Organic compounds -- part of the class 532-570 series – Organic compounds – Four or more ring nitrogens in the bicyclo ring system
Reexamination Certificate
2000-03-23
2003-11-04
Horlick, Kenneth R. (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Four or more ring nitrogens in the bicyclo ring system
Reexamination Certificate
active
06642380
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a novel fluorescent group-containing carbodiimide compound precursor and a fluorescent group-containing carbodiimide compound. More specifically, it relates to a fluorescent group-containing carbodiimide compound precursor that contains a halogen atom or a sulfonic acid group, which enables easy and efficient bonding of a fluorescent group and a method for producing it, as well as a fluorescent group-containing carbodiimide compound which has at least one group selected from a carboxyl group, a sulfo group, a phosphono group and a phospho group, which enables easy and efficient introduction of a fluorescent group into nucleic acids and proteins, a method for producing it and an analysis method utilizing it.
As methods for introducing a fluorescent substance into nucleic acids, there have been known, for example, introduction of a fluorescent substance into a nucleic acid by using a nucleotide bonded to a fluorescent substance through an enzymatic reaction and so forth (Japanese Patent Application Application Laid-open No. 6-271599), bonding of streptavidin, to which a fluorescent substance has already been bonded, to a biotin-labeled nucleic acid, a reaction of a fluorescent substance containing an amine-reactive group with a nucleic acid bonded to an amino-linker and so forth. In addition, there are also methods other than the above, such as utilization of a fluorescent group-containing carbodiimide compound which is highly reactive to nucleic acid bases (Japanese Patent Application Application Laid-open No. 10-287870).
However, among the aforementioned methods, the method described in Japanese Patent Application Laid-open No. 6-271599, the method utilizing biotin labeling and the method utilizing amino-linker bonding have drawbacks that it is not possible to introduce the fluorescent substance into naturally occurring nucleic acids, and the operations are complicated. The fluorescent group-containing carbodiimide compound exhibit good solubility in water because of the existence of quaternary ammonium salt in its molecular structure. However, in order to utilize a fluorescent substance with low solubility in water, such as a fluorescent substance having such a large electron system that it should have absorption in the near-infrared region, further improvement of the water solubility has been desired.
SUMMARY OF THE INVENTION
The object of the present invention is to provide a novel fluorescent group-containing carbodiimide compound, which enables efficient and easy introduction of fluorescent groups into proteins and nucleic acids including naturally occurring nucleic acids, and exhibits sufficient water solubility, and to provide a fluorescent group-containing carbodiimide compound precursor, which can be easily and efficiently bonded to a fluorescent group, and is suitable for the production of the aforementioned fluorescent group-containing carbodiimide compound.
The inventors of the present invention earnestly studied in order to achieve the aforementioned object. As a result, they found that a fluorescent group-containing carbodiimide compound having at least one group selected form a carboxyl group, a sulfo group, a phosphono group and a phospho group with sufficient water solubility could be produced by starting from a carbodiimide compound having a halogen atom or a sulfonic acid group, and that a fluorescent group could be easily and efficiently introduced into nucleic acids including naturally occurring nucleic acids and proteins by utilizing the aforementioned fluorescent group-containing carbodiimide compound. Thus, they accomplished the present invention.
The present invention provides the followings.
(1) A fluorescent group-containing carbodiimide compound precursor having a halogen atom or a sulfonic acid group which is represented by the following general formula (I):
B—Y
3
—N═C═N—Y
2
—W—Y
1
—[A]
n
—Y
4
—X (I)
wherein,
X represents a halogen atom or a sulfonic acid group;
A represents a functional group which is selected from the group consisting of —CH
2
—, —NHCO—, —CONH—, —O—, —S—, —NR
1
— wherein R
1
represents a linear, cyclic or branched saturated or unsaturated aliphatic hydrocarbon group having 1-20 carbon atoms, —NR
2
R
3
— wherein R
2
and R
3
independently represent a hydrogen atom, a linear or branched saturated or unsaturated aliphatic hydrocarbon group having 1-20 carbon atoms, or a cycloalkyl group, an aryl group or an aralkyl group which may have a substituent, provided that when one of R
2
and R
1
is a hydrogen atom, the other represents a linear or branched saturated or unsaturated aliphatic hydrocarbon group having 1-20 carbon atoms, or a cycloalkyl group, an aryl group or an aralkyl group which may have a substituent, or R
2
and R
3
may be bonded to each other to form as a whole a nitrogen-containing heterocyclic group which may contain an oxygen atom, —COO—, —OCO—, —NHSO
2
—, —NHC(S)NH— and —SO
2
NH—;
n represents 0 or 1;
W represents a direct bond or a quaternary onium group;
Y
1
, Y
2
, Y
3
and Y
4
each independently represent a functional group represented by the general formula (II):
—(CH
2
)
p
—(L)
r
—(CH
2
)
q
— (II)
wherein, L represents a functional group selected from the group consisting of —CH
2
—, —NHCO—, —CONH—, —O—, —S—, —NR
1
— wherein R
1
has the same meaning as defined for the formula (I), —NR
2
R
3
— wherein R
2
and R
3
have the same meanings as defined for the formula (I), —COO—, —OCO—, —NHSO
2
—, —NHC(S)NH— and —SO
2
NH—; p and q each independently represent an integer of from 0 to 20, and r represents 0 or 1;
B represents a hydrogen atom or a monovalent organic group being either the same as or different from —W—Y
1
—[A]
n
—Y
4
—X in the formula (I); and
any of the functional groups represented by B, Y
1
, Y
2
, Y
3
, Y
4
, A or W may have a group selected from a carboxyl group, a sulfo group, a phosphono group or a phospho group which have substitution of an alkali metal, an alkaline earth metal or a basic group containing a nitrogen or phosphorus atom.
(2) A fluorescent group-containing carbodiimide compound having at least one group selected from a carboxyl group, a sulfo group, a phosphono group and a phospho group which have substitution of an alkali metal, an alkaline earth metal or a basic group containing a nitrogen or phosphorus atom, which is represented by the following general formula (III):
wherein,
B, Y
1
, Y
2
, Y
3
, Y
4
, A, X, W and n have the same meanings as defined for the general formula (I);
F represents a fluorescent group;
Q represents either a tertiary or quaternary nitrogen atom, or a tertiary or quaternary phosphorus atom;
R
4
and R
5
each independently represent a hydrogen atom, a linear or branched saturated or unsaturated aliphatic hydrocarbon group having 1-20 carbon atoms, or a cycloalkyl group, an aryl group or an aralkyl group which may contain a substituent, provided that when one of R
4
and R
5
is a hydrogen atom, the other represents a linear or branched saturated or unsaturated aliphatic hydrocarbon group having 1-20 carbon atoms, or a cycloalkyl group, an aryl group or an aralkyl group which may contain a substituent, or R
4
and R
5
may be bonded to each other to form a nitrogen-containing heterocyclic group or a phosphorus-containing heterocyclic group, which may contain an oxygen atom, as —Q
+
R
4
R
5
—;
Y
5
has the same meaning as defined for Y
1
, Y
2
, Y
3
and Y
4
;
at least one functional group selected from B, Y
1
, Y
2
, Y
3
, Y
4
, Y
5
, A, W, R
4
, R
5
and F has at least one group selected from a carboxyl group, a sulfo group, a phosphono group and a phospho group which have substitution of an alkali metal, an alkaline earth metal or a basic group containing a nitrogen or phosphorus atom.
(3) The fluorescent group-containing carbodiimide compound precursor according to (1), wherein at least one functional group selected from B, Y
1
, Y
2
, Y
3
, Y
4
, A and W in the formula (I) has at least one group selected from a carboxyl
Kimura Naoki
Shiohata Namiko
Yoshikawa Yoko
Horlick Kenneth R.
Kilpatrick & Stockton LLP
Nisshinbo Industries Inc.
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