scholarly journals INFLUENCE OF CHEMICAL STRUCTURE ON THE RATE OF AZO COUPLING AND ITS SIGNIFICANCE IN HISTOCHEMICAL METHODOLOGY

1959 ◽  
Vol 7 (1) ◽  
pp. 50-65 ◽  
Author(s):  
MARVIN M. NACHLAS ◽  
THEODORE P. GOLDSTEIN ◽  
DAVID H. ROSENBLATT ◽  
MARVIN KIRSCH ◽  
ARNOLD M. SELIGMAN
Keyword(s):  
Aromatic Amines ◽  
Diazonium Salt ◽  
Tissue Sections ◽  
Coupling Techniques ◽  
Diazonium Ions ◽  
Coupling Rate ◽  
Diazonium Ion ◽  
Hydroxy Compounds ◽  
Positive Groups ◽  
Electro Negativity

Reliability of enzymatic localization in tissue sections by the simultaneous coupling techniques is dependent to a great extent upon the speed of coupling. Therefore, the influence on coupling rate of the structure of the diazonium ion and of the coupling component was studied. Electro-negative groups in the diazonium ion increase the rate of coupling, while the same groups in the coupling component decrease the rate. Electro-positive groups in the coupling component accelerate coupling, but slow it when present in the diazonium ion. The relation of coupling rate and electro-negativity of the substituents in the diazonium ion follows Hammett's equation (8). Although the relations is linear on coupling with aromatic hydroxy compounds, it is not so with aromatic amines. The most active diazonium ions showed no increase in coupling rate with aromatic amines. This suggests that for those enzymes hydrolyzing an ester link, increase of coupling rate might be accomplished by modifying the structure of either the coupling component used in the substrate or the diazonium salt. However, for enzymes splitting amide linkages, the only possibility of improving the localization is by modifying the structure of the coupling component.

Kinetics of reactions of 1,2,4-triazole-3-diazonium ions with phenol and with hydroxyl ion

1985 ◽  
Vol 50 (3) ◽  
pp. 658-674
Author(s):  
Vladimír Macháček ◽  
Josef Kořínek ◽  
Daniela Kreuzigová ◽  
Vojeslav Štěrba
Keyword(s):  
Main Reaction ◽  
Basic Medium ◽  
Phenyl Derivative ◽  
Rate Limiting Step ◽  
Diffusion Controlled ◽  
Hydroxyl Ion ◽  
Diazonium Ions ◽  
Diazonium Ion ◽  
Hydroxy Compounds ◽  
Intramolecular Hydrogen

5-Methyl- and 5-phenyl-l,2,4-triazole-3-diazonium ions (IIIa, IIIb) react with undissociated phenol in diluted hydrochloric acid. At pH > 1 the reactions with phenolate ions become kinetically significant, their bimolecular rate constants approaching those of the diffusion-controlled reactions. At the same time, the diazonium ions are dissociated into the dipolar ions IIIb and IVb. At pH > 4 (for the phenyl derivative) and pH > 5 (for the methyl derivative), the reaction of the dipolar ions with phenolate ion becomes the main reaction path. The rate constant of the reaction of the dipolar ion IIIb with hydroxyl ion (3 . 103 l mol-1 s-1) is comparable with that of the analogous reaction of benzenediazonium ion, but the reverse reaction of the dipolar ion IIIb is slower by about 5 orders of magnitude, and the pKA value of the diazo hydroxide formed is higher by about 4 units than that of benzenediazo hydroxide. The high stability of the heterocyclic diazo hydroxide and its low acidity are explained by formation of a strong intramolecular hydrogen bond. In sodium hydroxide solutions, the (Z)-diazotate IIId initially formed is transformed into the (E)-isomer (the corresponding half-life being about 10 h) which, in contrast to the (Z)-isorner, does not practically react with aromatic hydroxy compounds in basic medium. The transformation of the (E)-isomer to the diazonium ion is general acid-catalyzed reaction. Transformation of nitrosamine into diazo hydroxide is suggested to be the rate-limiting step of this reaction in solutions of pH < 5.

scholarly journals The Aminoalkylation of Aromatic Amines by the Leuckart Reaction Catalyzed by Hydroxy Compounds. II

1951 ◽  
Vol 71 (8) ◽  
pp. 834-839
Author(s):  
Masao Tomita ◽  
Shojiro Uyeo ◽  
Daisuke Sato ◽  
Hirokazu Otaya ◽  
Ichiro Kanayama ◽  
...  
Keyword(s):  
Aromatic Amines ◽  
Hydroxy Compounds

scholarly journals The Aminoalkylation of Aromatic Amines by the Leuckart Reaction Catalyzed by Hydroxy Compounds. VI

1951 ◽  
Vol 71 (8) ◽  
pp. 846-849 ◽  
Author(s):  
Masao Tomita ◽  
Shojiro Uyeo ◽  
Yasumasa Mori
Keyword(s):  
Aromatic Amines ◽  
Hydroxy Compounds

scholarly journals The Aminoalkylation of Aromatic Amines by the Leuckart Reaction Catalyzed by Hydroxy Compounds. III

1951 ◽  
Vol 71 (8) ◽  
pp. 839-841
Author(s):  
Masao Tomita ◽  
Shojiro Uyeo ◽  
Hirokazu Otaya ◽  
Ichiro Kanayama ◽  
Yasumasa Mori ◽  
...  
Keyword(s):  
Aromatic Amines ◽  
Hydroxy Compounds

Reactions of 4-Amino-3-penten-2-one and Its NSubstituted Derivatives with Diazonium Ions

1995 ◽  
Vol 60 (8) ◽  
pp. 1367-1379 ◽  
Author(s):  
Vladimír Macháček ◽  
Alexandr Čegan ◽  
Aleš Halama ◽  
Olga Rožňavská ◽  
Vojeslav Štěrba
Keyword(s):  
Coupling Reaction ◽  
Butyl Alcohol ◽  
Azo Compounds ◽  
Coupling Reactions ◽  
Azo Coupling ◽  
Alcohol Water ◽  
Diazonium Ions ◽  
Coupling Rate ◽  
Reaction Orders ◽  
Intramolecular Hydrogen

Azo coupling reactions of benzenediazonium salts with substituted 4-amino-3-penten-2-ones take place at the C-3 atom. 1H and 13C NMR spectroscopy has been used to study the structure of both the starting enaminones and coupling products. In CDCl3, 3-(4-chlorophenylhydrazono)-2-(4-methylphenylimino)-4-pentanone exists in hydrazo form whereas 4-amino-3-(4-chlorophenylazo)-3-penten-2-one is present as a mixture of two azo compounds differing probably in the arrangement of the intramolecular hydrogen bond. The azo coupling reaction kinetics have been studied in acetate buffers and methanol-water or tert-butyl alcohol-water mixtures. The coupling rate has been found independent of pH and buffer concentration. The reaction orders with respect to the starting compounds have been determined and the reaction mechanism is suggested. Linear dependence has been found between log kobs and substituent constants according to the Hammett or Yukawa-Tsuno equations.

scholarly journals Use of tissue-free glycol methacrylate sections as semi-permeable membranes: a simple way to shorten incubation times and to improve localization in enzyme histochemistry.

1989 ◽  
Vol 37 (2) ◽  
pp. 173-176 ◽  
Author(s):  
P O Gerrits ◽  
R W Horobin ◽  
M J Hardonk
Keyword(s):  
Alkaline Phosphatase ◽  
Azo Dye ◽  
Enzyme Histochemistry ◽  
Diazonium Salt ◽  
Histochemical Staining ◽  
Semipermeable Membranes ◽  
Large Molecules ◽  
Glycol Methacrylate ◽  
Tissue Sections ◽  
Antibody Staining

Placing 2-microns sections of tissue-free glycol methacrylate on top of tissue sections is a simple way of forming semipermeable membranes to enhance enzyme histochemical staining. For demonstrating alkaline phosphatase in glycol methacrylate-embedded kidney by a standard azo dye method, such membranes enabled incubation times to be reduced to 1-2 hr, with azo dye reaction product being more crisply localized as compared to sections stained without membranes. Such effects are possible because the membranes are highly permeable to small molecules (e.g., substrate and diazonium salt), slightly permeable to molecules of moderate size (e.g., the final reaction product), and impermeable to large molecules (e.g., alkaline phosphatase and other tissue biopolymers). The implications of these findings for enzyme histochemistry and for enzyme-labeled antibody staining are discussed.

scholarly journals Mechanisms and products of azo coupling in histochemical protease procedures based on primary aromatic amines as unspecific moieties.

1990 ◽  
Vol 38 (9) ◽  
pp. 1295-1300 ◽  
Author(s):  
H G Frank
Keyword(s):  
Azo Dyes ◽  
Aromatic Amines ◽  
Elevated Temperatures ◽  
Test Tube ◽  
Aromatic Nucleus ◽  
Diazonium Salts ◽  
Amino Group ◽  
Tissue Sections ◽  
Primary Aromatic Amines ◽  
Methylated Amines

It is presumed that the azo dyes generated by histochemical protease reactions are formed by substitution of a reactive aromatic carbon. They are referred to as dyes of the C-azo series. To confirm this assumption, the absorption spectra between 330 and 630 nm of azo dyes resulting from coupling between various aromatic amines of the aniline and naphthylamine series and the diazonium salts Fast Blue B and Fast Garnet GBC were studied in test tube experiments. Some of the amines were blocked by methylation to prevent coupling either at the amino group (N-methylated) or at the aromatic nucleus (C-methylated). Coupling was performed in buffered aqueous solutions of the diazonium salts. For analysis the azo dyes were dissolved in dimethylformamide. For acid rearrangement these solutions were acidified and incubated at elevated temperatures. After detection of dipeptidyl peptidase IV in tissue sections using Gly-Pro-4-methoxy-2-naphthylamine as substrate, the resulting dye was extracted and compared with the test tube compounds. All aromatic amines yielded azo dyes. Dyes extracted from sections and those test tube compounds derived from unmethylated or C-methylated amines showed almost identical spectral maxima, whereas dyes formed by N-methylated amines yielded different spectra. Acid rearrangement did not influence the spectral maxima of the N-methylated amine-derived dyes. Dyes resulting from C-methylated amines were destroyed. The results indicate that under histochemical conditions diazonium salts react primarily with the liberated free amino group but not with the aromatic nucleus of the unspecific moiety. Therefore, it is proposed that the formula of the final reaction product in naphthylamine-based protease histochemistry should be given as an N-azo dye, e.g., as a triazene.

On the involvement of diazonium ions in the photorearrangement of azoxybenzene

1977 ◽  
Vol 55 (3) ◽  
pp. 383-392 ◽  
Author(s):  
Nigel J. Bunce
Keyword(s):  
Excited State ◽  
Light Intensity ◽  
Substrate Concentration ◽  
Product Distribution ◽  
Weak Base ◽  
Rearrangement Product ◽  
Azoxy Compounds ◽  
Diazonium Ions ◽  
Azoxy Compound ◽  
Diazonium Ion

Photolysis of aromatic azoxy compounds in non-basic solvents in the presence of 2-naphthol leads quite generally to azonaphthols in addition to the normal photorearrangement product. Free diazonium ions are implicated as the precursors of the azonaphthols, both the latter and the usual photorearrangement product being derived from the same excited state of the azoxy compound. Variation of the solvent, light intensity, substrate concentration, and percent reaction all cause changes in the product distribution. Such changes can be accommodated by postulating an intermediate which can either afford the normal rearrangement product by loss of a proton to a weak base, or expel a diazonium ion when no base is present.

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