d, 1H, J=10.4 Hz), 7.12-7.20 (m, 4H), 7.20-7.32 (m, 9H), 7.40-7.58 (m, 8H), 8.05 (d, 2H, J=7.6 Hz), 8.07-8.10 (m, 1H), 8.68-8.80 (m, 1H), 9.50-9.76 (m, 2H).

13C-NMR (100 MHz, CDCl3): δ 12.44, 12.53, 14.14, 22.70, 26.08, 26.12, 29.32, 29.38, 29.43, 29.60, 29.68, 29.73, 30.35, 31.94, 36.47, 36.51, 36.63, 36.67, 44.56, 44.84, 47.02, 48.64, 48.78, 52.81, 53.10, 60.93, 64.23, 65.34, 65.69, 69.19, 73.56, 74.90, 74.97, 75.34, 79.53, 79.61, 79.97, 80.79, 108.27, 110.92, 111.25, 122.82, 123.26, 126.65, 128.00, 128.22, 128.29, 128.57, 128.63, 129.16, 132.47, 132.57, 133.49, 133.92, 134, 74, 134.80, 140.75, 140.78, 142.97, 143.00, 149.31, 149.68, 149.82, 150.12, 150.89, 151.40, 152.65, 152.77, 152.92, 152.94, 163.62, 165.02, 165.23, 165.82, 165.84, 167.56, 167.63.

TOF/MS (ESI): calcd for C111H165N10O14PNa [M+Na]+1916.2142. found 1916.2216.

Synthesis of (4-(((6-(N4-benzoylcytosine-1-yl)-4-tritylmorpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(N2-isobutyrylguanine-9-yl)morpholin-2-yl)methyl 2-((3,4,5-Tris(octadecyloxy)benzoyl)oxy)acetate

Example 6-4

(1) Synthesis of (6-(N2-isobutyrylguanine-9-yl)-4-tritylmorpholin-2-yl)methyl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (3.00 g, 97%) was obtained from 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetic acid (1.97 g, 2.00 mmol) and N-(9-(6-(hydroxymethyl)-4-tritylmorpholin-2-yl))-6-oxo-6,9-dihydro-1H-purine-2-yl)isobutylamide (1.51 g, 2.60 mmol) according to the procedure of Example 2-1.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.10-1.60 (m, 97H), 1.70-1.90 (m, 7H), 2.64 (sept, 1H), 3.18 (d, 1H, J=12.0 Hz), 3.45 (d, 1H, J=11.6 Hz), 3.95-4.15 (m, 6H), 4.13-4.25 (m, 2H), 4.37-4.46 (m, 1H), 4.72 (d, 1H, J=16.0 Hz), 4.84 (d, 1H, J=16.0 Hz), 5.89 (dd, 1H, J=2.2, 9.8 Hz), 7.06-7.37 (m, 12H), 7.39-7.55 (m, 5H), 7.61 (s, 1H), 8.18 (s, 1H), 11.92 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ 14.14, 18.93, 19.03, 22.71, 26.07, 26.12, 29.33, 29.37, 29.42, 29.60, 29.67, 29.74, 30.35, 31.94, 36.64, 49.04, 52.24, 60.90, 65.03, 69.25, 73.58, 74.22, 80.36, 108.38, 121.30, 123.27, 126.69, 127.96, 129.18, 136.12, 143.08, 147.32, 147.42, 152.95, 155.36, 165.98, 167.61, 177.85.

TOF/MS (ESI): calcd for C96H148N6O10Na [M+Na]+1568.1155. found 1568.1176.

Example 6-5

(2) Synthesis of (6-(N2-isobutyrylguanine-9-yl)morpholin-2-yl)methyl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate trifluoroacetic Acid Salt

The title compound (2.45 g, 96%) was obtained from (6-(N2-isobutyrylguanine-9-yl)-4-tritylmorpholin-2-yl)methyl 2-((3,4,5-tris(octa decyloxy)benzoyl)oxy)acetate (2.78 g, 1.80 mmol) according to the procedure of Example 2-2.

1H-NMR (400 MHz, CDCl3): δ 0.87 (t, 9H, J=6.8 Hz), 1.00-1.53 (m, 96H), 1.69-1.88 (m, 6H), 2.72-2.88 (m, 1H), 3.19-3.38 (m, 1H), 3.44-3.63 (m, 1H), 3.75-4.13 (m, 8H), 4.25-4.63 (m, 3H), 4.69-4.94 (m, 2H), 6.06-6.31 (m, 1H), 7.20-7.30 (m, 3H), 7.87-8.06 (m, 1H), 10.19-10.63 (brs, 1H), 12.19-12.63 (brs, 1H).

13C-NMR (100 MHz, CDCl3): δ 14.12, 18.92, 22.70, 26.09, 26.16, 29.35, 29.38, 29.47, 29.63, 29.68, 29.75, 29.79, 30.04, 30.38, 31.94, 36.17, 42.86, 60.73, 63.62, 69.21, 71.44, 73.57, 108.29, 115.13, 118.04, 123.10, 143.09, 148.28, 152.95, 156.00, 165.93, 167.50, 180.39.

TOF/MS (ESI): calcd for C77H134N6O10—CF3COOH [M−H]−1416.0012, found 1415.9960.

Example 6-6

(3) Synthesis of (4-(((6-(N4-benzoylcytosine-1-yl)-4-tritylmorpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(N2-isobutyrylguanine-9-yl)morpholin-2-yl)methyl 2-((3,4,5-Tris(octadecyloxy)benzoyl)oxy)acetate

The title compound (2.87 g, 91%) was obtained from (6-(N2-isobutyrylguanine-9-yl)morpholin-2-yl)methyl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate trifluoroacetic acid salt (2.27 g, 1.60 mmol) and (6-(N4-benzoylcytosine-1-yl)-4-tritylmorpholin-2-yl)methyldimethylphosphoramidochloridate (3.35 g, 4.80 mmol) according to the procedure of Example 6-3.

1H-NMR (400 MHz, CDCl3): δ 0.88 (t, 9H, J=6.8 Hz), 1.00-1.56 (m, 96H), 1.69-1.88 (m, 9H), 2.38-2.97 (m, 8H), 3.06-3.69 (m, 5H), 3.75-4.56 (m, 12H), 4.69-4.94 (m, 2H), 5.34-5.63 (m, 1H), 6.28-6.50 (m, 1H), 7.13-8.00 (m, 23H), 8.69-8.94 (brs, 1H), 9.31-9.63 (brs, 1H), 10.06-10.31 (brs, 1H), 12.13-12.31 (m, 1H).

13C-NMR (100 MHz, CDCl3): δ 14.13, 19.01, 19.04, 19.17, 19.22, 22.70, 26.07, 26.12, 29.32, 29.37, 29.43, 29.60, 29.67, 29.73, 29.98, 30.35, 31.94, 36.17, 36.27, 36.64, 36.68, 36.74, 36.78, 45.00, 45.08, 46.96, 47.43, 48.25, 48.76, 52.48, 60.82, 60.90, 64.27, 65.84, 69.20, 69.22, 73.57, 74.31, 74.51, 75.41, 79.30, 80.47, 81.36, 81.59, 108.31, 121.25, 122.00, 123.26, 123.33, 126.67, 126.73, 127.73, 127.99, 128.80, 128.98, 132.71, 133.07, 133.27, 136.19, 137.10, 142.90, 143.01, 147.88, 148.00, 148.09, 152.89, 152.93, 155.79, 155.90, 165.79, 165.91, 167.56, 167.58, 178.67, 179.37.

TOF/MS (ESI): calcd for C114H170N11O15PNa [M+Na]+1987.2513. found 1987.2521.

7. Confirmation of Tag Removal

Example 7-1

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)thymidine

A mixture solution of 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)-N2-isobutyryl deoxyguanosine-3′-yl)phosphoryl)thymidine-3′-yl 2-((3,4,5-tris(octadec yloxy)benzoyl)oxy)acetate (233 mg, 0.10 mmol) in THF (9 mL)-2-propanol (1 mL) was cooled to 0° C. In cool, 4 mol/L-lithium borohydride-THF solution (0.125 m L, 0.50 mmol) was added and stirred at the same temperature for 30 min. An aqueous ammonium chloride solution was added to the reaction solution, and then extracted 3 times with dichloromethane. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, and then the filtrate was concentrated. The solid precipitated by su spending the concentrated residue in acetonitrile was removed by filtration, and the filtrate was concentrated. A portion (2.5 mL) of the di methyl sulfoxide-methanol mixture solution (1:1, 5 mL) of the concentrated residue was purified by preparative chromatography in 5 portions to give the title compound (64 mg, 95%) as a white solid.

Preparative chromatographic conditions: Column MightysilRP-18 GP (5 μm, 20 ϕ×250 mm) manufactured by Kanto Chemical Co., Ltd., flow rate 5 mL/min, mobile phase acetonitrile-water (gradient 0-30 min (ratio of acetonitrile 50→60%)

1H-NMR (600 MHz, DMSO-d6): δ 1.12 (d, 6H, J=6.0 Hz), 1.42 (d, 3H, J=7.2 Hz), 1.74 (d, 3H, J=2.8 Hz), 2.07 (s, 1H), 2.30-2.41 (m, 3H), 2.60-2.90 (m, 6H), 3.18-3.29 (m, 2H), 3.73 (d, 6H, J=4.2 Hz), 4.00-4.43 (m, 12H), 4.97 (s, 1H), 5.10 (s, 1H), 5.51-5.52 (m, 1H), 6.15-6.25 (m, 3H), 6.88 (d, 4H, J=9.0 Hz), 7.20-7.50 (m, 11H), 8.17 (q, 1H, J=1.8 Hz), 11.33-12.07 (m, 3H).

13C-NMR (151 MHz, DMSO-d6): δ 10.56, 10.88, 17.66, 33.65, 35.78, 52.00, 53.86, 56.34, 61.48, 61.80, 65.49, 66.58, 68.73, 76.58, 77.11, 81.63, 82.11, 82.52, 82.87, 84.98, 90.73, 108.72, 112.08, 116.98, 119.14, 125.69, 126.46, 126.77, 128.53, 133.81, 134.00, 134.26, 134.70, 136.15, 143.26, 147.04, 147.32, 149.14, 153.53, 156.99, 162.50, 168.14, 178.89.

TOF/MS (ESI): calcd for C61H69N11O21P2Na [M+Na]+1376.4. found 1376.6.

Example 7-2

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)thymidine-3′-yl)phosphoryl)-N6-benzoyldeoxyadenosine

The title compound (55 mg, 75%) was obtained as a yellow solid from 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-N2-isobutyldeoxyguanosine-3′-yl)phosphoryl)thymidine-3′-yl)phosphoryl)-N6-benzoyldeoxyadenosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (244 mg, 0.10 mmol) according to the procedure of Example 7-1.

1H-NMR (600 MHz, DMSO-d6): δ 1.17-1.30 (m, 7H), 1.74-1.79 (m, 3H), 1.97-2.30 (m, 2H), 2.45-2.51 (m, 2H), 2.68-2.76 (m, 1H), 2.79-2.83 (m, 1H), 2.96 (dt, 4H, J=6.4, 12.4 Hz), 3.03-3.12 (m, 1H), 3.22 (t, 1H, J=11.3 Hz), 3.77 (s, 6H), 3.93 (s, 1H), 4.07-4.38 (m, 11H), 5.07-5.45 (m, 5H), 6.05-6.45 (m, 3H), 6.85 (t, 4H, J=10.7 Hz), 7.24 (d, 5H, J=8.9 Hz), 7.29 (t, 2H, J=7.6 Hz), 7.36 (d, 2H, J=7.6 Hz), 7.49-7.56 (m, 3H), 7.65 (t, 1H, J=7.2 Hz), 7.90 (dt, 1H, J=3.0, 8.1 Hz), 8.07 (d, 2H, J=7.6 Hz), 8.13-8.16 (m, 1H), 10.89-12.13 (m, 3H).

13C-NMR (151 MHz, DMSO-d6): δ 12.52, 14.58, 19.33, 35.33, 36.13, 37.02, 55.48, 60.34, 62.73, 63.24, 64.06, 67.31, 68.33, 70.66, 77.30, 79.12, 82.64, 83.37, 84.09, 84.74, 86.25, 113.58, 118.69, 121.00, 123.54, 127.24, 128.19, 128.72, 128.94, 130.23, 132.68, 133.50, 134.55, 135.77, 136.46, 137.81, 139.68, 145.08, 148.56, 149.12, 150.82, 155.31, 158.56, 161.08, 164.11, 165.03, 180.61.

TOF/MS (ESI): calcd for C68H72N14O20P2Na [M+Na]+1489.4. found 1489.4.

Example 7-3

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)-N4-benzoyldeoxycytidine

The title compound (57 mg, 79%) was obtained as a white solid from 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)-N4-benzoyldeoxycytidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (243 mg, 0.10 mmol) according to the procedure of Example 7-1.

1H-NMR (600 MHz, DMSO-d6): δ 1.09-1.12 (m, 6H), 1.42 (s, 3H), 1.75-2.10 (m, 4H), 2.61-2.95 (m, 7H), 3.15-3.25 (m, 3H), 3.70-3.77 (m, 7H), 4.08-4.36 (m, 11H), 5.09 (d, 2H, J=41.9 Hz), 5.32 (d, 2H, J=44.0 Hz), 6.18-6.29 (m, 3H), 6.85-6.88 (m, 4H), 7.20-7.22 (m, 5H), 7.29 (q, 2H, J=6.9 Hz), 7.34 (d, 2H, J=7.6 Hz), 7.41-7.53 (m, 4H), 7.86 (d, 2H, J=7.6 Hz), 8.19 (dt, 1H, J=4.4, 8.2 Hz), 8.84 (q, 1H, J=6.4 Hz), 11.34-12.05 (m, 3H).

13C-NMR (151 MHz, DMSO-d6): δ 12.17, 19.35, 27.86, 35.35, 55.56, 57.07, 63.19, 63.61, 67.29, 68.60, 70.86, 78.19, 78.93, 82.62, 83.40, 83.86, 84.14, 86.67, 110.47, 113.78, 118.59, 118.88, 120.91, 127.38, 128.02, 128.69, 130.24, 131.86, 134.55, 135.51, 135.97, 137.92, 144.97, 148.76, 149.02, 150.84, 154.36, 155.25, 158.69, 164.09, 165.95, 166.18, 180.60.

TOF/MS (ESI): calcd for C67H72N12O21P2Li [M+Li]+1449.5. found 1449.6.

Example 7-4

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine

The title compound (63 mg, 93%) was obtained as a white solid from 5′-O-((2′-cyanoethoxy)(5′-O-(2′-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl)phosphoryl)-N2-isobutyldeoxianosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (232 m g, 0.10 mmol) according to the method of Example 7-1.

1H-NMR (600 MHz, DMSO-d6): δ 1.12 (d, 6H, J=6.2 Hz), 1.42 (d, 3H, J=7.6 Hz), 1.73 (d, 3H, J=2.7 Hz), 2.07 (s, 1H), 2.32-2.43 (m, 3H), 2.61-2.91 (m, 6H), 3.20-3.29 (m, 2H), 3.73 (d, 6H, J=3.8 Hz), 4.04-4.43 (m, 12H), 4.97 (s, 1H), 5.10 (s, 1H), 5.51-5.52 (m, 1H), 6.13-6.26 (m, 3H), 6.88 (d, 4H, J=8.9 Hz), 7.23 (d, 5H, J=7.6 Hz), 7.30 (t, 2H, J=6.2 Hz), 7.36 (d, 2H, J=8.2 Hz), 7.43-7.47 (m, 2H), 7.89-8.18 (m, 1H), 11.33-12.07 (m, 3H)

13C-NMR (151 MHz, DMSO-d6): δ 10.46, 10.84, 17.69, 17.83, 33.61, 35.32, 36.22, 53.86, 61.50, 61.98, 65.53, 66.44, 68.89, 75.88, 77.15, 80.92, 81.78, 82.16, 82.47, 82.92, 83.51, 85.00, 108.75, 108.90, 112.08, 116.92, 119.11, 125.71, 126.48, 126.77, 128.55, 133.80, 134.02, 134.33, 134.53, 136.24, 143.27, 146.96, 147.20, 149.17, 153.63, 157.01, 162.41, 178.92.

TOF/MS (ESI): calcd for C61H69N11O21P2Na [M+Na]+1376.4. found 1376.6.

Example 7-5

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-thymidine-3′-yl)phosphorothioyl)-thymidine

A mixture solution of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-thymidine-3′-yl)phosphorothioyl)-thymidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (189 mg, 0.10 mmol) in THF (9 m L)-2-propanol (1 mL) was cooled to 0° C. In cool, 4 mol/L-lithium borohydride-THF solution (0.125 m L, 0.50 mmol) was added and stirred at the same temperature for 30 min. An aqueous ammonium chloride solution was added to the reaction solution, and then extracted 3 times with dichloromethane. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, and then the filtrate was concentrated. The solid precipitated by suspending the concentrated residue in acetonitrile was removed by filtration, and the filtrate was concentrated. The concentrated residue was purified by column chromatography (silica gel, eluent: chloroform-methanol) to give the title compound (86 mg, 93%) as a white solid.

1H-NMR (600 MHz, CDCl3): δ 1.46 (d, 3H, J=15.8 Hz), 1.91 (d, 3H, J=8.2 Hz), 2.21-2.79 (m, 6H), 3.39-3.49 (m, 2H), 3.79 (d, 6H, J=3.4 Hz), 4.08-4.52 (m, 8H), 5.26-5.31 (m, 1H), 6.25-6.41 (m, 2H), 6.85 (dd, 4H, J=2.7, 8.9 Hz), 7.22-7.38 (m, 9H), 7.58 (d, 1H, J=24.1 Hz), 9.45-10.50 (m, 2H).

13C-NMR (151 MHz, CDCl3): δ 11.90, 12.57, 19.43, 38.92, 39.90, 55.30, 62.59, 63.36, 67.70, 71.03, 80.38, 84.66, 85.46, 87.28, 111.32, 111.97, 113.38, 116.54, 127.28, 128.00, 128.11, 130.06, 135.00, 135.80, 144.09, 150.64, 150.92, 158.74, 163.98.

TOF/MS (ESI): calcd for C44H48N5O13PSNa [M+Na]+940.3. found 940.2.

Example 7-6

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-2′-fluorodeoxyuridine-3′-yl)phosphoryl)-2′-fluoro-N2-isobutyryldeoxyguanosine

A mixture solution of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-2′-fluorodeoxyuridin-3′-yl)phosphoryl)-2′-fluoro-N2-isobutyryldeoxyguanosine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (0.397 g, 0.200 mmol) in THF (27 mL)-2-propanol (3 mL) was cooled to 0 to −10° C. In cool, 4 mol/L-lithium borohydride-THF solution (0.10 m L, 0.400 mmol) was added and stirred at the same temperature for 20 min. 10% aqueous ammonium chloride (4 mL) was added to the reaction solution, and then the mixture was separated, and the organic layer was washed with 10% brine. The organic layer was dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, and then the filtrate was concentrated. The solid precipitated by adding methanol to the concentrated residue was removed by filtration, and the filtrate was concentrated and dried. The concentrated residue was purified by column chromatography (spherical neutral silica gel, eluent: dichloromethane-methanol) to give the title compound (0.137 g, 67%) as a white solid.

1H-NMR (400 MHz, CDCl3): δ 1.16-1.26 (m, 6H), 2.19 (s, 2H), 2.45-2.87 (m, 3H), 3.44-3.66 (m, 2H), 3.72-3.75 (m, 6H), 3.92-4.18 (m, 2H), 4.25-4.50 (m, 4H), 4.89-5.55 (m, 6H), 5.85-6.00 (m, 2H), 6.80-6.82 (m, 4H), 7.15-7.30 (m, 7H), 7.33-7.37 (m, 2H), 7.71-7.82 (m, 2H), 10.05-10.42 (brs, 1H), 10.55-10.87 (brs, 1H), 12.36 (s, 1H).

13C-NMR (100 MHz, CDCl3): δ 18.75, 18.85, 19.01, 19.07, 19.38, 19.45, 36.01, 50.81, 55.26, 55.28, 60.65, 62.85, 67.86, 80.26, 81.13, 87.13, 87.19, 87.53, 87.88, 90.59, 92.53, 102.62, 113.33, 116.74, 121.28, 127.26, 128.05, 128.19, 130.22, 134.80, 134.91, 134.96, 135.07, 140.41, 144.01, 144.11, 148.00, 148.06, 148.50, 148.55, 150.44, 155.70, 158.76, 163.72, 180.33, 180.39.

TOF/MS (ESI): calcd for C47H49N8O14F2PNa [M+Na]+1041.2972. found 1041.2975.

Examples 7-7

Synthesis of N4-benzoyl-5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-2′-O-methyluridine-3′-yl)phosphoryl)-2′-O-methylcytidine

Mixture solutions of N,N-diisopropylethylamine (0.065 g, 0.50 mmol) in THF (13.5 mL) and 2-propanol (1.5 mL) were cooled from 0 to −10° C. In cool, 4 mol/L-lithium borohydride-THF solution (0.125 mL, 0.50 mmol) was added and stirred at the same temperature for 10 min. A mixture solution of N4-benzoyl-5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-2′-O-methyluridine-3′-yl)phosphoryl)-2′-O-methylcytidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (0.200 g, 0.10 mmol) in THF (0.9 mL)-2-propanol (0.1 mL) was then added and stirred at the same temperature for 0.5 hours. 10% aqueous ammonium chloride (2 mL) was added to the reaction solution, and then the mixture was separated, and the organic layer was washed with 10% brine. The organic layer was dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, and then the filtrate w as concentrated. The solid precipitated by adding methanol to the concentrated residue was removed by filtration, and the filtrate was concentrated and dried. The concentrated residue was purified by column chromatography (spherical neutral silica gel, eluent: dichloromethane-methanol) to give the title compound (0.055 g, 53%) as a white solid.

1H-NMR (400 MHz, CDCl3): δ 2.00-2.27 (brs, 2H), 2.55-2.74 (m, 1H), 2.80 (t, 1H, J=6.2 Hz), 3.14-3.41 (m, 1H), 3.44-3.92 (m, 14H), 4.03-4.64 (m, 7H), 5.12 (q, 1H, J=5.6 Hz), 5.27 (t, 1H, J=8.0 Hz), 5.87-6.05 (m, 2H), 6.78-6.89 (m, 4H), 7.19-7.64 (m, 12H), 7.76-8.16 (m, 4H), 9.05-9.42 (brs, 1H), 9.50-9.87 (brs, 1H).

13C-NMR (100 MHz, CDCl3): δ 19.54, 19.56, 19.61, 19.64, 55.28, 55.32, 58.80, 58.82, 58.93, 61.30, 61.34, 62.38, 62.43, 62.68, 62.73, 66.03, 66.08, 66.41, 66.46, 67.85, 67.95, 74.30, 74.34, 74.42, 74.47, 81.32, 81.41, 81.49, 81.58, 81.64, 81.72, 82.17, 82.99, 83.08, 86.58, 86.79, 87.46, 87.48, 89.42, 89.51, 96.78, 102.69, 102.71, 113.39, 116.35, 116.48, 127.37, 127.42, 127.80, 127.85, 128.12, 128.25, 128.29, 128.57, 128.90, 128.93, 130.22, 130.27, 130.30, 131.01, 132.94, 133.02, 133.14, 133.17, 134.72, 134.75, 134.82, 134.84, 139.51, 139.66, 143.90, 143.97, 144.26, 150.43, 154.73, 158.81, 158.85, 162.82, 162.86, 163.22, 166.72.

TOF/MS (ESI): calcd for C51H53N6O16PNa [M+Na]+1059.3153. found 1059.3202.

Examples 7-8

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-2′-O-(tert-butyldimethylsilyl)uridine-3′-yl)phosphoryl)-2′-O-(tert-butyldimethylsilyl)uridine

A mixture solution of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)-2′-O-(tert-butyldimethylsilyl)uridine-3′-yl)phosphoryl)-2′-O-(tert-butyldimethylsilyl)uridine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)ox y)acetate (0.420 g, 0.20 mmol) in THF (27 mL)-2-propanol (3 mL) was cooled to 0 to −10° C. In cool, 4 mol/L-lithium borohydride-THF solution (0.25 m L, 1.00 mmol) was added and then stirred at the same temperature for 1.5 h. 10% aqueous ammonium chloride (4 mL) was added to the reaction solution, and then the mixture was separated, and the organic layer was washed with 10% brine. The organic layer was dried over anhydrous sodium sulfate, filtered to re move sodium sulfate, and then the filtrate was concentrated. Thesolid precipitated by adding methanol to the concentrated residue was re moved by filtration, and the filtrate was concentrated and dried. The concentrated residue was purified by column chromatography (spherical neutral silica gel, eluent: dichloromethane-methanol) to give the title compound (0.135 g, 60%) as a white solid.

1H-NMR (400 MHz, CDCl3): δ 0.11-0.15 (m, 12H), 0.89-0.93 (m, 18H), 1.66-1.84 (brs, 2H), 2.50-2.77 (m, 3H), 3.46-3.67 (m, 2H), 3.80 (s, 6H), 3.98-4.55 (m, 9H), 4.92-4.96 (m, 1H), 5.23-5.27 (m, 1H), 5.61-5.75 (m, 2H), 5.97-6.00 (m, 1H), 6.84-6.87 (m, 4H), 7.14-7.45 (m, 10H), 7.77-7.87 (m, 1H), 9.00-9.50 (brs, 2H).

13C-NMR (100 MHz, CDCl3): δ −5.14, −4.98, −4.96, −4.90, −4.82, −4.76, −4.74, −4.70, −4.68, 17.96, 18.02, 18.06, 19.54, 19.60, 19.67, 25.56, 25.60, 25.66, 25.69, 55.31, 62.02, 62.29, 62.34, 62.52, 62.57, 62.62, 67.06, 67.12, 67.32, 69.38, 69.54, 70.49, 70.93, 73.61, 74.57, 74.63, 74.71, 74.88, 81.55, 81.60, 81.73, 81.84, 81.93, 82.01, 87.37, 87.65, 87.85, 87.95, 88.05, 91.58, 91.81, 93.18, 93.89, 102.60, 102.67, 102.76, 102.82, 102.86, 113.41, 116.26, 116.30, 127.41, 127.45, 128.13, 128.17, 128.21, 130.16, 130.23, 130.29, 130.34, 134.65, 134.69, 134.72, 134.75, 139.67, 139.75, 140.36, 140.40, 141.65, 141.77, 143.99, 144.03, 149.97, 150.09, 150.19, 150.40, 150.55, 150.75, 158.83, 158.88, 162.93, 163.00, 163.18.

TOF/MS (ESI): calcd for C54H72N5O16Si2PNa [M+Na]+1156.4148, found 1156.4148.

Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine

Examples 7-9

(1) Synthesis from 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl 3-((3,4,5-tris(octadecyloxy)benzoyl)oxy)propionate

The title compound (0.136 g, 76%) was obtained as a white solid from 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl 3-((3,4,5-tris(octadecyloxy)benzoyl)ox y)propionate (0.377 g, 0.200 mmol) according to the method of Examples 7-8.

1H-NMR (400 MHz, CDCl3): δ 1.41 (d, 3H, J=5.4 Hz), 1.87 (d, 3H, J=2.0 Hz), 2.19-2.27 (m, 1H), 2.34-2.48 (m, 2H), 2.62-2.77 (m, 3H), 3.39 (t, 1H, J=7.2 Hz), 3.47-3.52 (m, 1H), 3.78 (s, 6H), 4.07-4.55 (m, 8H), 5.12-5.18 (m, 1H), 6.18-6.24 (m, 1H), 6.35-6.40 (m, 1H), 6.84 (d, 4H, J=8.8 Hz), 7.21-7.31 (m, 8H), 7.32-7.38 (m, 2H), 7.51-7.58 (m, 1H), 9.92-10.14 (m, 2H).

13C-NMR (100 MHz, CDCl3): δ 11.78, 12.45, 12.49, 19.59, 19.67, 19.71, 38.83, 39.01, 39.61, 39.67, 55.31, 62.45, 62.50, 62.60, 62.65, 63.31, 67.74, 70.84, 80.08, 84.40, 84.50, 85.60, 87.30, 87.36, 111.23, 111.34, 111.91, 112.00, 113.37, 116.49, 116.74, 127.32, 128.10, 130.11, 130.14, 134.98, 135.01, 135.04, 135.19, 136.00, 144.01, 144.06, 150.69, 151.00, 151.05, 158.80, 163.96, 164.16.

TOF/MS (ESI): calcd for C44H48N5O14PNa [M+Na]+924.2833. found 924.2836.

Examples 7-10

(2) Synthesis from 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl 2-(N-methyl-3,4,5-tris(octadecyloxy)benzamide)acetate

A mixture solution of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl 2-(N-methyl-3,4,5-tris(octadecyloxy)benzamido)acetate (189 mg, 0.10 mmol) in THF (9 mL)-2-propanol (1 mL) was cooled to 0° C. In cool, 4 mol/L-lithium borohydride-THF solution (0.125 m L, 0.50 mmol) was added and stirred at the same temperature for 30 min. An aqueous ammonium chloride solution was added to the reaction solution, and then extracted 3 times with dichloromethane. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, and then the filtrate was concentrated. The solid precipitated by suspending the concentrated residue in acetonitrile was removed by filtration, and the filtrate was concentrated. The concentrated residue was purified by column chromatography (silica gel, eluent: chloroform-methanol) to give the title compound (89 mg, 98%) as a white solid.

TOF/MS (ESI): calcd for C44H48N5O14PNa [M+Na]+924.3. found 924.2.

Example 7-11

(3) Synthesized from 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl 3-(3,4,5-tris(octadecyloxy)benzamide)propionate

A mixture solution of 5′-O-((2-cyanoethoxy)(5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl 3-(3,4,5-tris(octadecyloxy)benzamide)propionate (189 mg, 0.10 mmol) in THF (9 mL)-2-propanol (1 mL) was cooled to 0° C. In cool, 4 mol/L-lithium borohydride-THF solutions (0.125 mL, 0.50 mmol) were added, and the n stirred at room temperature for 1 hours. An aqueous ammonium chloride solution was added to the reaction solution, and then extracted 3 times with dichloromethane. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, and then the filtrate was concentrated. The solid precipitated by suspending the concentrated residue in acetonitrile was removed by filtration, and the filtrate was concentrated. The concentrated residue was purified by column chromatography (silica gel, eluent: chloroform-methanol) to give the title compound (74 mg, 82%) as a white solid.

TOF/MS (ESI): calcd for C44H48N5O14PNa [M+Na]+924.3. found 924.2.

Example 7-12

Synthesis of (6-(N6-benzoyladenine-9-yl)-4-trityl-morpholin-2-yl)methyl P-(2-(hydroxymethyl)-6-thymine-1-yl)morpholino)-N,N-dimethylphosphonoamidate

The title compound (0.114 g, 61%) was obtained as a white solid from (4-((6-(N6-benzoyladenine-9-yl)-4-tritylmorpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(thymine-1-yl)morpholin-2-yl)methyl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (0.379 g, 0.20 mmol) according to the procedure of Examples 7-7.

1H-NMR (400 MHz, CDCl3): δ 0.87-1.95 (m, 6H), 2.01 (s, 1H), 2.45-2.64 (m, 7H), 3.06-4.04 (m, 9H), 4.43-4.46 (m, 1H), 5.52 (d d, 1H, J=2.4, 10.4 Hz), 6.41 (dt, 1H, J=2.4, 10.0 Hz), 7.05-7.64 (m, 19H), 8.03 (d, 2H, J=8.4 Hz), 8.09 (s, 1H), 8.71-8.80 (m, 1H), 9.69-9.78 (m, 1H), 10.28 (s, 1H), 10.75-11.05 (brs, 1H).

13C-NMR (100 MHz, CDCl3): δ 12.41, 12.45, 36.54, 36.58, 36.62, 36.66, 44.61, 44, 82, 47.13, 48.68, 48.78, 52.80, 52.85, 62.72, 62.78, 65.46, 65.61, 75.07, 75.14, 75.45, 75.52, 78.06, 78.13, 78.27, 78.33, 79.60, 79.65, 80.01, 80.49, 110.96, 111.03, 122.47, 122.78, 126.65, 128.00, 128.24, 128.41, 128.49, 128.62, 129.17, 132.61, 132.69, 133.48, 133.64, 134.86, 134.96, 140.94, 149.54, 149.75, 149.82, 150.02, 151.23, 151.42, 152.66, 152.75, 163.75, 163.79, 165.25.

TOF/MS (ESI): calcd for C48H51N10O8PNa [M+Na]+949.3527. found 949.3545.

Example 7-13

Synthesis of (6-(N4-benzoylcytosine-1-yl)-4-tritylmorpholin-2-yl) methyl P-(2-(hydroxymethyl)-6-(N2-isobutyrylguanine-9-yl)morpholino)-N,N-dimethylphosphonoamidate

The title compound (0.052 g, 52%) was obtained as a white solid from (4-(((6-(N4-benzoylcytosine-1-yl)-4-tritylmorpholin-2-yl)meth oxy)(dimethylamino)phosphoryl)-6-(N2-isobutyrylguanine-9-yl)morpholin-2-yl)methyl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (0.197 g, 0.10 mmol) according to the methods of Examples 7-7.

1H-NMR (400 MHz, CDCl3): δ 0.78-1.55 (m, 9H), 2.37-2.95 (m, 8H), 3.08-3.94 (m, 9H), 4.13-4.43 (m, 2H), 5.42-5.54 (m, 1H), 6.22-6.38 (m, 1H), 7.05-7.80 (m, 19H), 7.93 (d, 2H, J=7.2H z), 9.58-9.92 (brs, 1H), 10.64-10.86 (brs, 1H), 12.05-12.21 (br s, 1H).

13C-NMR (100 MHz, CDCl3): δ 13.91, 14.12, 19.03, 19.06, 19.28, 22.69, 25.76, 28.55, 29.36, 29.70, 31.72, 31.92, 35.99, 36.05, 36.62, 36.65, 36.69, 44.63, 44.92, 47.15, 47.56, 48.44, 48.79, 52.26, 52.42, 61.85, 62.58, 62.73, 65.66, 66.15, 71.09, 71.69, 75.40, 75.86, 75.91, 78.19, 79.73, 80.56, 81.37, 81.76, 97.87, 120.56, 120.92, 126.65, 127.96, 128.04, 128.70, 128.83, 128.92, 129.13, 131.00, 132.21, 132.97, 133.02, 133.14, 136.66, 137.43, 144.02, 144.54, 147.92, 147.98, 148.00, 154.99, 155.57, 155.62, 162.69, 163.09, 167.24, 179.62, 179.86.

TOF/MS (ESI): calcd for C51H56N11O9PNa [M+Na]+1020.3898. found 1020.3942.

Examples 7-14

Confirmation of De-Tagging of 8 Mer

(1) Synthesis of 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(N4-benzoyldeoxycytidine-3′-yl)phosphoryl)-N6-benzoyldeoxyadenosine-3′-yl)phosphoryl)thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)-N4-benzoyldeoxycytidine-3′-yl)phosphoryl)-N6-benzoyldeoxyadenosine-3′-yl)phosphoryl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (8 Mer)

Under argon atmosphere, 5-benzylthio-1H-tetrazole (0.38 g, 1.96 mmol) was added to a suspension of thymidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate (1.19 g, 0.98 mmol) and 5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl-phosphoroamidite (1.46 g, 1.96 mmol) in dichloromethane (10 mL), and stirred at room temperature for 1.5 hours. Next, pyridine (0.93 g, 11.77 mmol), water (0.11 g, 5.89 m mol), and iodine (1.25 g, 4.91 mmol) were added, and then stirred at room temperature for 4 hours. 1.2 mol/L-ascorbic acid aqueous solution (3 mL) was added to the reaction solution, and then stirred at room temperature for 15 minutes. The solid precipitated by dropping acetonitrile (24 mL) to the reaction solution was filtered. The solid was washed with acetonitrile-dichloromethane mixture solvent, and then dried under reduced pressure at 50° C. to obtain a DMTr (2.22 g)

Next, pyrrole (0.33 g, 4.91 mmol) and trifluoroacetic acid (0.13 g, 1.18 mmol) were added to a suspension of DMTr (1.83 g, 0.98 mmol) in dichloromethane (15 mL), and then stirred at room temperature for 1.5 hours. The solid precipitated by dropping acetonitrile (37 mL) and methanol (18 mL) to the reaction solution was filtered. The solid was washed with acetonitrile-methanol-dichloromethane mixture solvent, and then dried under reduced pressure at 50° C. to obtain a 2 mer (1.48 g).

The same procedure as was repeated 6 times to obtain 2.94 g of 8 mer.

TOF/MS (ESI): m/z 2106.3 [M+2H]2+, 1403.9 [M+3H]3+.

Example 7-15

(2) De-Tagging of 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy) (5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(N4-benzoyldeoxycytidine-3′-yl)phosphoryl)-N6-benzoyldeoxyadenosine-3′-yl)phosphoryl)thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)-N4-benzoyldeoxycytidine-3′-yl)phosphoryl)-N6-benzoyldeoxyadenosine-3′-yl)phosphoryl)thymidine-3′-yl)phosphoryl)thymidine-3′-yl 2-((3,4,5-tris(octadecyloxy)benzoyl)oxy)acetate

N,N-Diisopropylethylamine (65 mg, 0.50 mmol) was added to a mixture solution of dichloromethane (0.4 mL), THF (0.2 mL) and 2-propanol (0.04 mL), and then cooled to 0 to −10° C. Next, 4 mol/L-lithium borohydride-THF solution (0.125 m L, 0.50 mmol) was added, and then stirred at the same temperature for 20 minutes to prep are a reducing reagent. The reducing reagents were added to a mixture of 8 mer (84 mg, 0.02 mmol) in dichloromethane (4.2 mL)-THF (1.8 mL)-2-propanol (0.4 mL) previously cooled to 0 to −10° C., and then the mixture was stirred at the same temperature for 40 minute s. 10% aqueous ammonium chloride (5 mL) was added to the reaction solution, and then was extracted with dichloromethane. The organic layer was washed with brine, and then the organic layer was concentrated to dryness. The precipitated solid was collected by suspending the concentrated residue in dichloromethane. The solid was washed with dichloromethane and dried in vacuo, and then 5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy)(5′-O-((2-cyanoethoxy) (N4-benzoyldeoxycytidine-3′-yl)phosphoryl)-N6-benzoyldeoxyadenosine-3′-yl)phosphoryl)thymidine-3′-yl)phosphoryl)-N2-isobutyryldeoxyguanosine-3′-yl)phosphoryl)-N4-benzoyldeoxycytidine-3′-yl)phosphoryl)-N6-benzoyldeoxyadenosine-3′-yl)phosphoryl)thymidine-3′-yl)phosphoryl)thymidine (25 mg) was obtained.

TOF/MS (ESI): m/z 1621.9 [M+2H]2+, 1081.6 [M+3H]3+, 811.2 [M+H]4+.

8. Comparison of the Rate of Progression of De-Tagging

Example 8

5′-O-(4,4′-Dimethoxytrityl)thymidine-3′-yl 2-((3,4,5-tris(octadec yloxy)benzoyl)oxy)acetate (abbreviated as A-Tag-dT-DMTr) and 3,4,5-tris(octadecyloxy)benzyl 5′-O-(4,4′-dimethoxytrityl)thymidine-3′-yl succinate (abbreviated as S-Tag-dT-DMTr) were compared for the rate of progression of de-tagging under ammonia basic conditions as shown below.

<De-Tagging Conditions>

A-Tag-dT-DMTr or S-Tag-dT-DMTr 50 μmol

28% aqueous ammonia 2.0 mL

Ethanol 0.5 mL

THF 0.5 mL

Reaction temperature 35° C.

<Formation Rate of dT-DMTr (Tag-Free Form)>

TABLE 11 hr2 hr3 hr4 hr5 hrA-Tag-dT-DMTr34%74%91%98%100%S-Tag-dT-DMTr0%2%3%4%5%
<HPLC Condition>

Columns: Inertsil WP 300 Diol (5 μm, 4.6 ϕ×150 mm)

Flow rate: 1.0 mL/min

Mobile phase: n-hexane, CHCl3-MeOH (1/1)

Gradient: 0-11 min; 5 to 30% (CHCl3-MeOH)

Detection method: UV (λ=254 nm)

Retention time: A-Tag-dT-DMTr: Rt=4.23 min, S-Tag-dT-DMTr: Rt=4.17 min, dT-DMTr: Rt=9.40 min

Production rate: (dT-DMTr)/[(A-Tag-dT-DMTr or S-Tag-dT-DMTr)+(dT-DMTr)]

As described above, we confirmed that A-Tag is more rapidly de-Tagged than succinate esters in common deprotection conditions for oligonucleic acid synthesis.

9. Summary of Synthesis Examples

Hereinafter, those summarizing the synthesis examples in each of the above-described examples will be described.

9.1 Synthesis of the 3 Mer

TABLE 2Synthesis of the 3 mersequenceYield (%)5′ → 3′B1B2B3abcdtotalTGTTGBuT95%91%98%90%76%GTAABzTGBu96%92%97%94%81%TGCCBzGBuT97%97%99%89%83%TTGGBuTT98%98%94%89%80%

As shown in Table 2 above, various Tag-conjugated 3 mer DNA oligomers could be obtained in high yields.

9.2 Synthesis of the Dimer

TABLE 3Synthesis of the dimersequenceYield (%)5′ → 3′XYB1B2abctotalTT (P = S)HSTT95%91%96%83%UU (TBS)OTBSOUU68%97%92%61%UC (OMe)OMeOCBzU91%99%70%63%UG (F)FOGBuU96%98%93%87%

As shown in Table 3 above, various Tag-conjugated oligonucleotides (dimers) were obtained in high yields.

9.3 Synthesis of the Dimer

TABLE 4Synthesis of the dimersequenceYield (%)5′ → 3′ZnB1B2abctotalTTO2TT18%96%17%TTN—Me1TT78%98%96%73%TTN—H2TT60%99%95%56%

As shown in Table 4 above, various Tag-conjugated oligonucleotides (dimers) were obtained in high yields.

9.4 Synthesis of the Dimer

TABLE 5Synthesis of the dimersequenceYield (%)5′ → 3′B1B2abctotalTATABz98%97%59%56%GCGBuCBz97%96%91%85%

As shown in Table 5 above, various Tag binding oligonucleotides (dimers) were obtained in high yields.

9.5 Synthesis and Deprotection of 20 Mers

By using the alkoxyphenyl derivative of the present invention and the Tagged protected nucleotide, the Tagged protected oligonucleotide (20 mer) could be easily obtained. Moreover, by removing Tag moieties as well as protecting groups, oligonucleotides could be conveniently obtained.

9.6 De-Tag of the 3 Mer

TABLE 6de-Tag of the 3 mersequence5′ → 3′B1B2B3ReductantBaseYieldTGTTGBuTLiBH4—95%GTAABzTGBuLiBH4—75%TGCCBzGBuTLiBH4—79%TTGGBuTTLiBH4—93%

As shown in Table 6 above, Tag moieties of various Tagged protected oligonucleotides (3 mers) were removed, and each protected oligonucleotide (3 mer) could be obtained in high yield.

9.7 De-Tag of the Dimer

TABLE 7de-Tag of the dimersequence5′ → 3′XYB1B2ReductantBaseYieldTT (P = S)HSTTLiBH4—93%UU (TBS)OTBSOUULiBH4—60%UC (OMe)OMeOCBzULiBH4—53%UG (F)FOGBuULiBH4—67%

As shown in Table 7 above, the Tag moieties of the various Tagged protected oligonucleotides (dimers) were removed and each protected oligonucleotide (dimer) was obtained in high yield.

9.8 De-Tag of the Dimer

TABLE 8de-Tag of the dimersequence5′ → 3′ZnB1B2ReductantBaseYieldTTO2TTLiBH4—76%TTN—Me1TTLiBH4—98%TTN—H2TTLiBH4—82%

As shown in Table 8 above, the Tag moieties of the various Tagged protected oligonucleotide (dimers) were removed and each protected oligonucleotide (dimer) was obtained in high yield.

9.9 De-Tag of the Dimer

TABLE 9de-Tag of the dimersequence5′ → 3′B1B2ReductantBaseYieldTATABzLiBH4DIPEA61%GCGBuCBzLiBH4DIPEA52%

As shown in Table 9 above, the Tag moieties of the various Tagged protected oligonucleotides (dimers) were removed and each protected oligonucleotide (dimer) was obtained in high yield.

INDUSTRIAL APPLICABILITY

The alkoxyphenyl derivative of the present invention is available for the production of the Tagged protected nucleosides and Tagged protected nucleotides and the like. Also, a method for producing an oligonucleotide, such as a Tagged protected nucleoside and a Tagged protected nucleotide of the present invention, and a method for selective Tag moiety removal of a Tagged protected nucleoside or a Tagged protected nucleotide are available for the synthesis of protected oligonucleotides, particularly in liquid phase synthesis (including pseudo-liquid phase synthesis). All of these are very useful compounds and methods for nucleic acid drug development, biological mechanism elucidation, and the like.