Novel chromogenic substrates with metal chelating properties for the histochemical detection of peroxidasic activity, derived from 3-amino-9-ethylcarbazole (AEC) and 3,6-diamino-9-ethylcarbazole.
For staining of peroxidase activity routinely employed 3-amino-9-ethylcarbazole 1 (AEC) was chemically modified in order to obtain chromogenic enzyme substrates with improved staining properties. In conclusion of systematically structure/staining considerations of a series of novel substrates, it can be generalized that the performance of traditional chromogenic peroxidase amine-substrates is accessible an considerably improvement in terms of sensitivity and adaptibility for various application purposes (solubility and color of the reaction product, electron dense and osmiophilic properties, ...) by attachment of chelating N-benzyl-moieties making available highly efficient the well known metal catalytic effect in a proposed intramolecular way. Thus, the model compounds 3(arylmethyl)amino-9-ethyl-carbazole 4 and 3,6-bis-(arylmethyl)amino-9-ethyl-carbazole 7 were synthesized by condensation of 3-amino-9-ethylcarbazole 1 (AEC) or the corresponding 3,6-diamine 5 with aromatic aldehydes 2. The resulting Schiff bases 3 and 6 were subsequently reduced with sodium borohydride. The obtained benzylamines 4 and 7 were examined as chromogenic substrates: 1) qualitatively in test tube experiments concerning color, precipitation behavior and solubility of the precipitates, 2) quantitatively by means of electroblotted dilution series of horseradish peroxidase, and finally in a biological environment for the localization of endogenous peroxidasic activity 3) in native cryotome tissues of Wistar rats. 4) The usefulness of the new approach for electron microscopy was revealed, too. Thus the discrimination between internum and externum of specific granules after osmium tetroxide contrastate was higher if compared with results obtained by the Karnovsky protocol. The wide spread variation of substitution patterns of the novel reagents gave reason for structure-reactivity considerations and ongoing leading structures. The stereochemical and electronic factors as well as competing reaction pathways governing the reaction course are briefly discussed. In addition, the metal associating reagents are highly effective in oxidative side-coupling reactions with aromatic amine or phenol-additives exemplified here by means of 4-amino-N,N-diphenylamine. The reagents 4 and 7 are obtainable in a simple in situ synthesis, too, offering in principle a 'chemical construction unit'. The demonstrated approach is of general interest for bioanalytical applications offering an access to potentially novel chromogens and electron opaque markers for the detection of peroxidasic activity/hydroperoxides or related redox enzyme systems.
Krieg
R
R
Institute of Anatomy/Anatomy II, Friedrich Schiller University Jena, Germany. rkrieg@mti-n.uni-jena.de
Halbhuber
K J
KJ
Oehring
H
H
D000818:Animals; D002227:Carbazoles / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D002614:Chelating Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011232:Chemical Precipitation; D002863:Chromogenic Compounds / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D003116:Color; D003594:Cytoplasmic Granules / Q000201:enzymology / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D004804:Eosinophils / Q000166:cytology / Q000201:enzymology / Q000648:ultrastructure; D006651:Histocytochemistry / Q000379:methods*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007422:Intestines / Q000166:cytology / Q000201:enzymology; D008297:Male; D008854:Microscopy, Electron; D015394:Molecular Structure; D009195:Peroxidase / Q000032:analysis* / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D013270:Stomach / Q000166:cytology / Q000201:enzymology
D002227:Carbazoles; D002614:Chelating Agents; D002863:Chromogenic Compounds; D006861:Hydrogen Peroxide; D009195:Peroxidase