Reagents, diazo

In the presence of proton-donative organic solvents (alcohols), aliphatic amines do not react with diazonium, whereas aromatic amines form mainly triazenes and also para-aminoazo compounds, which subsequently interact slowly with an excess of diazo reagent via N-coupling and form disazo derivatives. 

In the presence of aprotonic organic solvents, both aromatic and aliphatic amines interact with 4-nitrophenyldiazonium in the same way. The first stage yields fast in corresponding triazenes. At the second stage, irrespective of initial amine nature, triazenes interact with an excess of diazo reagent and fonu l,3-bis(4-nitrophenyl)-triazene. Triazenes of aliphatic amines transform fast as well. In case of aromatic amines, the second stage yield depends on the inductive constants of substituents in an azo component. 

The resulting diazo reagents undergo a wide variety of reactions including those of interest in the manufacture of azo dyes and pharmaceuticals. With primary aliphatic amines the course of the reaction is different N2 is quantitatively evolved and alcohols usually result ... 

Bilirubin. The most widely used method for bilirubin continues to be the method which couples bilirubin with the diazo reagent and reads the color formed (38). Unfortunately the sensitivity of this procedure is such that a lower limit of approximately 10 microliters of specimen is a limitation on the use of this procedure. 

B) Synthetic BC and excess B are coupled with [ S]sulfanilic acid diazo reagent in presence of ethanol azo derivatives are extracted in presence of cold carrier pigments, separated chro-matographically and counted by radio scanning... 

C) Solutions of standard BDC and of unknown are treated with [ S]sulfanilic acid diazo reagent the conjugated azo derivatives are purified, then hydrolyzed the derived unconjugated azo pigments are further purified, then counted... 

To obtain blank values it is not suflScient to treat a duplicate incubation mixture with so-called diazo-blank reagent (NaN02 omitted from the diazo reagent), as is common practice in assaying serum or bile (H12). Contributions (b)-(d) would be obtained with the test, not with the control mixture. A control incubation mixture, treated with diazo reagent in parallel with the text mixture, will adequately measure contributions (b)-(f). Whether endogenous conjugate formation in the incubation control (contribution a) is suppressed completely or not, must depend on the preference of the reader (Sections 3.1.4 and 3.2). 

The only valid alternative is to choose the diazo reagent and the reaction conditions in such a way that diazo-coupling is reasonably complete. However, promotion of coupling, e.g., by increasing the concentration of... 

Azo Derivatives of Bile Pigments The instability of pigments I and II has made their isolation a difficult task. Attempts to characterize them by indirect means were therefore undertaken and a study was made of the different pigments formed in the coupling reaction of bile pigments with diazo reagents. 

In Westheimer s original photoaffinity labeling experiment, diazoacetyl-chymotrypsin was photolysed. Later, Westheimer s group made the improved diazo reagents listed in Table 3.1. 

While azides were far more susceptible to reduction by dithiols, the rates of reduction of a diazotrifluoropropionyl derivative by dithiothreitol, (3-mercaptoethanol, cysteine, and reduced glutathione did not differ widely. Thioglycolic acid was however a poor reductant and it was suggested that it should be used to replace (3-mercaptoethanol or DTT when diazo reagents are used. The reduction may be monitored by TLC or by a 500-fold increase in the absorbance at 260 nm. 

The stability of a prospective diazo reagent in the dark should further be ensured by a careful consideration of its structural features. A problem that was encountered with A6-diazomalonoyl cAMP was the Dimroth rearrangement (Fig. 3.7). 

In another successful case, Hexter and Westheimer (1971) were able to locate 5% of the total radioactivity in Tyr-146 after irradiation of [14C]diazoacetylchymotrypsin. The reaction is actually intermolecular, occurring in chymotrypsin dimers. Westheimer s group have determined the structure of several of the modified amino acids derived from the photolysis of proteolytic enzymes acylated with diazo reagents. Such data is not available for other photoaffinity reagents. Knowing that O-carboxy-methyl tyrosine was an expected insertion product Hexter and Westheimer (1971) were able to show that of the two Tyr residues in the chymotrypsin B chain only Tyr-146, the C-terminal residue, was modified. If the nature of the modified amino acid had not been known it would have been considerably more difficult to pin-point the site of photolabeling. 

Scheme II/6. Ring expansions of cycloalkanones by diazo reagents.

Mechanism The mechanism of this reaction is assumed to proceed via a metallocarbene-phosphorane. The Fe(III) porphyrin is reduced in situ by diazo reagents to the catalyti-cally active Fe(II) porphyrin. The activation of ketones by acids operates possibly through protonation of the carbonyl oxygen, which would render the carbonyl group a stronger electrophile towards reaction with phosphorane (Scheme 4.57). 

Reaction with Diazo Reagents. The classification of the acid proteases on the basis of their pH optima as suggested by Hartley (43) was useful in the absence of other information on the nature of active site residues. Since Hartley s proposal, however, it has been discovered— first with porcine pepsin—that in the presence of Cu ions these enzymes can be specifically inactivated with diazotized dipeptide esters. An ester linkage is formed between one specific aspartic acid side chain and the inhibitor (50). 

In 1958 Wachstein and Lobel (W5) reported the results of analysis of 6350 patients whose first morning specimens of urine were tested with diazo reagent. Of the total, only 24 yielded a positive reaction with the paper and the tube test this positivity resulted from 3-hydroxykynurenine in the urine of only 11 of these patients. 

T = 37°C and pH =10) or nonaqueous conditions in vacuo (24h, T=75°C and LpH =10) tested negative with Pauly s diazo reagent (31) in both cases, weakly ninhydrin positive for the nonaqueous sample and ninhydrin negative for the aqueous sample, showing that the reactions proceeded to completion. 

The diazo method described by Jendrassik and Grof in 1938 and later modified by Doumas and colleagues gives results for serum total bilirubin that are reproducible and reliable. In this procedure, an aqueous solution of caffeine and sodium benzoate serves as the accelerator. Studies on the mechanism by which the caffeine-benzoate solution facilitates the reaction of unconjugated bilirubm with the diazo reagent have provided strong, albeit indirect, evidence that... 

Because only conjugated bihrubin is excreted in urine, its presence indicates conjugated hyperbilirubinemia. The most commonly used method for detecting bilirubin in urine involves the use of a dipstick impregnated with a diazo reagent. Dipstick methods can detect bilirubin concentrations as low as 0.5mg/dL. 

The azido/n.v(dicthykirnino)phosphoniurn bromide appears to be an exceptionaly safe diazo reagent stable to shock, friction, heating, and even flame.It reacts smoothly with diethyl 1-(ethoxycarbonyl)methylphosphonate in Et2O at room temperature in the presence of t-BuOK or another alkoxide to give diethyl 1-ethoxycarbonyl-l-diazomethylphosphonate in 70% yield (Scheme 8.32). 5 5 ° Mild diazo transfer has recently been achieved in excellent yields (85%) through the use of para-acetamidobenzenesulfonyl azide at O C ... 

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