Phosphoramidite Compound And Method For Producing Oligo-Rna

• US 20070282097A1
• Filed: 08/25/2005
• Published: 12/06/2007
• Est. Priority Date: 08/26/2004
• Status: Active Grant

• Current Assignee: Nippon Shinyaku Company Limited
• Original Assignee: Nippon Shinyaku Company Limited
• Inventors: Ohgi, Tadaaki, Ishiyama, Kouichi, Masutomi, Yutaka
• Granted Patent: US 8,691,970 B2
• Time in Patent Office: Days
• US Class Current: 536/23.100
• CPC Class Codes: C07C 255/13 containing cyano groups and etherified hydroxy groups bound to the carbon skeleton
• CPC Class Codes: C07H 19/067 with ribosyl as the saccharide radical
• CPC Class Codes: C07H 19/167 with ribosyl as the saccharide radical
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
• CPC Class Codes: Y02P 20/582 Recycling of unreacted starting or intermediate materials

Abstract

An object of the present invention is to provide a useful and novel phosphoramidite compound for the synthesis of oligo-RNA.
A phosphoramidite compound represented by general formula (1), wherein:

• • B_X represents a nucleobase optionally having a protecting group; and
  • R¹ is a substituent represented by general formula (2), wherein:
  • R¹¹, R¹² and R¹³ are the same or different and each represents hydrogen or alkoxy;
  • R^2a and R^2b are the same or different and each represents alkyl, or R^2a and R^2b taken together with the adjacent nitrogen atom may form a 5- to 6-membered saturated amino cyclic group, the amino cyclic group optionally having an oxygen or sulfur atom as a ring-composing member in addition to the adjacent nitrogen atom; and
  • WG¹ and WG² are the same or different and each represents an electron-withdrawing group.

Claims

1. :

A phosphoramidite compound represented by general formula (1),

Wherein;

B_X represents a nucleobase optionally having a protecting group;

R¹ is a substituent represented by general formula (2),

wherein;

R¹¹, R¹², and R¹³ are the same or different and each represents hydrogen or alkoxy;

R^2a and R^2b are the same or different and each represents alkyl, or R^2a and R^2b taken together with the adjacent nitrogen atom may form a 5- to 6-membered saturated amino cyclic group, the amino cyclic group optionally having an oxygen or sulfur atom as a ring-composing member in addition to the adjacent nitrogen atom; and

WG¹ and WG² are the same or different and each represents an electron-withdrawing group.

2. :

The phosphoramidite compound of claim 1, wherein WG¹ is cyano.

3. :

A method for producing an oligoribonucleotide represented by general formula (3), comprising reacting the phosphoramidite compound of claim 1,

wherein;

each B represents independently adenine, guanine, cytosine, uracil, thymine or a modified form thereof;

each R represents independently H or hydroxyl, and at least one of the R groups is hydroxyl;

Z represents H or a phosphate group; and

n represents an integer in the range from 1 to 100.

4. :

The method for producing the oligoribonucleotide (3) of claim 3, which comprises the following steps;

(A) producing a compound represented by general formula (5) by removing the 5′

-hydroxyl group by reacting an acid with a compound represented by general formula (4),

wherein;

n is the same as defined above;

each B_X represents independently a nucleobase optionally having a protecting group; and

R¹ is a substituent represented by general formula (2),

wherein;

R¹¹, R¹² and R¹³ are the same or different and each represents hydrogen or alkoxy;

each WG² represents an electron-withdrawing group; and

each R⁴ represents independently H, acyloxy or a substituent represented by general formula (6),

wherein;

WG¹ represents an electron-withdrawing group; and

E represents acyl or a substituent represented by general formula (7),

wherein;

Q represents a single bond or a substituent represented by general formula (8),

wherein;

WG² is the same as defined above; and

T represents H, acyloxy, or the substituent represented by the above general formula (6), with the proviso that either E or T is a substituent (7),

(B) producing a compound represented by general formula (9) by condensing a nucleic acid monomer compound with the compound produced in step (A) using an activating agent,

wherein;

B_X, E, n, R¹, R⁴, T and WG² are the same as defined above,

(C) capping the 5′

-hydroxyl group of the unreacted compound (5) in step (B),

wherein;

B_X, E, n, R⁴, T and WG² are the same as defined above; and

R⁵ represents methyl or phenoxymethyl,

(D) converting a phosphorous group into a phosphate group by reacting an oxidizing agent with compound (9), which is produced in step (B),

wherein;

B_X, E, n, R¹, R⁴, T and WG² are the same as defined above,

(E) cleaving the compound (11) produced by step (D) from the solid support, and then deprotecting each nucleobase and each 2′

-hydroxyl group,

wherein;

B, B_X, E, n, R, R¹, R⁴, T, WG² and Z are the same as defined above,

(F) removing the 5′

-hydroxyl group of the compound (12) produced by step (E),

wherein;

B, n, R, R¹ and Z are the same as defined above; and

(G) isolating and purifying the oligoribonucleotide (3) produced in step (F).

5. :

The method for producing the oligoribonucleotide (3) of claim 4 having a desired chain-length by repeating steps (A) to (D).

6. :

The method for producing an oligoribonucleotide of claim 4, comprising the addition of alkylamine, amidine, thiol, a thiol derivative or mixtures thereof in step (E).

7. :

An ether compound represented by general formula (13),

wherein;

L represents halogen, an arylthio group, an alkyl sulfoxide group or an alkylthio group; and

WG¹ represents an electron-withdrawing group.

8. :

The ether compound of claim 7, wherein WG¹ is cyano.

9. :

A method for producing a phosphoramidite compound represented by general formula (1);

wherein;

B_X represents a nucleobase optionally having a protecting group; and

R¹ represents a substituent represented by general formula (2),

wherein;

R¹¹, R¹² and R¹³ are the same or different and each represents hydrogen or alkoxy;

R^2a and R^2b are the same or different and each represents alkyl, or R^2a and R^2b taken together with the adjacent nitrogen atom may form a 5- to 6-membered saturated amino cyclic group, the amino cyclic group optionally having an oxygen or sulfur atom as a ring-composing member in addition to the adjacent nitrogen atom; and

WG¹ and WG² are the same or different and each represents an electron-withdrawing group, wherein the method comprises the steps of;

(a) producing a nucleoside derivative represented by general formulas (15) and (15′

), wherein an ether-type protecting group which can be removed under neutral conditions is introduced to 2′

-hydroxyl group, by allowing an alkylating reagent to act on a nucleoside derivative represented by general formula (14),

wherein;

B_X represents a nucleobase optionally having a protecting group; and

R¹ and WG¹ are the same as defined above,

(b) isolating and purifying the nucleoside derivative (15) produced by step (a),

(c) separately from step b, producing a ribonucleic acid compound represented by general formula (17), wherein an ether-type protecting group which can be removed under neutral conditions is introduced to the 2′

-hydroxyl group, by allowing an alkylating reagent to act on a ribonucleic acid compound represented by general formula (16),

wherein;

B_X and WG¹ are the same as defined above; and

A represents a silicon substituent represented by general formula (18a) or (18b),

wherein;

R⁶ represents alkyl,

(d) separately from steps (a) to (c), producing a ribonucleic acid compound represented by general formula (19) by allowing dimethylsulfoxide, acetic acid and acetic anhydride to act on the ribonucleic acid compound (16),

wherein;

A and B_X are the same as defined above,

(e) producing a ribonucleic acid compound represented by general formula (17), wherein an ether-type protecting group which can be removed under neutral conditions is introduced to the 2′

-hydroxyl group, by allowing an alcohol compound represented by general formula (20), an acid and a reagent for halogenating a sulfur atom to act on a nucleoside derivative (19) produced in step (d),

wherein;

A, B_X and WG¹ are the same as defined above,

(f) producing a ribonucleic acid compound represented by general formula (21) by removing the protecting groups of the 3′

- and 5′

-hydroxyl group of the ribonucleic acid compound (17) produced by step (c) or step (e),

wherein;

A, B_X and WG¹ are the same as defined above,

(g) producing a ribonucleic acid compound (15) by introducing a protecting group (R¹), which can be removed under acidic conditions, to the 5′

-hydroxyl group of the ribonucleic acid compound (21) produced by step (f),

wherein;

B_X, R¹ and WG¹ are the same as defined above; and

X³ represents halogen; and

(h) producing a phosphoramidite compound represented by general formula (1) by phosphoramiditing the 3′

-hydroxyl group by allowing a phosphoramiditing reagent and if necessary an activating agent to act on nucleoside derivative (15) produced by step (b), or step (g),

wherein;

B_X, R¹ and WG¹ are the same as defined above;

R^2a and R^2b are the same or different and each represents alkyl, or R^2a and R^2b taken together with the adjacent nitrogen atom may form a 5- to 6-membered saturated amino cyclic group, the amino cyclic group optionally having an oxygen or sulfur atom as a ring-composing member in addition to the adjacent nitrogen atom; and

WG² represents an electron-withdrawing group.

10. :

The method for producing a phosphoramidite compound according to claim 9, wherein the alkylating reagent is an ether compound represented by general formula (13).

wherein;

L represents halogen, an arylthio group, an alkyl sulfoxide group or an alkylthio group; and

WG¹ represents an electron-withdrawing group.

11. :

The method for producing a phosphoramidite compound according to claim 9, wherein WG¹ is cyano.

12. :

The method for producing a phosphoramidite compound according to claim 9, wherein the phosphoramiditing reagent is a compound represented by general formula (22) or (23),

wherein;

R^2a and R^2b are the same or different and each represents alkyl, or R^2a and R^2b taken together with the adjacent nitrogen atom may form a 5- to 6-membered saturated amino cyclic group, the amino cyclic group optionally having an oxygen or sulfur atom as a ring-composing member in addition to the adjacent nitrogen atom;

WG² represents an electron-withdrawing group; and

X¹ represents halogen.

13. :

The method for producing a phosphoramidite compound according to claim 9, wherein the activating agent is 1H-tetrazole, 5-ethylthiotetrazole, 5-benzilmercapto-1H-tetrazole, 4,5-dichloroimidazole, 4,5-dicyanoimidazole, benzotriazole triflate, imidazole triflate, pyridinium triflate, N,N-diisopropylethylamine or 2,4,6-collidine/N-methylimidazole.

14. :

A ribonucleic acid compound represented by general formula (21),

wherein;

B_X represents a nucleobase optionally having a protecting group; and

WG¹ represents an electron-withdrawing group.

15. :

The ribonucleic acid compound of claim 14, wherein WG¹ is cyano.

16. :

A ribonucleic acid compound represented by general formula (15),

wherein;

B_X represents a nucleobase optionally having a protecting group;

R¹ is a substituent represented by general formula (2),

wherein;

R¹¹, R¹² and R¹³ are the same or different and each represents hydrogen or alkoxy; and

WG¹ represents an electron-withdrawing group.

17. :

The ribonucleic acid compound according to claim 16, wherein WG¹ is cyano.

18. :

A ribonucleic acid compound represented by general formula (17),

wherein;

B_X represents a nucleobase optionally having a protecting group;

A represents a silicon substituent represented by general formula (18a) or (18b),

wherein;

R⁶ represents alkyl;

and

WG¹ represents an electron-withdrawing group.

19. :

The ribonucleic acid compound according to claim 18, wherein WG¹ is cyano.