Compositions – Organic luminescent material containing compositions
Reexamination Certificate
1997-11-14
2001-01-09
Huang, Evelyn Mei (Department: 1612)
Compositions
Organic luminescent material containing compositions
C546S102000, C546S104000
Reexamination Certificate
active
06171520
ABSTRACT:
TECHNICAL FIELD
This invention relates to a reagent for labeling SH groups, which makes use of an acridine compound containing a maleimide group. More specifically, the present invention is concerned with an SH-labeling reagent using as a labeling substance an acridine compound, its production process and a labeling method making use of the same. The acridine compound contains therein a maleimide group, which binds to SH groups contained in or easily introducible into an analyte such as an amino acid, a protein or the like, and produces chemiluminescence.
BACKGROUND ART
Acridinium esters, acridine compounds, are useful as chemiluminescent labeling substances for their possession of high efficiencies of luminescence. Use of such an acridinium ester as a chemiluminescent label in an immunoluminescent analysis for a clinical test is disclosed, for example, in European Patent Publication No. 82636 and U.S. Pat. No. 4,745,181.
Known acridinium esters include those having hydrophilic structures such as those containing sulfonium ions at ends [Japanese Patent Application Laid-Open (Kokai) No. HEI 5-255264], those containing hydrazonium ions in spacer portions [Japanese Patent Application Laid-Open (Kokai) No. HEI 5-255263], and those obtained by substituting a carboxyl group for the methyl group or the like of acridinium [Japanese Patent Application Laid-Open (Kokai) No. HEI 6-228102]. These acridinium esters having hydrophilic structures are practically intended to label amino groups, and are considered to be compounds suitable for labeling amino acids, proteins and the like and hence for use in immunoluminescent analyses for clinical tests.
Incidentally, analytes in immunoluminescent analyses for clinical tests are mostly amino acids and proteins. As these substances contains many amino groups, the above-described known acridinium compounds have a significant advantage in that labeling can be easily performed.
In contrast, they label amino groups contained abundantly in amino acids and proteins. As a result, there are many site to be labeled. This has led to problems in the uniformity and reproducibility of labeling, the problem of insolubilization of analytes such as antibody proteins, and a problem that, when labeling is effected to an antibody, a labeling compound binds to amino groups located at antigen recognition sites and its function as an antibody is reduced or lost. When an analyte is a low-molecular substance, that is, contains only a limited number of amino groups, it may be possible to control a labeling reaction and to readily perform labeling at a constant molar ratio. However, when an analyte is a high-molecular substance such as an antibody protein and the number of amino groups cannot be precisely determined, there is a drawback that conditions for permitting labeling at a constant molar ratio have to be provisionally ascertained through repeated trial and error.
Incidentally, to furnish a compound as a chemiluminescent labeling reagent for practical use, it is essential that the compound assures an easy labeling reaction and does not result in luminescence or decomposition under labeling conditions. Counterparts to be labeled by an acridinium ester vary widely, led by low-molecular compounds such as amino acids and including even high-molecular compounds such as enzymes and antibodies. When amino groups of an analyte are relied upon as described above, an imide group is often introduced into an acridinium ester to make it bind to amino groups as disclosed in the publications referred to above. When a binding reaction to amino groups is performed using this imide group, the reaction proceeds efficiently under alkaline conditions. However, an acridinium compound is an unstable compound so that it results in luminescence or decomposition under alkaline conditions. Efficient performance of labeling while retaining luminescent activity therefore requires mutually contradictory reaction conditions and is not conveniently feasible.
It has accordingly been desired to find out a labeling compound capable of binding to an analyte such as an amino acid or protein under mild conditions in a chemiluminescent labeling method useful in an immunoluminescent analysis or the like and having sufficient binding force while possessing high specificity, and further to provide a method for stably and accurately detecting the analyte by making use of the labeling compound.
DISCLOSURE OF THE INVENTION
To solve the above-described problems, the present inventors first hinted upon using, as groups to be labeled, SH groups instead of amino groups used to date because SH groups have good reactivity although they are generally not abundantly contained in amino acids, proteins or the like. The present inventors have then found that an acridinium compound with a maleimide group introduced therein can be advantageously used as an SH-labeling reagent for labeling such SH groups, leading to the completion of the present invention.
An object of the present invention is therefore to provide an SH-labeling reagent comprising an acridine compound represented by the following formula (I):
wherein
A represents the following group:
—(CH
2
)
m1
—
or
—(CH
2
)
m2
—Q—(CH
2
)
n
—
in which Q represents a group —S
+
RX
−
—, a group —N
+
RR
1
X
−
— wherein R
1
represents an alkyl group having 1 to 6 carbon atoms or an aryl group, a group
wherein R
2
and R
3
may be the same or different and are each independently a group —(CH
2
)
k
— (k: a number of 1 to 3) or —O(CH
2
CH
2
O)
l
— (l: a number of 1 to 3),
m1 stands for a number of 1 to 6,
m2 denotes a number of 0 to 2,
n means a number of 1 to 2;
R represents an alkyl group having 1 to 6 carbon atoms or an aryl group; and
X
−
represents an anion.
Another object of the present invention is to provide an SH-labeling reagent comprising an intermediate for the above-described acridine compound, that is, an acridine compound represented by the following formula (II):
wherein
A′ represents the following group:
—(CH
2
)
m1
—
or
—(CH
2
)
m2
—Q′—(CH
2
)
n
—
in which Q′ represents a group —S—, a group —NR
1
— wherein R
1
represents an alkyl group having 1 to 6 carbon atoms, a group
wherein R
2
and R
3
may be the same or different and are each independently a group —(CH
2
)
k
— (k: a number of 1 to 3) or —O(CH
2
CH
2
O)
l
— (l: a number of 1 to 3), and
m1, m2 and n have the same meanings as defined above.
A further object of the present invention is to provide processes for the preparation of the acridine compounds represented by the formulas (I) and (II), respectively.
A still further object of the present invention is to provide a method for labeling an analyte by using the above formula (I) or (II).
BEST MODE FOR CARRYING OUT THE INVENTION
The compound represented by the formula (I), which pertains to the present invention, can be prepared, for example, in accordance with one of the following Processes 1 or 2.
Process 1
Among acridine compounds according to the present invention, each compound (Ia) in which A is a group —(CH
2
)
m1
—, wherein m1 has the same meaning as defined above, can be obtained by reacting an alkylating agent (III) with a compound represented by the formula (IIa) [hereinafter called the “intermediate (IIa)”] in a manner known per se in the art in accordance with the following reaction formula.
wherein X represents an eliminative group readily convertible into an anion, and R and m1 have the same meanings as defined above.
Illustrative of the alkylating agent (R—X) employed in the above reaction are alkyl halides such as methyl iodide, ethyl bromide and ethyl iodide, methyl trifluoromethanesulfonate, methyl fluorosulfonate, methyl methanesulfonate, and methyl p-toluene sulfonate. Accordingly, X
−
in the formula (Ia) may most typically be a halogen ion such as I
−
or Br
−
, CF
3
SO
3
−
, FSO
3
−
, CH
3
SO
3
−
, or p-CH
3
C
6
H
4
SO
3
−
, and R is an alkyl group having 1 to 6 carbon atoms such as methyl or ethyl
Eto Hiromichi
Imai Kazuhiro
Kotsugai Takeshi
Narita Tadashi
Huang Evelyn Mei
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
SS Pharmaceutical Co., Ltd.
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