Fluorescent nucleotides and labeling methods using the same

• US 20030113755A1
• Filed: 07/17/2002
• Published: 06/19/2003
• Est. Priority Date: 07/19/2001
• Status: Active Grant

• Current Assignee: Fujifilm Corporation (Fujifilm Holdings Corporation)
• Original Assignee: Fuji Photo Film Co Limited (Fujifilm Holdings Corporation)
• Inventors: Kojima, Masayoshi, Takeuchi, Kazuya, Inomata, Hiroko, Nishigaki, Junji, Nakamura, Kouki
• Granted Patent: US 7,087,744 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: C09B 23/0066 the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)
• CPC Class Codes: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: C09B 23/086 more than five >CH- groups
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

Fluorescent nucleotides useful for labeling nucleic acids which are represented by the formula: X—Y—Z wherein X represents a residue of natural or non-natural nucleotide and the like and binds to Y at a basic moiety of the residue; Y represents a divalent bridging group or a single bond; Z represents a monovalent group derived from a compound represented by the formula (I) wherein R¹ to R⁴ represent an alkyl group; V¹ to V⁶ represent a hydrogen atom or a substituent; L¹ to L⁷ represent a methine group; W represents an oxygen atom, a sulfur atom, —C(R³)(R⁴)— or —N(R⁵)— wherein R³ to R⁵ represents an alkyl group; Q represents a nitrogen atom or —C(V⁷)— wherein V⁷ represents a hydrogen atom or a monovalent substituent; M represents a counter ion; m represents a number required to neutralize the charge of the molecule; s, t and u represent 0 or 1 or the like, and binds to Y at a reactive group existing in R¹ or R².

Claims

1. A fluorescent nucleotide represented by the formula:

X—

Y—

Z wherein,

X represents a residue of a natural or non-natural nucleotide, an oligonucleotide, a polynucleotide, or a derivative thereof which binds to Y at a basic moiety of said residue;

Y represents a divalent bridging group or a single bond;

Z represents a monovalent group derived from;

a compound represented by the following general formula (I);

 

wherein, R¹, R², R³, and R⁴ independently represent a substituted or unsubstituted alkyl group, R³ and R⁴ may bind to each other to form a saturated or unsaturated ring;

V¹, V², V³, V⁴, V⁵, and V⁶ represents a hydrogen atom or a substituent, V¹ and V², V² and V³, V⁴ and V⁵, and/or V⁵ and V⁶ may bind to each other to form a saturated or unsaturated ring;

L¹, L², L³, L⁴, L⁵, L⁶ and L⁷ independently represent a substituted or unsubstituted methine group;

W represents an oxygen atom, a sulfur atom, —

C(R³)(R⁴)—

or —

N(R⁵)—

wherein R³, R⁴, and R⁵ independently represent a substituted or unsubstituted alkyl group;

Q represents a nitrogen atom or —

C(V⁷)—

wherein V⁷ represents a hydrogen atom or a monovalent substituent and may bind to V⁶ to form a saturated or unsaturated ring;

M represents a counter ion;

m represents a number required to neutralize the charge of the molecule;

s represents 0 or 1;

t represents 0 or 1; and

u represents 0 or 1;

a compound represented by the following general formula (II);

 

wherein, V¹, V², V³, R¹, R², R³, R⁴, L¹, L², L³, L⁴, L⁵, L⁶, L⁷, W, M, m, s, t, and u have the same meanings as defined above;

V⁸ represents a hydrogen atom or a monovalent substituent, provided that, when W represents —

N(R⁵)—

or —

C(R³)(R⁴)—

, V⁸ may bind to a substituent on W to form a saturated or unsaturated ring;

or

a compound represented by the following general formula (III);

 

wherein, V¹, V², V³, R¹, R², R³, R⁴, L¹, L², L³, L⁴, L⁵, L⁶, L⁷, W, M, m, s, t, and u have the same meanings as defined above;

E represents a nitrogen atom or —

C(R⁶)═

(R⁶ represents a hydrogen atom or a monovalent substituent;

V⁹ represents a hydrogen atom or a monovalent substituent and V⁹ may bind to R⁶ to form a saturated or unsaturated ring,

and Z binds to Y at a reactive group existing in R¹ or R².

2. The fluorescent nucleotide according to claim 1, wherein at least one of R¹ and R² is an alkyl group substituted with an active ester group in Y, wherein said active ester group can covalently bond to an amino group, a hydroxyl group, or a thiol group.

3. The fluorescent nucleotide according to claim 1, wherein at least one of R¹ and R² is an alkyl group substituted with a carboxyl group in Y.

4. The fluorescent nucleotide according to claim 1, wherein X is a residue of a nucleotide or a derivative thereof.

5. The fluorescent nucleotide according to claim 1, wherein X is a residue of a nucleotide or a derivative thereof selected from the group consisting of:

(1) a nucleotide selected from the group consisting of AMP, ADP, ATP, GMP, GDP, GTP, CMP, CDP, CTP, UMP, UDP, UTP, TMP, TDP, TTP, 2-Me-AMP, 2-Me-ADP, 2-Me-ATP, 1-Me-GMP, 1-Me-GDP, 1-Me-GTP, 5-Me-CMP, 5-Me-CDP, 5-Me-CTP, 5-MeO—

CMP, 5-MeO—

CDP, and 5-MeO—

CTP wherein Me represents a methyl group, MeO represents a methoxy group, and a numeral before Me or MeO represents a substituting position,

(2) a nucleotide selected from the group consisting of a deoxynucleotide and a dideoxynucleotide corresponding to the nucleotide defined in (1); and

(3) a nucleotide derivative derived from the nucleotide defined in (1) or (2).

6. The fluorescent nucleotide according to claim 1, wherein Y represents —

CH2—

, —

CH═

CH—

, —

C═

C—

, —

CO—

, —

O—

, —

S—

, —

NH—

, or a bridging group consisting of a combination thereof wherein a hydrogen atom on the bridging group may be replaced with other substituent.

7. The fluorescent nucleotide according to claim 1, wherein Y represents an aminoallyl group.

8. A method for producing a fluorescence-labeled nucleic acid which comprises a step of performing a nucleic acid synthesis reaction by using a nucleic acid synthetase, a template nucleic acid, and the fluorescent nucleotide according to claim 1.

9. The method according to claim 8, wherein the nucleic acid synthesis reaction comprises one or more reactions selected from the group consisting of a reverse transcription reaction, terminal transferase reaction, reactions of random prime method, PCR, and nick translation.

10. A nucleic acid probe or a primer labeled with the fluorescent nucleotide according to claim 1.

11. An agent for detecting a nucleic acid which comprises the nucleic acid probe or the primer according to claim 10.

12. The agent according to claim 11 which is used for diagnosis of a disease.

13. A kit for detecting a nucleic acid, which comprises:

(1) the fluorescent nucleotide according to claim 1;

(2) a nucleic acid synthetase; and

(3) a buffer.

14. An azamethine dye represented by the general formula (I) defined in claim 1.

15. An azamethine dye represented by the general formula (II) defined in claim 1.

16. An azamethine dyes represented by the general formula (III) defined in claim 1.