Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2000-01-11
2001-05-08
Oswecki, Jane (Department: 1626)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C548S542000, C548S545000, C548S547000, C560S043000
Reexamination Certificate
active
06229020
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to new reagents for preparation of caged compounds, and new caging methods using the same.
2. Related Background Art
For investigating the mechanism of action of a substance in an organism, it is necessary to quantitatively measure the rise and fall of this substance within a biological system in a short period of time. Further, it is important to observe various changes following the substance introduced into the system.
On the other hand, biological reactions are mostly very fast, and a plurality of reactions usually progress at the same time while complicatedly relating to each other. Therefore, when the above-mentioned substance is added from the outside, the process of its diffusing within the system rather becomes the rate-determining step, thereby often making it hard to clearly grasp the subsequent reaction to be determined in practice.
SUMMARY OF THE INVENTION
For overcoming such a problem, various methods have been proposed as to a method of rapidly adding a target substance. As one of such methods, a method using a technique based on irradiation with light, i.e., so-called caged reagent, has been reported. In general, this method comprises the steps of introducing into a biological system a caged compound (which refers to a compound in which a so-called caging group is introduced or a compound labeled with a caging group) in which a specific protective group protects an active part of a physiologically active substance to be traced; verifying that this substance has sufficiently diffused to a point of application; and liberating the protective group (caging group) upon irradiation with light, so as to release the target substance, thereby making it possible to trace the reaction caused by the substance. This protective group is characterized in that it can attain deprotection (release of the protective group) upon irradiation with light alone, it can achieve deprotection very fast, and it allows the light irradiation to be narrowed to only a specific part as necessary. The present invention provides new reagents capable of preparing caged compounds having such a function with a new structure by a simple reaction. Also, the present invention provides methods of yielding caged compounds by introducing a caging group into a target physiologically active substance by using these new reagents.
As a result of diligent studies, the inventor has succeeded in finding new reagents which can prepare caged compounds having such an excellent function by a simple reaction. Also, the inventor has succeeded in establishing methods of caging various compounds by using these new reagents, thereby accomplishing the present invention.
Namely, a new reagent for preparation of caged compounds in accordance with the present invention is a compound having, as a basic structure, an N-succinimidyl aminocinnamate structure expressed by the following formula 1:
where X and Y may be identical or different, each representing one kind selected from the group consisting of a hydrogen atom, a halogen atom, an alkyl group having a carbon number from 1 to 4, an alkyloxy group having a carbon number from I to 4, a benzo group, and an alkylamino group having a carbon number from 1 to 4; R
1
represents one kind selected from the group consisting of a hydrogen atom, an alkyl group having a carbon number from 1 to 4, and a group expressed by the following formula 2:
where R
6
represents one kind selected from the group consisting of an alkyl group having a carbon number from 1 to 4, a phenyl group, and an alkylsilyl group having a carbon number from 1 to 4, and n represents an integer from 0 to 2;
R
2
and R
3
may be identical or different, each representing one kind selected from the group consisting of a hydrogen and an alkyl group having a carbon number from 1 to 4; and Z represents one kind selected from the group consisting of a hydrogen atom and SO
3
M, where M represents one kind selected from the group consisting of a hydrogen atom, an alkali metal, and an alkaline-earth metal.
As preferable examples of the new reagent for preparation of caged compounds in accordance with the present invention, those having the respective structures shown in the following formulae 4 to 17 can be noted specifically:
Another new reagent for preparation of caged compounds in accordance with the present invention is a compound having, as a basic structure, a nitrophenyl ester structure expressed by the following formula 3:
where X and Y may be identical or different, each representing one kind selected from the group consisting of a hydrogen atom, a halogen atom, an alkyl group having a carbon number from 1 to 4, an alkyloxy group having a carbon number from 1 to 4, a benzo group, and an alkylamino group having a carbon number from 1 to 4; R
1
represents one kind selected from the group consisting of a hydrogen atom, an alkyl group having a carbon number from l to 4, and a group expressed by the following formula 2:
where R
6
represents one kind selected from the group consisting of an alkyl group having a carbon number from 1 to 4, a phenyl group, and an alkylsilyl group having a carbon number from 1 to 4, and n represents an integer from 0 to 2; and
R
2
and R
3
may be identical or different, each representing one kind selected from the group consisting of a hydrogen and an alkyl group having a carbon number from 1 to 4.
As preferable examples of the new reagent for preparation of caged compounds in accordance with the present invention, those having the respective structures shown in the following formulae 18 to 23 can be noted specifically:
On the other hand, the methods in accordance with the present invention are caging methods in which the above-mentioned reagents for preparation of caged compounds are reacted with compounds having various functional groups (physiologically active substances such as amino acids) , so as to prepare caged compounds.
FIGS. 1A and 1B
show caging methods of compounds having various functional groups, in which the reagents for preparation of caged compounds in accordance with the present invention are used.
FIG. 2
shows a photolysis reaction of a caged compound obtained. As shown in
FIGS. 1A and 1B
, N-succinimidyl group and p-nitrophenyl group easily react with various functional groups under mild reaction conditions. Preferable examples of the above-mentioned functional groups include amino group, hydroxide group, phenol group, thiol group, carboxylic acid group, and the like. The caged compounds obtained by the methods of the present invention are stable in the dark since the double bond of cinnamate group is kept in a trans (E) form.
REFERENCES:
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Sheehan et al., “The Use of Water-Soluble and Basic Carbodimides in Peptide Synthesis”, Contribution from the Department of Chemistry, Massachusetts Institute of Technology, vol. 21, Received Dec. 23, 1965, pp. 439-441.
Artico et al., “Heterocycles with a Benzothiadiazepine Moiety—1. Synthesis of Pyrrolo[1,2-b]-s-Triazolo[3,4-d] [1,2,5]Benzothiadiazepine 5,5,-Dioxide” Synthetic Communications, 22(10) (1992) pp. 1433-1439.
Sato
Laboratory of Molecular Biophotonics
Leydig , Voit & Mayer, Ltd.
Oswecki Jane
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