Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2002-05-21
2004-11-30
McKane, Joseph K. (Department: 1626)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Heterocyclic carbon compounds containing a hetero ring...
C514S414000, C544S144000, C548S455000
Reexamination Certificate
active
06825195
ABSTRACT:
This application claims priority of Japanese Patent Application No. 2001-164905, filed May 31, 2001 and Japanese Patent Application No. 2002-95074, filed Mar. 29, 2002.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel fluorescent group-containing carbodiimide compound and a process for producing the compound. More specifically, the present invention relates to a fluorescent group-containing carbodiimide compound, which is capable of efficiently introducing a fluorescent group into a nucleic acid or a protein in a simple manner, and to a process for producing the compound through which a fluorescent group can be efficiently coupled to a carbodiimide compound in a simple manner.
2. Description of the Related Art
As a method of introducing a fluorescent substance into a nucleic acid, several methods are known in the art, for example, a method of introducing a fluorescent substance into a nucleic acid using a fluorescent substance-bound nucleotide with the aid of an enzyme or the like (JP-A-6-271599); a method of biding a fluorescent substance-bound streptavidin to a biotin-labeled nucleic acid; and a method of reacting a fluorescent substance containing an amino reactive group with an amino linker-bound nucleic acid. In addition to those methods, there are known a method that utilizes a fluorescent group-containing carbodiimide compound indicative of an extremely high reactivity to a nucleic acid base (JP-A-10-287870), and the like.
Of those, however, the method disclosed in JP-A-6-271599, the method using the biotin label, and the method using the amino linker binding have disadvantages in that the fluorescent substances cannot be introduced into a naturally-occurring nucleic acid, and complicated operation is required. In the method (disclosed in JP-A-10-287870) in which a fluorescent group-containing carbodiimide compound is used, the presence of a quaternary ammonium in the molecule provides the comparatively favorable solubility to water. However, there may be cases where, in case of introducing a carbodiimide group into a polycyanine compound, the method of JP-A-10-287870 suffers a problem of difficulty. Thus, there is a need to newly investigate a simplified synthesis method.
SUMMARY OF THE INVENTION
The present invention has been made under the above-mentioned circumstances, and therefore, has an object to provide a novel fluorescent group-containing carbodiimide compound, by which fluorescent group can be efficiently introduced into a nucleic acid including a naturally-occurring nucleic acid and also efficiently introduced into a protein in a simple manner, while retaining sufficient solubility to water.
Another object of the present invention is to provide a method, which is appropriate to the manufacture of the fluorescent group-containing carbodiimide compound, and more specifically to a process for producing a fluorescent group-containing carbodiimide compound, with which a fluorescent group-containing compound and a carbodiimide compound are efficiently bound together in a simple manner.
For solving the above-mentioned problems in the art, the inventors of the present invention have conducted intensive investigation for solving the above-mentioned problems, and finally found out a fluorescent group-containing carbodiimide compound having a chemical structure with an ester linkage, represented by the following formula (1).
Thus, the present invention is following:
(1) There is provided a fluorescent group-containing carbodiimide compound, comprising a chemical structure with an ester linkage, represented by the general formula (1):
Y
1
—N═C═N—Y
2
—Q—Y
3
—F (1)
wherein each of Y
1
, Y
2
, and Y
3
is a functional group selected from the group consisting of —CH
2
—, —NHCO—, —CONH—, —O—, —S—, —NR
1
— (R
1
is a straight-chain, circular, or branched saturated or unsaturated aliphatic hydrocarbon group having from 1 to 20 carbon atoms), —NR
2
R
3
— (R
2
and R
3
are independently a hydrogen atom, a straight-chain, circular, or branched saturated or unsaturated aliphatic hydrocarbon group having from 1 to 20 carbon atoms, or a cycloalkyl group, an aryl group, or an aralkyl group, each of which may have a substituent, or if either R
2
or R
3
is a hydrogen atom, then the remainder is a straight-chain, circular, or branched saturated or unsaturated aliphatic hydrocarbon group having from 1 to 20 carbon atoms, or a cycloalkyl group, an aryl group, or an aralkyl group, each of which may have a substituent, and furthermore, R
2
and R
3
may be coupled together to form a nitrogen-containing heterocyclic group which may include oxygen as a whole), —COO—, —OCO—, —NHSO
2
—, —NHC(S)NH—, —SO
2
NH—, and other functional groups represented by the general formula (2):
—(CH
2
)p—(L)r—(CH
2
)q— (2)
(where L is a functional group selected from the group consisting of —CH
2
—, —NHCO—, —CONH—, —O—, —S—, —NR
1
—, —NR
2
R
3
—, —COO—, —OCO—, —NHSO
2
—, —NHC(S)NH—, and —SO
2
NH—; each of p and q is one of integers of from 0 to 20, while r is zero (0) or one (1), and R
1
, R
2
, and R
3
are same as those of the general formula (1));
Q is an ester linkage; and
F is a fluorescent group made of a cyanine dye represented by one of the general formulas (3-1), (3-2), and (3-3),
(where m is one of integers of from 1 to 3, while n is one (1), each of J
1
, J
2
, J
3
, and J
4
is a functional group independently selected from the group consisting of H, OH, COOH, a sulfonic group, a sulfonate group, and an ester, amide, ether, alkyl, alkane, alkene, alkyne, allyl, and amino groups, and J
1
and J
2
are different functional groups) where
each of the functional groups represented by Y
1
, Y
2
, Y
3
, and F may include a group selected from the group consisting of carboxyl groups, sulfonyl groups, and phosphonyl groups, which are substituted with alkali metals, alkaline earth metals, or basic groups containing nitrogen or phosphorus, respectively, and also Y
1
, Y
2
, and Y
3
may be the same functional group or different functional groups.
(2) There is provided a fluorescent group-containing carbodiimide compound according to (1), in which at least one of the functional groups represented by Y
1
, Y
2
, Y
3
, and F includes a group selected from the group consisting of carboxyl groups, sulfonyl groups, and phosphonyl groups, which are substituted with alkali metals, alkaline earth metals, or basic groups containing nitrogen or phosphorus, respectively.
(3) There is provided a process for producing a fluorescent group-containing carbodiimide compound according to (1) comprising the steps of: mixing a carbodiimide compound represented by the general formula (4) with a fluorescent group-containing compound represented by the general formula (5) to form an ester linkage, in which the general formula (4) is:
Y
1
—N═C═N—Y
2
—ZZ
1
(4)
(wherein Y
1
and Y
2
represent the same as those of the general formula (1), respectively, and ZZ
1
represents carboxylate, sulfonate, or phosphonate); and
the general formula (5) is:
X—Y
3
—F (5)
(wherein Y
3
and F represent the same as those of the general formula (1), respectively, and X represents halogen).
(4) There is provided a process for producing a fluorescent group-containing carbodiimide compound according to (2), further comprising the steps of: selecting at least one of the carboiimide compound represented by the general formula (4) and the fluorescent group-containing compound represented by the general formula (5) so as to include a functional group which can be selected from the group consisting of carboxyl groups, sulfonyl groups, and phosphonyl groups, which are substituted with alkali metals, alkaline earth metals, or basic groups containing nitrogen or phosphorus, respectively, and reacting the carboiimide compound represented by the general formula (4) with the fluorescent group-containing compound represented by the general formula (5).
(5) There is provided a process for producing a fluorescent group-containing carbodiimide comp
McKane Joseph K.
Nisshinbo Industries Inc.
Shameem Golam M M
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