Acetylation solution

Add 150 ]xL of the acetylating solution [triethylamine-acetic anhydride (2 1, v/v)] into the Eppendorf tube and allow to stand for about 30 min at room temperature. Concentrate the reaction mixture under an N2 gas flow at about 40 °C. Add 1 mL of 0.1N HCl to the residue and extract the solution with 3 x 0.5 mL of n-hexane-ethyl acetate (4 1, v/v). Concentrate the organic phase under an N2 gas flow at about 40 °C. 

Prepare the acetylation solution for immediate and one-time use. Acetylate the slides by soaking for 10 min, and rinse in distilled water for 2 min (see Note 1). 

Acetylation of slides prevents nonspecific, electrostatic binding of DNA probe to the cells. It is important that the acetylation solution be prepared immediately before use, with stirring, and that it not be reused. Prepare as follows add O.IM triethanolamine to a Coplin jar with a magnetic stirring bar. As stirring proceeds, add sufficient acetic anhydride to give a final concentration of 0.25% (v/v). 

Although the acetylation of alcohols and amines by acetic anhydride is almost invariably carried out under anhydrous conditions owing to the ready hydrolysis of the anhydride, it has been shown by Chattaway (1931) that phenols, when dissolved in aqueous sodium hydroxide solution and shaken with acetic anhydride, undergo rapid and almost quantitative acetylation if ice is present to keep the temperature low throughout the reaction. The success of this method is due primarily to the acidic nature of the phenols, which enables them to form soluble sodium derivatives, capable of reacting with the acetic... 

When the phenol contains a carboxylic acid group, e.g., m- or p-hydroxy-benzoic acid, the acetylated derivative will of course remain in solution as the sodium salt, but is precipitated when the solution is subsequently acidified. Salicylic acid, however, cannot be acetylated under these conditions. 

Dissolve 10 g. of salicylic acid (o-hydroxybenzoic acid) in 7 ml. of dry pyridine contained in a too ml. conical flask. Then without delay (since this solution if allowed to stand tends to become a semi-solid mass) run in 7 5 ml. (8 3 g.) of acetyl chloride, adding about i ml. of the chloride at a time, and shaking the mixture continuously during the addition. The heat of the reaction causes the temperature of the mixture to rise rapidly ... 

Add a few drops of the distillate to an aqueous silver nitrate solution containing some dilute nitric acid and warm gently no silver chloride should be precipitated, indicating the complete absence of unchanged acetyl chloride. 

Acyl halides, both aliphatic and aromatic, react with the sodium derivative, but the product depends largely on the solvent used. Thus acetyl chloride reacts with the sodium derivative (E) suspended in ether to give mainly the C-derivative (t) and in pyridine solution to give chiefly the O-derivative (2). These isomeric compounds can be readily distinguished, because the C-derivative (1) can still by enolisation act as a weak acid and is therefore... 

Action of silver nitrate. Acidify 2 ml. of aqueous AgNOj solution with dil. HNO3 and add the acid chloride drop by drop with shaking. Acetyl chloride and benzoyl chloride give a precipitate of AgCl. Filter, wash with water, and then with methylated spirit to remove any benzoic acid the AgCl remains. 

Acetylation. Proceed as in 2 (p. 373). Pour the final acetylation mixture into 10 ml. of water, and add 10% NaOH solution, with stirring, until no more anilide is precipitated (acetyl-monoethylaniline is very soluble even in dil. acetic acid acetyUdiphenylamine readily separates without the addition of alkali). Filter, wash with water and recrystallise. 

Z>) Benzoylation. Proceed exactly as for benzoylation of amines (Test 3 (a), p.374), but use a suspension of the finely ground nitroaniline in the 10% NaOH solution. This preparation of the benzoyl derivatives is rarely necessary, however, as the above acetylation proceeds very satisfactorily. (M.ps., p. 550.)... 

The excess of unchanged acetic anhydride is then hydrolysed by the addition of water, and the total free acetic acid estimated by titration with standard NaOH solution. Simultaneously a control experiment is performed identical with the above except that the alcohol is omitted. The difference in the volumes of NaOH solution required in the two experiments is equivalent to the difference in the amount of acetic add formed, i.e., to the acetic acid used in the actual acetylation. If the molecular weight of the alcohol is known, the number of hydroxyl groups can then be calculated. 

Method A. In a 500 ml. round-bottomed flask, fitted with a reflux condenser attached to a gas trap (Fig. II, 13, 8), place 59 g. of succinic acid and 117-5 g. (107-5 ml.) of redistilled acetyl chloride. Reflux the mixture gently upon a water bath until all the acid dissolves (1-2 hours). Allow the solution to cool undisturbed and finally cool in ice. Collect the succinic anhydride, which separates in beautiful crystals, on a Buchner or sintered glass funnel, wash it with two 40 ml. portions of anhydrous ether, and dry in a vacuum desiccator. The yield of succinic anhydride, m.p. 118-119°, is 47 g. 

A. Maleic acid. Assemble the apparatus shown in Fig. Ill, 28, 1. Place 45 g. of dry mahc acid in the 200-250 ml. distilling flask and cautiously add 63 g. (57 ml.) of pure acetyl chloride. Warm the flask gently on a water bath to start the reaction, which then proceeds exothermically. Hydrogen chloride is evolved and the malic acid passes into solution. When the evolution of gas subsides, heat the flask on a water bath for 1-2 hours. Rearrange the apparatus and distil. A fraction of low boiling point passes over first and the temperature rises rapidly to 190° at this point run out the water from the condenser. Continue the distillation and collect the maleic anhydride at 195-200°. Recrystallise the crude maleic anhydride from chloroform (compare Section 111,93) 22 g. of pure maleic anhydride, m.p. 54°, are obtained. 

The procedure is not usually applicable to aminosulphonic acids owing to the interaction between the amino group and the phosphorus pentachloride. If, however, the chlorosulphonic acid is prepared by diazotisation and treatment with a solution of cuprous chloride in hydrochloric acid, the crystalline chlorosulphonamide and chlorosulphonanilide may be obtained in the usual way. With some compounds, the amino group may be protected by acetylation. Sulphonic acids derived from a phenol or naphthol cannot be converted into the sulphonyl chlorides by the phosphorus pentachloride method. 

Reflux 1 g. of the sulphonamide with 2-5 ml. of acetyl chloride for 30 minutes if solution is not complete within 5 minutes, add up to 2-5 ml. of glacial acetic acid. Remove the excess of acetyl chloride by distillation on a water bath, and pour the cold reaction mixture into water. Collect the product, wash with water and dissolve it in warm sodium bicarbonate solution. Acidify the Altered solution with glacial acetic acid Alter oflF the precipitated sulphonacetamide and recrystaUise it from aqueous alcohol. 

The disadvantages attending the use of acetic anhydride alone are absent when the acetylation is conducted in aqueous solution according to the following procedure. The amine is dissolved in water containing one equivalent of hydrochloric acid, slightly more than one equivalent of acetic anhydride is added to the solution, followed by enough sodium acetate to neutralise the hydrochloric acid, and the mixture is shaken. The free amine which is liberated is at once acetylated. It must be pointed out that the hydrolysis of acetic anhydride at room temperature is extremely slow and that the free amine reacts much more readily with the anhydride than does the water this forms the experimental basis for the above excellent method of acetylation. 

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 ... 

Excellent results may be obtained by conducting the acetylation in aqueous solution (cf. Section IV,45). Dissolve 0-5 g. of the amine in 2N hydrochloric acid, and add a little crushed ice. Introduce a solution of 5 g. of hydrated sodium acetate in 25 ml. of water, followed by 5 ml. of acetic anhydride. Shake the mixture in the cold until the smell of acetic anhydride disappears. Collect the solid acetyl derivative, and recrystallise it from water or dilute alcohol. 

Crystalline derivatives, suitable for identification and characterisation are dealt with in Section IV, 114, but the preparation of the following, largely liquid, derivatives will be described in the following Sections. When phenols are dissolved in aqueous sodium hydroxide solution and shaken with acetic anhydride, they undergo rapid and almost quantitative acetylation if the temperature is kept low throughout the reaction. This is because phenols form readily soluble sodium derivatives, which react with acetic anhydride before the latter undergoes appreciable hydrolysis, for example ... 

Dissolve 0 01 mol (or 1 g. if the molecular weight is unknown) of the compound in 5 ml. of 3A sodium hydroxide solution, add 10-20 g. of crushed ice followed by 1-5 g. (1-5 ml.) of acetic anh3 dride. Shake the mixture vigorously for 30-60 seconds. The acetate separates in a practically pure condition either at once or after acidification by the addition of a mineral acid. Collect the acetyl derivative, and recrystallise it from hot water or from dilute alcohol. 

Phenols, unlike amines, cannot be acetylated satisfactorily in aqueous solution acetylation proceeds readily with acetic anhydride in the presence of a little concentrated sulphuric acid as catalyst. Salicylic acid (o-hydroxy-benzoic acid) upon acetylation yields acetylsalicylic acid or aspirin ... 

Bemoyl chloride may replace acetyl chloride as a class reagent it possesses the advantage that it is only very slowly decomposed by cold water and consequently may be employed for detecting alcohols even in aqueous solution. The reaction is usually carried out in aqueous solution containing sufficient caustic alkali to decompose any excess of benzoyl chloride into the water-soluble alkali benzoate (Schotten - Baumann reaction compare Section IV,52). The benzoyl esters formed are insoluble in water ... 

Reduction of a nitro compound to a primary amine. In a 50 ml. round-bottomed or conical flask fitted with a reflux condenser, place 1 g. of the nitro compound and 2 g. of granulated tin. Measure out 10 ml. of concentrated hydrochloric acid and add it in three equal portions to the mixtiue shake thoroughly after each addition. When the vigorous reaction subsides, heat under reflux on a water bath until the nitro compound has completely reacted (20-30 minutes). Shake the reaction mixture from time to time if the nitro compound appears to be very insoluble, add 5 ml. of alcohol. Cool the reaction mixture, and add 20-40 per cent, sodium hydroxide solution imtil the precipitate of tin hydroxide dissolves. Extract the resulting amine from the cooled solution with ether, and remove the ether by distillation. Examine the residue with regard to its solubility in 5 per cent, hydrochloric acid and its reaction with acetyl chloride or benzene-sulphonyl chloride. 

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