Tetrafluoroazidoaniline and method of making and using the same

Details Tetrafluoroazidoaniline and method of making and using the same Tetrafluoroazidoaniline and method of making and using the same

2000-03-14

2001-06-26

Richter, Johann (Department: 1621)

Organic compounds -- part of the class 532-570 series

Organic compounds

Azides

C548S303100

Reexamination Certificate

active

06252096

ABSTRACT:

FIELD OF INVENTION
The present invention relates to a fluorinated aryl azide and the method of making and using the same as an photoaffinity probe and photoreactive group. In particular, the fluorinated aryl azide is 4-azidotetrafluoroaniline or an alkyl, acyl or sulfonamide derivative thereof.
BACKGROUND OF THE INVENTION
Perfluorophenyl azides have been used extensively in the biochemistry and molecular biology fields as photoaffinity probes to study protein structure and function. In addition, aryl azides have been used in optical lithography as photoresists. A photoaffinity probe is a labeling reagent that has a photoactive moiety such as an azido or diazo group that forms a stable covalent bond with a targeted protein by CH insertion.
For example, an azide labeling reagent can be attached to an active site of an enzyme molecule. When the labeled enzyme is exposed to ultraviolet light, there is a loss of nitrogen and the formation of a highly reactive intermediate, a singlet nitrene, which reacts rapidly with a nearby protein molecule such that the protein forms a covalent bond with the enzyme.
Para-substituted pentafluorophenyl azides are among the most popular labeling agents used as photoaffinity probes. Their popularity stems from the fact that non-fluorinated arylazides do not form the highly reactive singlet nitrene intermediate upon photoactivation, rather they form an electrophilic dehydroazepine intermediate with a very different and undesirable reactivity. A variety of these azides have been synthesized in accordance with the following reaction:
wherein X is an electron withdrawing group such as CN, CONH
2
, CHO, CO
2
CH
3
, COCH
3
and NO
2
(Keana et al., J. Org. Chem., Vol. 55, No. 11, pp. 3640-3647 (1990)). All of the prior art perfluorophenyl azide photoaffinity labeling agents have electron-withdrawing groups para to the azido functionality. There is a need for a photoaffinity labeling agent having a chemically reactive electron donating group para to the azido functionality. There is also a need for photoreactive crosslinking agents which are hetero-bifunctional having a chemically reactive electron donating group para to the azido functionality.
Photoreactive crosslinking reagents are important tools for determining the proximity of two sites on a molecule or between two molecules. These probes can be employed to define relationships between two reactive groups on a protein molecule, on a ligand and its receptor, or on separate biomolecules within an assembly. In the latter case, photoreactive crosslinking reagents can potentially reveal interactions among proteins, nucleic acids, and membranes in live cells. The general scheme for defining spatial relationships usually involves photoreactive crosslinking reagents that contain a chemically reactive group as well as a photoreactive group. These crosslinkers are first chemically reacted with one molecule, for example a receptor ligand, and then this modified molecule is coupled to a second molecule, for example the ligand's receptor, using UV illumination. Depending on the reactive properties of the chemical and photoreactive groups, these crosslinkers can be used to couple like or unlike functional groups. Fluorinated aryl azides are useful in these processes because they generate nitrenes, thereby producing more C—H insertion products than the simple aryl azides.
Simple aryl azides may be initially photolyzed to electron-deficient aryl nitrenes that rapidly ring-expand to form dehydroazepines thereby producing molecules that tend to react with nucleophiles rather than form C—H insertion products. Photolysis products of the fluorinated aryl azides are clearly aryl nitrenes and undergo characteristic nitrene reactions such as C—H bond insertion with high efficiency.
A photocrosslinking agent known in the art is 4-azido-2,3,5,6-tetrafluorobenzyl amine. This benzylic amine has a pKa of approximately 9. The crosslinking reagents of the present invention have an anilino nitrogen, and not a benzylic amine, and have a pKa closer to −1 pKa units. In addition, the nitrogen in the prior art crosslinking agent is one carbon further away from the amino functionality than in the 4-tetrafluoroaniline azide. Because hetero-bifunctional crosslinking probes have demonstrated utility for determining the proximity of two sites on a molecule or between two different molecules, the 4-tetrafluoroaniline azide molecule allows the generation of probes that are an atom shorter than current probes, thus allowing shorter distances to be probed.
SUMMARY OF THE INVENTION
The object of this invention is to provide new photoaffinity labeling agents having a chemically reactive electron-donating amino group para to the azido functionality. Another object of the invention is to provide a photoaffinity labeling agent which will extend the range of existing hetero-bifunctional crosslinking reagents. A further object of the invention is to provide a compound, 4-azidotetrafluoroaniline, and its alkyl, acyl and sulfonamide derivatives, having a chemically reactive electron-donating amino group para to the azido functionality. A still further object of the invention is to provide a method of making 4-azidotetrafluoroaniline, or its alkyl, acyl or sulfonamide derivative from a stable carbamate intermediate. Still another object of the invention is to provide for a method of using 4-azidotetrafluoroaniline, or its alkyl, acyl or sulfonamide derivative as a photoaffinity labeling agent. The other objects and characteristics of the present invention will become apparent from the further disclosure of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to a novel 4-tetrafluoroaniline azide and to the mono- and di-substituted alkyl, acyl, or sulfonamide derivatives thereof, including mono-alkyl-mono-sulfonyl derivatives, and the method of making and using the same as a photoaffinity probe. In particular, the compounds of the invention have the following formula:
wherein R′ and R″ are independently hydrogen, C
1
-C
8
alkyl, C
1
-C
8
allylic, unsubstituted benzyl, or benzyl substituted with up to five substituents selected from the group consisting of NO
2
, N
3
, NH
2
, NHR′″, N(R′″)
2
, N(R′″)
3
+
, halogen, and C
1
-C
8
alkyl where R′″ is hydrogen, C
1
-C
8
alkyl, C
1
-C
8
allylic, or an unsubstituted benzyl. The most preferred compound is the unsubstituted compound, 4-azidotetrafluoroaniline (I) having the following formula:
The most preferred derivatives of 4-azidotetrafluoroaniline are N-iodo-actamido-4-tetrafluoroaniline azide and 1-(4-azido-tetrafluoroaniline)-dansylsulfonamide.
When 4-azidotetrafluoroaniline (I) or its alkyl, acyl or sulfonamide derivative is irradiated with ultraviolet light in the presence of cyclohexane in accordance with the following reaction scheme, three primary products are formed: 1,4-diaminotetrafluorobenzene (II), cyclohexylaminotetrafluorobenzene (III) and azotetrafluorobenzene (IV). The reaction is exemplified below with respect to 4-azidotetrafluoroaniline.
The formation of C—H insertion product (III) indicates that 4-azidotetrafluoroaniline forms a singlet nitrene upon photolysis.
The novel 4-azidotetrafluoroaniline compound is made by forming a stable carbamate intermediate and then converting the carbamate to 4-azidotetrafluoroaniline. Two embodiments are disclosed.
In the first embodiment, a stable carbamate is formed via a modified Curtius rearrangement by transforming 4-azidotetrafluorobenzoic acid into an intermediate acid chloride, converting the acid chloride into an acyl azide, and further converting the acyl azide into an isocyanate by thermal rearrangement. A stable carbamate is formed by reacting the isocyanate with an alcohol. The carbamate includes an acid labile group which is derived from the alcohol. The carbamate is then converted to 4-azidotetrafluoroaniline by removal of the acid labile group. The reaction scheme for the first embodiment is illustrated as follows:
wherein R

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