Purification of the Acetate Derivative

The acetate is dissolved in 1-2 ml of chloroform and the solution is dripped into 100 ml of magnetically stirred ether. The precipitated acetate is centrifuged off and washed with ether. 

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A large number of polyfructosans that have been reported from time to time by different authors have been investigated by Schlubach and his associates. In order to obtain polysaccharides of constant optical rotation, 100 to 300 precipitations from aqueous solution by the addition of alcohol were necessary. Fifty to 150 precipitations from chloroform solution with petroleum ether were required for purification of the acetate derivatives. These were methylated according to the procedure of Haworth and Straight,24 and upon hydrolysis partially methylated fructoses were obtained. 

Reaction of the imine-indoline products 157 and 158 with potassium borohydride in acetic acid resulted in rupture of the C-3 -C-7 bond and reduction of the resulting imonium function (Scheme 45). While the initial C-I6 -C-14 PARF imine-indolines 157 and 158 can be isolated, they are less stable on silica gel chromatography than the corresponding l -vincad-ifformine-derived C-16 -C-14 PREF compounds 145, and consequently purification of the vindoline coupling products and separation of dia-stereomers were best carried out at the indole-indoline stage (162, 163). 

This reaction proceeds in quantitative yield and requires no chromatographic purification. After protection of the hydroxyl function of 18 as the acetate derivative (19), the latter can be... 

Diacetate Derivative, Isopropylidene Formation, and Mass Spectrometry. A particularly facile route to proof of structure of the glyceryl ether components is to use a combination of (a) purification by thin-layer chromatography and (b) separation into individual species of the isopropylidene or acetate derivatives by gas-liquid chromatography. As an example, the experimental protocol for examination of the diacetates will be described at this point and later that of the isopropylidene derivative. Inherent in such an experimental approach is to have synthetic standards of high purity available. It is possible to accomplish this task, but it is not necessarily quick and neat. The synthesis... 

Quinazoline-4(3//)-thiones 13 are prepared in a very simple, one-step synthesis by treatment of 2-[(ethoxymethylene)amino] derivatives 12 with alcoholic sodium hydrosulfide. Numerous other hetero-fused pyrimidinethiones are also available by this procedure (Method Cyclization proceeds uniformly in very high yields, generally in excess of 90%. In some cases, since isolation and purification of (ethoxymethylene)amino derivatives results in the lowering of the overall yield, the one-step procedure, consisting of treatment of a 2-aminonitrile 11, either with a 1 1 mixture of triethyl orthofoimate and acetic anhydride or with triethyl orthoformate alone, to give an intermediate ethoxymethyleneamino derivative which, without isolation, is treated with an ethanolic solution of sodium hydrosulfide, is preferred for preparative purposes (Method B). In a few cases, conversion of the 2-aminonitrile to a 2-aminothioamide followed by cyclization with triethyl orthoformate (see p 47) competes favorably with the above procedure. 

Tilak et described the use of excess mixed carbonic anhydrides to force condensation reactions to completion followed by the destruction of the excess mixed anhydride via the addition of aqueous potassium hydrogencarbonate. Hydrolysis of the nnixed anhydride was rapid and the resulting protected dipeptide could be extracted into ethyl acetate in a high state of purity, leaving the excess amino acid derivative and the salts in the aqueous phase. Without further purification the protected dipeptide was N -deprotected and reacted with the next mixed anhydride, and the process repeated until the desired peptide was obtained. Beyerman et al. substantially expanded the scope of this procedure and named it the REMA method for peptide synthesis (Repetitive Excess Mixed Anhydride).P°1 These reaction conditions provide an excellent method to ensure complete reaction of the amine component as well as rapid reaction rates and minimal side products. However, care must be taken to ensure that the excess carboxylic acid component is soluble in sodium hydrogencarbonate solution, e.g. when Z-Asp(OBzl)-OH is the acid component, it is extracted into the ethyl acetate as the sodium salt along with the product. With the due precautions the yields of small peptides are so high that the method could be applied without purification of the intermediate products, that is, in a repetitive way. 

The conditions for this reaction are the same as those used by earlier workers to prepare mono dicarboxy pyrrole derivatives (3-jJ). Upon cooling of the acetic acid reaction solution the product, bis pyrrole derivative, crystallizes out and is recovered in sufficient purity to be used without further purification. Attempts to hydrolyze the bis(dicar-boethoxy pyrrole) derivatives in aqueous NaOH proved unsuccessful due to the high degree of insolubility. 

Hydrolysis. The anomeric acetate is the only hydrolyzable group in polyacetyl amino sugars on treatment with Si02-Me0H. Oxazolidinones such as those derived from proline are similarly cleaved, allowing for a very simple purification of the end products in a synthetic approach to a-alkylprolines. 

Mixtures containing nearly all possible partially methylated alditol acetates have been prepared as standards for the methylation analysis of cell-wall polysaccharides. Incomplete Hakamori methylation (1.e. limiting amounts of potassium dimsyl) of neutral monosaccharides with purification of the derivatives on a C g reversed-phase column was followed by standard hydrolysis, reduction, and acetylation. Relative capillary g.c. retention data was... 

Gas chromatography with flame ionization detection (GC-FID). Total MHPG, free and conjugated, was extracted from urine with ethyl acetate after enzymatic hydrolysis. Quantitative analysis was carried out with the GC-FID technique. Some difficulties were encountered in the purification of the urinary extracts and in choosing a derivative which would give satisfactory resolution on GC. The following efforts were made toward improving the feasibility and the specificity of the method. 

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