Diazapentalene derivatives as a specific reagent for thiol compo

Details Diazapentalene derivatives as a specific reagent for thiol compo Diazapentalene derivatives as a specific reagent for thiol compo

1996-01-31

1997-07-08

Richter, Johann

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

Organic compounds

Heterocyclic carbon compounds containing a hetero ring...

435 405, 436 63, 436120, 5483051, 5483121, 5483647, 548412, 548444, 548452, 548453, 5482624, 548252, 548253, 548255, 548260, 548261, 5482664, 5483625, 548119, 5483027, 548469, 548969, 546256, 5462774, 544333, 544242, 544294, 558307, C07D40304, C07D48702, C07D20952, C07F 902, G01N 3348, G01N 3300

Patent

active

056462956

DESCRIPTION:

BRIEF SUMMARY
This application is a 371 of PCT/EP95/01981 May 25, 1995.
The invention concerns diazapentalene derivatives and their use as a fluorochrome specific for thiol groups in particular for cell staining as well as a method for the fluorimetric detection of compounds containing thiol groups especially in cells.
Of numerous known fluorescent compounds that react with thiol groups only a few fulfil the condition that this reaction should be sufficiently selective.
An important area of application of fluorochromes that react selectively with thiol groups is in general the analysis of compounds which contain thiol groups (in particular proteins), for example the detection of such compounds in HPLC analysis, in thin layer chromatography or in gel electrophoresis.
A special area of application of such specific reagents for thiol groups is the staining of living cells. In this case the fluorochrome reagents that are specific for thiol groups must be able to penetrate into the cell in order to be able to react with the thiols present there and in particular with glutathione and cysteine residues. The content and under some circumstances also the position of thiols and in particular of glutathione in the cell can be determined by measuring the fluorescence of the cells stained in this manner. Since one of the functions of glutathione in the cell is to render mutagens or carcinogens harmless, the determination of the cellular content of glutathione also provides information on the protection of the cell against harmful influences. Furthermore cellular thiols, in particular glutathione, are involved in a number of other biochemical processes so that their cellular content can be associated with a number of pathological diseases (see for example J. Bio. Chem. 263, 17205).
After the cells have been incubated with a thiol-specific fluorochrome, the content of fluorescent thiol adducts in the cell is preferably determined by means of flow cytometry or by digital video imaging. Flow cytometry has the very advantageous property of being able to measure physiological parameters in a large number of living single cells. It even allows the determination of heterogeneities within a cell population. In this method the fluorescence of individual cells in a liquid current is determined by means of fluorescent optics preferably after excitation with an argon laser at 488 nm (Flow Cytometry and Sorting, M. R. Melamed et al. (eds) Wiley and Liss, 1990). A review of the determination of thiols in cells by flow cytometry is given by Rabinowitch et al. in Clinical Flow Cytometry, K. D. Bauer et al. (eds) Williams and Wilkins, 1993, page 505-534, Durant et al., Radiation Research 95, 456-470 (1983) and McLean Grogan, Guide to Flow Cytometry Methods, Marcel Dekker Inc., New York and Basel, (1990), page 171-176. These literature references also give a review of conventional fluorochrome reagents that are specific for thiol groups for flow cytometry. Maleimide derivatives and bromobimane are described in these references as being the most common.
Disadvantages of the fluorochrome thiol-specific reagents for cell staining described in the state of the art are that some of them have a high cell toxicity (bromobimane), can only penetrate into the cells with difficulty (fluorescein-5-maleimide, didansyl cystine), have an unfavourable excitation wavelength for the excitation wavelength especially of common argon lasers (bimane at 360-400 nm, 3-4-maleimidylphenyl-4-methyl-7-diethylamino-coumarin (CPM): at 395 nm, didansyl cystine: 335 nm), have slow addition kinetics (chlorobimane, CPM) and must therefore be used in high concentrations or they have an inadequate specificity for thiol groups (orthophthaldialdehyde). In addition most of the known compounds have a low photostability which is a disadvantage particularly when observing the cells over a long time period under a microscope e.g. under a mercury vapour lamp.
The object of the present invention was therefore to eliminate the disadvantages of the thiol-specific fluorochrome compounds of the stat

REFERENCES:
patent: 4914211 (1990-04-01), Jost et al.
"(Hetero)Pentalene Derivatives and Y-Shaped Carbodications", F. Closs et al, Synthetic Metals, 29(1989) E537-E544.
"Chemistry of Organic Compounds", 3rd Ed., Carl R. Noller (1965), p. 119.

Affiliated with

Also associated with

Rating

Diazapentalene derivatives as a specific reagent for thiol compo does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Diazapentalene derivatives as a specific reagent for thiol compo, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Diazapentalene derivatives as a specific reagent for thiol compo will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2409659