Amines, acetylation identification

In general, benzoylation of aromatic amines finds less application than acetylation in preparative work, but the process is often employed for the identification and characterisation of aromatic amines (and also of hydroxy compounds). Benzoyl chloride (Section IV, 185) is the reagent commonly used. This reagent is so slowly hydrolysed by water that benzoylation can be carried out in an aqueous medium. In the Schotten-Baumann method of benzoylation the amino compound or its salt is dissolved or suspended in a slight excess of 8-15 per cent, sodium hydroxide solution, a small excess (about 10-15 per cent, more than the theoretical quantity) of benzoyl chloride is then added and the mixture vigorously shaken in a stoppered vessel (or else the mixture is stirred mechanically). Benzoylation proceeds smoothly and the sparingly soluble benzoyl derivative usually separates as a solid. The sodium hydroxide hydrolyses the excess of benzoyl chloride, yielding sodium benzoate and sodium chloride, which remain in solution ... 

Primary and secondary amines are acylated by acid chlorides and anhydrides, in particular also by the chloride of benzene sulphonic add (p. 192). The preparation of acetanilide has already been described (pp. 125, 128). The acetyl- and benzoyl-derivatives of all the simpler primary amines of the benzene and naphthalene series are known, so that these derivatives can always serve for purposes of identification. 

Acetyl derivatives, which may even be prepared directly in the GC column by using a subsequent injection of acetic anhydride [68], are the most readily available and can be used for the rapid characterization or identification of amines. Marmion et al. [69] used acetyl derivatives for the determination of small amounts of 2-naphthylamine in 1-naphthylamine. 

Icmt catalyzes the methyl esterification of the prenylated cysteine residue after Reel has proteolyzed the -CaaX-containing proteins. The first step in identification of the minimal substrate for Icmt was through identification of AFC (Figure 9.2) as described above. Interestingly, farnesylcys-teine (FC), which is devoid of the acetyl substitution, was not a substrate but did possess some activity as an inhibitor [51], suggesting that the free amine of FC requires modification for catalytic turnover. Alterations in the stereochemistry about the FC backbone also appeared to be detrimental to substrate activity. The stereoisomer, d-AFC, was not a substrate for Icmt but was a modest mixed-type inhibitor of the enzyme. AFC-methyl ester (AFC-Me) was also reported to be a mixed-type inhibitor with respect to both l-AFC and -adenosylmethionine (SAM), the methyl donor, with Ki values of 41 and 73 pM, respectively [52,53] The farnesyl homocysteine homolog of AFC is not a substrate for the enzyme however, the racemic DL-homocysteine farnesyl derivative is in fact a weak inhibitor [40]. Similar to the results with racemic prenylcysteine, these data demonstrate that the linker between the carboxylate and thioether moieties is critical for substrate activity. 

Primary and secondary amines possess replaceable amino hydrogen atoms. With acid chlorides (RCOCl) or anhydrides, (RCO)jO, they yield crystalline derivatives which are very useful in their separation and identification. Benzoyl derivatives are often preferred to acetyl derivatives because the former are less soluble and have higher melting points further, benzoyl chloride, unlike acetyl chloride, is not ea.sily hydrolyzed in water, and hence the acylation can be carried out in aqueous solutions. Tertiary amines do not react and therefore can be obtained unchanged. 

It now seems incontestable that irradiation of phenyl azide at room temperature gives dehydroazepine. Once formed, dehydroazepine can react with itself and/or phenyl azide to give tarry polymer or with nucleophiles to give substituted 3f/-azepines. The rate of reaction of dehydroazepines with amines depends dramatically on substitution the 5-acetyl substituted compound reacts 10,000 times faster than does the 5-methoxy substituted dehydroazepine. At low concentration of phenyl azide, dehydroazepine itself has a lifetime of approximately 5 ms and, presumably, isomerizes to phenyl nitrene. We will have more to say about this point later. The achievement of positive structural identification of the reactive intermediate formed in the room temperature photolysis of phenyl azide permits the detailed characterization of phenyl azide photochemistry. Further consideration of this analysis will be aided by examination of the results from time-resolved experiments for other aryl azides. 

Macfarlane, R. G., Midgley, J. M., and Watson, D. G., Identification and quantification of //-acetyl metabolites of biogenic amines in the thoracic nervous system of the locust, Schistocerca gregaria, by gas chromatography negative-ion chemical ionization mass spectrometry, J. Chromalogr., 532, 13-25, 1990. 

As the boiling points of 0-, m-, and p-toluidine are, respectively, 199°, 200°, and 198°, it is impossible to identify these compounds by a determination of their boiling points. The identification can be effected, however, by a determination of the melting points of their acetyl derivatives, which are formed when the amines are treated with acetic anhydride. The acetyl derivatives of 0-, m-, and p-toluidine melt at 107°, 65°, and 147°, respectively. In order to identify a substance it is necessary to determine the physical properties of the substance and of at least one of its derivatives. The method used in the identification of the toluidines is an excellent example of this principle. 

When a compound has been shown to be an amine, and the class to which it belongs has been ascertained, its identification is completed by the determination of the melting point or boiling point of a compound prepared from it. Acetyl derivatives are frequently prepared in identifying amines, as many of them melt sharply and crystallize well from water. 

For the identification and subsequent characterization of hydroxy compounds as well as primary and secondary amines, by preparing their crystalline acetyl derivatives. 

Coenzyme A. CoA was discovered as a factor needed for the acetylation of choline and of aromatic amines. The isolation and identification of this material was one of the major advances of modem biochemistry. Not only is CoA of intrinsic interest as an active biochemical reagent, but it is essential for many diverse reactions that could not be studied until CoA and its many acylated forms became available. The study of... 

For the identification of amines acylation methods (acetylation, benzoylation, 3,5-dinitrobezoylation), reaction with p-toluenesulfonyl chloride, preparation of substituted thioureas, diazotization and coupling (for aromatic primary amines), and the preparation of salts (picrates, tetraphenylboron salts) are most commonly used. 

An indirect identification of amines consists in catalytic denitrogena-tion and subsequent identification of the resulting hydrocarbon by gas chromatography (32). Aung et al. (33) recommend the preparation of Schiff s bases with picolinaldehyde and the measurement of the rate of dissociation by reaction with ferrous salt. The half-time of the reaction is characteristic of each amine. Differences in rates of acetylation were used for the identification of amines in petroleum fractions (34). 

Formyl and acety derivatives of aliphatic amines are, as a rule, oils or low-melting substances and they are therefore not very suitable for identification. On the contrary, acetylated aromatic amines are very suitable for identification because they crystallize well and have sharp melting points distributed over a broad temperature range. Identification constants of practically all acetyl derivatives are known, which is an additional advantage. Acetyl derivatives are usually crystallized from water or alcohol, or a mixture of both, as well as from benzene, cyclohexane (or a mixture of these), or ethyl acetate. 

The advantages of benzoyl derivatives for identification are the same as those of acetyl derivatives. Benzoyl derivatives of lower aliphatic amines melt at low temperatures, and we therefore prefer the use of / -nitro- or... 

Capillary GC-MS is an extremely powerful approach, combining the high separation efficiency of the capillary GC column with the identification power of the MS in electron-ionization mode. However the applicability range of GC is limited to relatively volatile compounds. In order to widen the applicability range, precolumn analyte derivatization strategies are often applied to enhance the volatility of the analytes. Methylation, silylation and acetylation reactions are most often applied for the analysis of compounds with amine, (poly-) hydroxy and/or carboxylic acid functional groups. Furthermore, derivatization to pentafluorobenzyl derivatives is applied to enhance the sensitivity of analytes in electron-capture negative-ion chemical ionization. 

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