Nucleotide compound, nucleotide block oligonucleotide, and method for producing them

• US 6,380,378 B1
• Filed: 12/23/1999
• Issued: 04/30/2002
• Est. Priority Date: 12/24/1998
• Status: Expired due to Fees

• Current Assignee: Toagosei Company Limited
• Original Assignee: Toagosei Company Limited
• Inventors: Kitamura, Akinori, Yoshida, Tadao, Horie, Yoji, Uchida, Takayoshi
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US09/471,802
• Time in Patent Office: 859 Days
• Field of Search: 536/22.1, 536/25.33, 536/25.34, 536/25.4, 536/25.3, 536/25.31, 536/26.7, 536/26.8
• US Class Current: 536/25.3
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Novel nucleotide compounds represented by the formula (I)

wherein R¹ represents a protective group or a PEG bearing organic group; R², R^2′, R^2″, R³, R^3′ and R^3″ each represents a hydrogen atom or an alkyl, cycloalkyl, aryl or aralkyl group which may contain a hetero-atom; B¹, B², B³ and B⁴ each represents a base, if necessary, protected by a protective group common in nucleotide chemistry or by a PEG bearing organic group; X, X′ and X″ each represents an oxygen atom or a sulfur atom; Y represents an azolyl group, a mono- or di-alkylamino group or a saturated nitrogenous heterocyclic ring; A¹, A², A³ and A⁴ each represents a hydrogen atom, a hydroxyl group, an alkoxy group or a trialkylsilyloxy group; and m and n each represents 0 or an integer of 1 to 100. The nucleotide of the formula (I) can be used as it is in a reaction mixture for further reaction with 3′-O- and 5′-O-unprotected nucleoside or nucleotide of the formula (II) to yield a nucleotide block or oligonucleotide, and thus is useful as in situ DNA synthesis reagents.

Claims

1. A nucleotide compound represented by the following formula (I):

wherein R¹ represents a protective group or an organic group represented by the formula —

C(═

O)—

A′

—

(OCH₂CH₂)_kOCH³ in which k represents an integer of 3 or more, and A′

is a divalent organic group;

R², R^2′

, R³ and R^3′

each represents a hydrogen atom or an alkyl, cycloalkyl, aryl or aralkyl group which may contain a hetero-atom and R², R^2′

, R³ and R^3′

may be the same or different;

B¹ and B² each represents a base, optionally, protected by a protective group common in nucleotide chemistry or B′

which represents —

B^1′

—

C(═

O)—

A—

(OCH₂CH₂)_kOCH₃ in which B^1′

represents one of the groups represented by the

k represents an integer of 3 or more, A is a divalent group and represents an arylene group or an alkylene group having a straight or branched chain which may contain a heteroatom;

X represents an oxygen atom or a sulfur atom;

Y represents an azolyl group, a monoalkylamino group represented by HNR⁵ in which R⁵ is an alkyl group or a cycloalkyl group, a dialkylamino group or a saturated nitrogenous heterocyclic ring;

A¹ and A² each represents a hydrogen atom, a hydroxyl group, an alkoxy group or a trialkylsilyloxy group; and

n represents 0 or an integer of 1 to 100;

excluding the case wherein at least one of the combinations of R² and R³ and of R^2′

and R^3′

being a hydrogen atom and hydrogen atom, a hydrogen atom and a methyl group, a hydrogen atom and an ethyl group, a methyl group and a methyl group, a methyl group and an ethyl group or an ethyl group and an ethyl group.

2. A compound according to claim 1, wherein the sum of van der Waals volumes of R² and R³ and sum of van der Waals volumes of R^2′

and R^3′

in formula (I) are respectively 49 cubic angstroms or larger, and Y is an imidazolyl group, a 2-methylimidazolyl group, a 4-methylimidazolyl group or a triazolyl group.

3. A compound according to claim 1, wherein said compound is represented by the following formula (I-1):

wherein B¹ represents a base, optionally, protected by a protective group common in nucleotide chemistry; and

Y represents an azolyl group.

4. A compound according to claim 3, wherein the sum of van der Waals volumes of R^2′

and R^3′

in formula (I-1) is at least 49 cubic angstroms and Y is an imidazolyl group, a 2-methylimidazolyl group or a 4-methylimidazolyl group.

5. A compound according to claim 1, wherein said compound is represented by the following formula (I-2):

wherein B¹ represents a base, optionally, protected by a protective group common in nucleotide chemistry; and

Y represents either a monoalkylamino group represented by HNR⁵ in which R⁵ represents an alkyl group or a cycloalkyl group or a dialkylamino group.

6. A monoalkylamino type phosphoramidite compound according to claim 5, wherein the sum of van der Waals volumes of R^2′

and R^3′

in formula (I-2) is at least 49 cubic angstroms.

7. A compound according to claim 1, wherein said compound is represented by the following formula (I-3):

8. A compound according to claim 7, wherein the sum of Van der Waals volumes of R^2′

and R^3′

in formula (I-3) is at least 49 cubic angstroms.

16. A nucleotide compound according to claim 1, wherein said nucleotide excludes the combination of R² and R³ representing a hydrogen atom and a hydrogen atom, a hydrogen atom and a methyl group, a hydrogen atom and an ethyl group, a methyl group and a methyl group, a methyl group and an ethyl group or an ethyl group and an ethyl group.

18. A nucleotide compound according to claim 1, wherein the nucleotide excludes the combination of R^2′

and R^3′

from representing a hydrogen atom and a hydrogen atom, a hydrogen atom and a methyl group, a hydrogen atom and an ethyl group, a methyl group and a methyl group, a methyl group and an ethyl group or an ethyl group and an ethyl group.

21. A nucleotide according to claim 1, wherein said nucleotide R^2′

and R^3′

are selected from the group consisting of a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, a 1-methylbutyl group, a 1-ethylpropyl group, a cyclohexyl group, a n-nonyl group, 2-phenyletayl group, a 2-(methylthio)ethyl group, a phenyl group, a 1,1-diethyl-3-butenyl group and 1,1-dimethyl-2-phenylethyl group, provided that the combination of R^2′

and R^3′

does not represent a hydrogen atom and hydrogen atom, a hydrogen atom and a methyl group, a hydrogen atom and an ethyl group, a methyl group and a methyl group, a methyl group and an ethyl group or an ethyl group and an ethyl group.

9. A nucleoside compound represented by the following formula (I-3′

);

wherein R¹ represents a protective group or an organic group represented by the formula —

C(═

O)—

A′

—

(OCH₂CH₂)_kOCH₃ in which k represents an integer of 3 or more, R⁴ represents a hydrogen atom or a protective group commonly used in nucleotide chemistry, A is a divalent group and represents an arylene group or an alkylene group having a straight or branched chain which may contain a hetero-atom;

A¹ represents a hydrogen atom, a hydroxyl group, an alkoxy group or a trialkylsilyloxy group; and

B^1′

represents one of the groups represented by one of the following formula (1);

10. A method for producing a nucleotide block oligonucleotide represented by the following formula (IV) which comprises reacting a nucleotide derivative represented by formula (I-4) with a 3′

-O- and 5′

-O-unprotected nucleoside derivative or nucleotide derivative represented by formula (II) or reacting them in the presence of an optionally selected activating agent and oxidizing or sulfurizing a trivalent phosphorus atom of the resulting nucleotide represented by the following formula (III) to a pentavalent phosphorus atom;

wherein the above formulas (I-4), (II), (III) and (IV), R¹ represents a protective group or an organic group represented by the formula —

C(═

O)—

A′

—

(OCH₂CH₂)_kOCH₃ in which k represents an integer of 3 or more, and A′

represents a divalent organic group;

R², R^2′

, R^2″

, R³, R^3′

and R^3″

each represents a hydrogen atom or an alkyl, cycloalkyl, aryl or aralkyl group which may contain a hetero-atom and R², R^2′

, R^2″

, R³, R^3′

and R^3″

may be the same or different;

B¹, B², B³ and B⁴ each represents a base, optionally, protected by a protective group common in nucleotide chemistry or B′

which represents —

B^1′

—

C(═

O)—

A—

(OCH₂CH₂)_kOCH₃ in which B^1′

represents one of the groups represented by the formula (1);

k represents an integer of 3 or more, A is a divalent group and represents an arylene group or an alkylene group having a straight or branched chain which may contain a hetero-atom;

X, X¹ and X″

each represents an oxygen atom or a sulfur atom;

Y represents an azolyl group;

A¹, A², A³ and A⁴ each represents a hydrogen atom, a hydroxyl group, an alkoxy group or a trialkylsilyloxy group; and

m and n each represents 0 or an integer of 1 to 100;

excluding the case wherein at least one of the combinations of R² and R³, of R^2′

and R^3′

and of R^2″

and R^3″

being a hydrogen atom and a hydrogen atom, a hydrogen atom and a methyl group, a hydrogen atom and an ethyl group, a methyl group and a methyl group, a methyl group and an ethyl group or a ethyl group and ethyl group.

11. A method for producing a nucleotide block or an oligonucleotide according to claim 10, wherein the sum of van der Waals volumes of R² and R³, the sum of van der Waals volumes of R^2′

and R^3′

, and the sum of van der Waals volumes of R^2″

and R^3″

in the nucleoside derivative or nucleotide derivative are respectively at least 49 cubic angstroms, and X is an imidazolyl group, 2-methylimidazolyl group, 4-methylimidazolyl group or triazolyl group.

12. A nucleotide derivative represented by the formula (III) according to claim 10.

13. A nucleotide block or oligonucleotide represented by the formula (IV) according to claim 10.

14. A nucleotide derivative represented by the formula (III) according to claim 11.

15. A nucleotide block or oligonucleotide represented by the formula (IV) according to claim 11.

17. A method according to claim 10, wherein formula (III) excludes the combination of R² and R³ from representing a hydrogen atom and a hydrogen atom, a hydrogen atom and a methyl group, a hydrogen atom and an ethyl group, a methyl group and a methyl group, a methyl group and an ethyl group or an ethyl group and an ethyl group.

19. A method according to claim 10, wherein formula (III) excludes the combination of R^2′

and R^3′

from representing a hydrogen atom and a hydrogen atom, a hydrogen atom and a methyl group, a hydrogen atom and an ethyl group, a methyl group and a methyl group, a methyl group and an ethyl group or an ethyl group and an ethyl group.

20. A method according to claim 10, wherein formula (III) excludes the combination of R^2″

and R^3″

from representing a hydrogen atom and a hydrogen atom, a hydrogen atom and a methyl group, a hydrogen atom and an ethyl group, a methyl group and a methyl group, a methyl group and an ethyl group or an ethyl group and an ethyl group.