Benzophenoxazine dyes

Details Benzophenoxazine dyes Benzophenoxazine dyes

1999-01-15

2000-12-26

Riley, Jezia

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

Organic compounds

Chalcogen in the nitrogen containing substituent

544 1, 544 31, 514 1, C07D26534, C07D49800, C07D27936, C07D51300, A01N 6100

Patent

active

06166202&

DESCRIPTION:

BRIEF SUMMARY
Benzophenoxazine compounds provide a family of fluorescent dyes which can be used for labelling biological molecules in various applications. The phenoxazine dyes tend to have large Stokes' shifts of 80-100 nm. This is useful in simplifying the type of filtering needed to differentiate between excitation and emission wavelengths, and may enable sensitivity limits to be improved. The benzophenoxazines also appear to have particularly good photostability properties.
The benzophenoxazine dye Nile Red (9-diethylaminobenzo[a]phenoxazine-5-one) (FIG. 1) has been shown to be useful in the fluorescence detection of proteins. Binding of dye to protein has generally been accomplished through non-covalent, hydrophobic interactions. Fluorescence properties of the dye such as emission wavelength and intensity have been shown to be dependent on the polarity of the environment and this appears to be the reason for the different spectral properties on binding to different proteins. Its low fluorescence in water is thought to be due, at least in part, to the formation of dimers and higher aggregates, since there is a time-dependent decrease in intensity on addition to water.
In order to allow specific labelling of proteins and other biomolecules such as nucleic acids, attempts have been made to form covalent links, e.g. through the nitrogen at C5 on Nile Blue (5-amino-9-diethylaminobenzo[a]phenoxazine) (FIG. 1). Linkage to Nile Blue in this way, however, has been shown to be unsatisfactory because of the adverse effect this modification has on the dye's fluorescence properties (Mank et al., 1995. Anal. Chem., 67, 1742). Fluorescence quantum yield is reduced by a factor of 10 (from 27 to 2.50) and extinction coefficient is reduced by almost 20 times (from 75000 to 4000) (Karnes et al, 1995, Proc. SPIE-Int. Soc. Opt. Eng., 2388, 21).
Bhansali and Kook, Heterocycles, Volume 36, No 6, page 1239, 1993, describe the synthesis of benzophenoxazine compounds substituted at the 10 position for therapeutic applications by intercalating into DNA. It is also stated in U.S. Pat. No. 5,283,330 that the alanyl substituted compound can act as a protein dye. In neither of these cases was this class of compound covalently linked to the DNA or protein target, nor are the fluorescence properties of the compounds mentioned or described.
The benzophenoxazine ring numbering scheme is also shown in FIG. 1. This invention is concerned with novel derivatives of benzophenoxazines which have substituents at the 2 and/or 10 and/or the 9 position, which substituents do not cause a significant loss of fluorescence. Reactive groups at all three positions can be used for linkage to tracer molecules such as biomolecules or to introduce water solubilising functions in order to improve compatibility with aqueous systems or to change the fluorescence emission wavelength of the compound.
According to this invention there is provided a benzophenoxazine compound having the formula ##STR2## where X is O or NH or N-alkyl or N-aryl or N-alkenyl, alkenyl or alkyl or is -L-A, more O or N or S atoms, and sulfonate or phosphate, or a reactive group by means of which the benzophenoxazine compound may be covalently linked to a biomolecule or a group which enhances or reduces water solubility or provides electron donor or withdrawal properties to modify the spectral characteristics of the dye,
In these compounds, the 5-substituent X may be O as in Nile Red or NH as in Nile Blue, or may be N-alkyl, N-aryl or N-alkenyl as in related dyes such as oxazine 170.
At the 9-position, each N-substituent R.sup.1 and R.sup.2 is the same or different C1-C12 alkyl or aryl or alkenyl or is -L-A.
In at least one -L-A group, L is a linker chain of 0 to 20 carbon atoms and which may include O or N or S atoms. Preferably the chain is at least three atoms in length, so as to avoid possible steric inhibition of reactions, e.g. --OC.sub.5 H.sub.10 -- and --OCOC.sub.4 H.sub.8 -- and --C.sub.2 H.sub.4 CO--.
The compound contains at least one group A which is an amine or amide or --CN or alcohol o

REFERENCES:
patent: 4600775 (1986-07-01), Theodoropulos
patent: 4962197 (1990-10-01), Foley et al.
patent: 5283330 (1994-02-01), Bhansali
Alekseev, N.N. et al., "9-(dimethylamino)benzo(a)phenoxazin-5-one or its derivatives," Chemical Abstracts,101(25):774 (Dec. 17, 1984) (Abstract 230551p).
Bhansali, K.G. et al., "Synthesis and Characterization of a Series of 5H-Benzo[a]-phenoxazin-5-one Derivatives as Potential Antiviral/Antitumor Agents," Heterocycles, 36(6):1239-1251 (1993).
Karnes, H.T. et al., "Long-Wavelength Derivatization Reagents for use in Diode Laser Induced Fluorescence Detection," Proc. SPIE-Int. Soc. Opt. Eng., 2388:21-31 (1995).
Mank, A.J.G. et al., "Visible Diode Laser-Induced Fluorescence DEtection in Liquid Chromatography after Precolumn Derivation of Amines," Analytical Chemistry, 67(10):1742-1748 (May, 1995).
The Chemistry of Synthetic Dyes, Chapter 5, Part iiic, vol. 4, Venkataraman, Academic Press, New York, pp. 228-240 (1971).

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