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acetate mixed with a small amount of the acetylated glycoside. crystals are fractionally recrystallized from absolute ethyl alcohol' and 14 g of the pure orthoacetate derivative is obtained. It melts at 167° C and gives [a]20=-13° (chloroform, c=3). The material left after the separation of the orthoacetate consists of the acetylated beta glycoside and some of the orthoacetate. This mixture is separated by fractional crystallization, first from water and then from absolute alcohol. In these solvents the acetylated beta glycoside is slightly less soluble than the orthoacetate. The melting point of the pure methyl heptaacetyl-4-(6-d-glucosido)-8-d-mannopyranoside, of which about 2 g is obtained, is 178° C, and the rotation is [a] = -23.2° (chloroform, c=3).

A small quantity (0.5 g) of methyl heptaacetyl-4-(6-d-glucosido)-a-dmannoside is obtained from the original mother liquor after the orthoacetate and the acetylated beta glycoside have been separated. Methyl heptaacetyl-4-(8-d-glucosido)-a-d-mannopyranoside melts at 185° C and gives [a]2+29.3° (chloroform, c=2).

NOTES

This method may be used for the preparation of both orthoacetates and B-methyl glycosides. Orthoacetates are produced in good yield by application of the method to halogeno-acetates which have trans configurations for the halogen and adjacent acetyl groups [6].

The proportions of the products obtained vary with the experimental conditions. By conducting the reaction at the temperature of boiling methyl alcohol, Haworth, Hirst, Streight, Thomas, and Webb [28] found that the acetylated alpha glycoside crystallizes readily.

3 Prepared as described on page 514.

The orthoacetate derivatives are very reactive in the presence of traces of acids. Hence all solvents should be acid-free.

(k) METHODS FOR REMOVING ACETYL GROUPS

(1) CATALYTIC BARIUM METHYLATE METHOD. The catalytic barium methylate method originated by Isbell [29] is one of the most satisfactory methods for the deacetylation of the acetylated sugars and related substances. It resembles the sodium methylate method of Zemplén [30]. In both methods the alcoholate acts catalytically and merely facilitates the conversion of the acetyl groups to methyl acetate. Barium methylate is more convenient than sodium methylate because the barium may be removed readily and stock solutions may be purified before use by filtration to remove any barium hydroxide or carbonate. The reagent is prepared by adding powdered barium oxide to absolute methyl alcohol. The barium oxide reacts with the alcohol, forming barium methylate and barium hydroxide. The barium hydroxide is insoluble in the alcoholic solution and is separated by filtration. The strength of the filtered solution is ascertained by titration with 0.1 N aqueous sulfuric acid. The method is illustrated by the following example:

Deacetylation of methyl heptaacetyl-4-(B-d-glucosido)-B-d-mannopyranoside [26, 29].-Dry methyl heptaacetyl-4-(6-d-glucosido)-8-d-mannopyranoside (1.5 g) is dissolved in 30 ml of cold anhydrous methyl alcohol. Two milliliters of 0.4 N barium methylate solution in methyl alcohol is added, and the solution is allowed to stand in the refrigerator for 24 hours. After the solution has been tested for excess barium methyl

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ate, the barium is precipitated by the addition of an equivalent quantity of sulfuric acid, or by carbonation. The precipitate is removed by filtration and the filtrate evaporated to a thick sirup which is allowed to crystallize. The crystalline mass is triturated with absolute alcohol and after separation weighs about 0.7 g. Two recrystallizations from ethyl alcohol yield pure methyl 4-(8-d-glucosido)-8-dmannopyranoside hemihydrate, which melts at 229° C and gives [a] 20=-48.5° (water, c=4). Other acetylated sugar derivatives may be deacetylated in similar manner.

(2) RAPID DEACETYLATION WITH HOT SODIUM METHYLATE.-The acetyl-glycosides, acetyl-alcohols, and other acetylated products without a free reducing group are rapidly deacetylated by a small amount (one six-hundredth of the theoretical quantity) of sodium or barium methylate at the boiling point of the methyl alcoholic solution of the substance. The amount of sodium or barium acetate formed is so small that for ordinary purposes it is not separated. The method is advantageous for acetylated substances of low solubility, but it cannot be used for substances having a free reducing group or for those sensitive to alkali. The method is illustrated by the following example:

Deacetylation of pentaacetylsalicin [31].-To 80 ml of absolute methyl alcohol, 20 g of pentaacetylsalicin and 4 ml of 0.1 N sodium methylate solution are added and the mixture is boiled for 5 to 10 minutes on a water bath. The cool solution, upon standing overnight, deposits about 10 g of salicin (87 percent of theory), which melts at 201° C.

(3) DEACETYLATION WITH AN EXCESS OF ALKALI.-If the sugar derivative contains toluenesulfonyl or other groups which react stoichiometrically with barium or sodium methylate in methyl alcohol to form salts, an excess of alkali must be used. Barium hydroxide in aqueous solution [32], barium methylate in alcoholic solution [33], and sodium hydroxide in acetone [34] have been employed.

(1) QUANTITATIVE DETERMINATION OF ACETYL GROUPS

Since the percentages of carbon and hydrogen in the various acetyl derivatives of the carbohydrates differ only slightly, the number of acetyl groups in a carbohydrate derivative is usually determined by titration of the acetic acid formed either directly or indirectly by the hydrolysis of the acetyl groups. The determination may be conducted by several methods, which will be considered in three groups: (1) methods which depend on hydrolysis in alkaline solution, (2) methods which depend on hydrolysis in acid solution, and (3) methods which depend on the production and distillation of ethyl acetate.

The methods employing alkaline hydrolysis are applicable to the determination of acetyl groups in carbohydrate derivatives which under the conditions of the method do not give other acidic or basic decomposition products. By conducting the hydrolysis at 0° C, as described by Brauns [35], decomposition of the sugar is largely avoided and satisfactory results can be obtained with ketoses and other sugars which are relatively sensitive to alkalies. But in some cases, as for

Since moisture or acids in the original sirup will decompose the barium methylate, it is well to test the deacetylated mixture to see if an excess of barium methylate is present. This may be done by diluting a few milliliters with water and adding several drops of phenolphthalein solution. A definite red color indicates a sufficient excess. If the test is not positive, add more barium methylate solution to the deacetylation mixture and allow 24 hours more for the deacetylation to be completed.

example with tetraacetyl-d-ribosido-dihydroxyacetone [36], the alkali, even at 0° C, results in the formation of acidic decomposition products; hence, the results obtained by alkaline hydrolyses are considerably greater than the theoretical values. Certain orthoester groups are relatively stable to alkaline hydrolysis but are very sensitive to acid hydrolysis. Consequently the orthoester groups can be determined by the difference in the results obtained from hydrolyses in acid and in alkaline solution. In the event that the substance contains acetyl groups attached to nitrogen, the ethyl acetate method of Perkin [37] as modified by Freudenberg and Harder [38] gives the combined O-acetyl and N-acetyl groups, while the alkaline hydrolysis at 0° C gives only the O-acetyl [39]. In the ethyl acetate method the acetyl derivative is heated in acid solution with ethyl alcohol, the ethyl acetate which is formed is distilled and saponified with standard alkali. (1) DETERMINATION OF ACETYL GROUPS BY ALKALINE HYDROLYSIS. Method [37].-A 0.4-g sample of finely powdered tetraacetylfructose is shaken with 75 ml of 0.1 N sodium hydroxide at 0° C. The saponification is complete in 2 to 5 hours, when a clear solution is obtained. The excess of sodium hydroxide is titrated with 0.1 N sulfuric acid (phenolphthalein indicator). The difference in the amount of alkali added and the acid used in the titration gives the amount of acetic acid formed. The results usually agree with the theoretical value within 0.5 percent.

The more insoluble acetylated sugars and glycosides dissolve very slowly in aqueous sodium hydroxide and require a long time for saponification. This difficulty may be overcome by the following procedure, which uses acetone as a solvent [40]:

One-half gram of the substance is dissolved in 50 ml of acetone. The solution is cooled in an ice-and-salt mixture and 100 ml of 0.1 N aqueous potassium hydroxide is added dropwise. The solution is kept for 2 hours at the temperature of the ice-and-salt mixture. Then the excess alkali is titrated with 0.2 N hydrochloric acid. A blank control test is run and the results are corrected for its value.

(2) DETERMINATION OF ACETYL GROUPS BY ACID HYDROLYSIS. Method [41].-A half-gram sample of the finely powdered substance is mixed with 100 ml of 0.25 N sulfuric acid and boiled for 5 hours. under a reflux condenser. The solution is cooled and titrated with 0.1 N sodium hydroxide. The amount of acetic acid formed by the hydrolysis of the acetyl groups is equal to the difference between the amount of sulfuric acid added and the amount of alkali used.

(3) DETERMINATION OF ACETYL GROUPS BY THE ETHYL ACETATE METHOD.

Method [38].-The determination is made in the apparatus illustrated in figure 111. Flask A has a volume of 100 ml and B a volume of 150 ml. The tubes F and G must have a minimum inside diameter of 5 mm. The weighed sample (0.3 to 0.4 g), 30 ml of absolute alcohol, 5 g of p-toluenesulfonic acid, and a few boiling stones are introduced into flask A, and 10 ml of absolute alcohol is placed in flask B. Water is allowed to flow through the condensers C, D, and E. Flask B is cooled in an ice bath, and flask A is heated by a water bath at 100° C while its contents are allowed to reflux for 10 minutes. The bath temperature is lowered to 95° C (at which point it is kept during the subsequent operations), the reflux condenser, C, is emptied of

water, and the contents of flask A are allowed to distill for 15 minutes into flask B. Then 20 ml of absolute alcohol is introduced into flask A from the dropping funnel. The contents of flask A are refluxed for 10 minutes while water again flows through condenser C. Condenser C is then emptied and the material in flask A distilled into flask B for 10 minutes. With condenser C still empty, 20 ml of absolute alcohol is added dropwise to flask A over a period of 15 minutes while simultaneous distillation takes place. The distillation is continued

A

B

LE

FIGURE 111.-Apparatus for the determination of acetyl groups by the ethyl acetate method.

for 10 minutes. When the distillation is finished, 30 ml of 0.2 N sodium hydroxide 2 is added to flask B through the tube of condenser E. The temperature of flask A is kept at 80° C, the cold bath of flask B is replaced by a hot-water bath, and the contents of flask B are refluxed for 10 minutes. Flask B is again cooled and removed from the apparatus. After the contents have been diluted with 30 ml of water, they are titrated with 0.2 N sulfuric acid (phenolphthalein indicator). A blank control determination is run, and the titration is corrected for its value. Freudenberg and Harder state that the normal deviation from the theoretical value is -0.1 to +0.4 percent.

NOTES

1 The experimental conditions as given are suitable for most sugar derivatives. In case the sample contains acetyl groups attached to nitrogen, the temperature of the bath for the flask A is kept at 100° C during the entire esterification reaction and the distillations. It is changed only during the final saponification, which is carried out as described in the O-acetyl directions at 80° C. The first refluxing time is lengthened from 10 minutes to 45 minutes and the second from 10 minutes to 30 minutes.

2 The standardization of the sodium hydroxide solution is made by diluting a definite volume with an equal amount of water, adding 2 volumes of alcohol, and titrating with 0.2 N sulfuric acid, using phenolphthalein indicator.

(4) DETERMINATION OF ACETYL GROUPS AND HALOGEN IN HALOGENO-ACETYL DERIVATIVES.-The analytical results obtained by alkaline or acid hydrolysis of halogeno-acetyl sugars represent the total number of acid groups. However, a quantitative determination of the halogen may easily be made on the hydrolyzed solution. If alkaline saponification is employed, the excess alkali is titrated with standard nitric acid. The halogen in the solution is then determined in the conventional manner by precipitation as silver chloride, bromide, or iodide, or as calcium fluoride.

(m) REFERENCES

[1] C. S. Hudson, J. Ind. Eng. Chem. 8, 380 (1916).

[2] C. L. Jungius, Z. physik. Chem. 52, 101 (1905).

[3] E. Montgomery and C. S. Hudson, J. Am. Chem. Soc. 56, 2463 (1934). [4] C. S. Hudson and H. O. Parker, J. Am. Chem. Soc. 37, 1589 (1915).

[5] C. S. Hudson and J. M. Johnson, J. Am. Chem. Soc. 38, 1223 (1916).
[6] H. S. Isbell, Ann. Rev. Biochem. 9, 70 (1940).

[7] D. H. Brauns, Proc. Roy. Acad. Amsterdam 10, 563 (1907-1908).
[8] C. S. Hudson and D. H. Brauns, J. Am. Chem. Soc. 37, 2738 (1915).
[9] C. S. Hudson and D. H. Brauns, J. Am. Chem. Soc. 37, 1283 (1915).
[10] E. Pacsu, J. Am. Chem. Soc. 54, 3649 (1932).

[11] F. B. Cramer and E. Pacsu, J. Am. Chem. Soc. 59, 711, 1467 (1937).
[12] R. Behrend and P. Roth, Liebigs Ann. Chem. 331, 362 (1904); 353, 109
(1907).

[13] E. Fischer and R. Oetker, Ber. deut. chem. Ges. 46, 4029 (1913).

[14] W. W. Pigman and H. S. Isbell, J. Research NBS 19, 189 (1937) RP1021.

[15] Zd. H. Skraup and J. Koenig, Ber. deut. chem. Ges. 34, 1115 (1901).

[16] C. S. Hudson and J. K. Dale, J. Am. Chem. Soc. 37, 1280 (1915). [17] E. Erwig and W. Koenigs, Ber. deut. chem. Ges. 22, 2207 (1889).

[18] E. Erwig and W. Koenigs, Ber. deut. chem. Ges. 22, 1464 (1889). [19] D. H. Braums, J. Am. Chem. Soc. 48, 2784 (1926).

[20] J. K. Dale, J. Am. Chem. Soc. 37, 2745 (1915).

[21] C. S. Hudson and J. M. Johnson, J. Am. Chem. Soc. 37, 2748 (1915).

[22] R. M. Hann and C. S. Hudson, J. Am. Chem. Soc. 56, 2465 (1934).

[23] M. L. Wolfrom, J. Am. Chem. Soc. 51, 2188 (1929).

[24] M. L. Wolfrom, L. W. Georges, and S. Soltzberg, J. Am. Chem. Soc. 56, 1794 (1934).

[25] F. B. Cramer and E. Pacsu, J. Am. Chem. Soc. 59, 1148 (1937).

[26] H. S. Isbell, BS J. Research 7, 1115 (1931) RP392.

[27] E. Pacsu and F. V. Rich, J. Am. Chem. Soc. 55, 3022 (1933).

[28] W. N. Haworth, E. L. Hirst, H. Streight, H. Thomas, and J. Webb, J. Chem.

Soc. 1930, 2643.

[29] H. S. Isbell, BS J. Research 5, 1185 (1930) RP253.

[30] G. Zemplén, Ber. deut. chem. Ges. 59, 1258 (1926).

[31] G. Zemplén and E. Pacsu, Ber. deut. chem. Ges. 62, 1613 (1929).

[32] C. S. Hudson and D. H. Brauns, J. Am. Chem. Soc. 38, 1216 (1916).

[33] W. Weltzien and R. Singer, Liebigs Ann. Chem. 443, 104 (1925).

[34] A. Kunz and C. S. Hudson, J. Am. Chem. Soc. 48, 1982 (1926).

[35] D. H. Brauns, Verslag Koninkl. Akad. Wetensch. Amsterdam, 577, (1908). [36] C. W. Klingensmith and W. L. Evans, J. Am. Chem. Soc. 61, 3012 (1939). [37] A. G. Perkin, J. Chem. Soc. 87, 107 (1905).

[38] K. Freudenberg and M. Harder, Liebigs Ann. Chem. 433, 230 (1923).

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