3

carbon, allowed to stand overnight, filtered, and evaporated to a 75-percent sirup (n=1.477) which crystallizes readily. The crystals are separated and washed, first with 1:1 aqueous ethyl alcohol and then with 95-percent ethyl alcohol. The yield is about 90g. A review of the literature is given by Harding [3].

Recrystallization.-Three parts of rhamnose hydrate are dissolved with 2 parts of hot water. After a small quantity of a decolorizing carbon is added, the solution is filtered and seeded with crystalline rhamnose. Crystallization takes place in the course of several hours at room temperature, during which time the mixture is preferably kept in motion. The crystals are separated and washed, first with 75-percent and then with 95-percent aqueous alcohol. The first crop of crystals is about 60 percent of the crude product. By concentrating the mother liquors in vacuo, additional crystalline sugar is obtained.

The modification of the sugar obtained under these conditions is a-l-rhamnose monohydrate. În 4-percent aqueous solution this substance exhibits [a]2.2-8.6° and at equilibrium +8.2°.

NOTES

1 Lemon flavine is a commercial yellow dyestuff prepared from black oak bark and may be obtained from J. S. Young and Company, Hanover, Pa.

2 The solution may be heated over a direct flame, provided a suitable stirring device is used. Foaming may be reduced by the occasional addition of an antifoam agent, such as capryl alcohol.

3 If crystallization does not take place spontaneously, add seed crystals. If crystallization still does not commence, extract the aqueous solution with 95percent alcohol, discard the residue, and evaporate the extractions to a sirup of about 75 percent of total solids, seed, and allow to stand until crystallization takes place.

REFERENCES

[1] C. Liebermann and O. Hörmann, Liebigs Ann. Chem. 196, 323 (1879). [2] C. F. Walton, Jr., J. Am. Chem. Soc. 43, 127 (1921).

[3] T. S. Harding, Sugar 25, 82 (1923).

20. d-RIBOSE

Method.-[1, 2, 3, 4] The following specific directions for the preparation of ribose have been found convenient and effective in this laboratory.

Hydrolysis of nucleic acid.-Five hundred grams of commercial yeast nucleic acid,1 100 g of magnesium oxide, and 3 liters of distilled water are placed in a 4-liter Pyrex beaker and mixed until a uniform, smooth suspension is obtained free from lumps. The mixture in the 4-liter beaker or in a 5-liter short-necked flask is placed in an autoclave and heated to 145° C (60 pounds steam pressure) for 4 hours with continuous stirring. The maximum working range of temperature lies between approximately 120° and 155° C [5].

3

The pressure in the autoclave is then reduced as quickly as possible without causing the reaction mixture to boil over. As soon as the pressure is reduced, the autoclave is opened and the boiling-hot reaction mixture filtered through a little decolorizing carbon on a large Büchner funnel into a 4-liter filtering flask.5 The copious precipitate of magnesium-containing phosphates is packed down and washed with 1 to 11⁄2 liters of boiling water, making the total volume of filtrate and washings between 3.5 and 4 liters.

Separation of guanosine. The filtrate from the hydrolysis of nucleic acid is allowed to stand overnight in a stoppered 4-liter Erlenmeyer flask at room temperature, or preferably in the icebox to permit the guanosine to crystallize out as completely as possible.

The crude guanosine is filtered off and washed on the filter with cold water followed by alcohol to facilitate drying. The yield is 70 to 80 g of crude air-dried guanosine.

6

Purification of guanosine.-The crude guanosine is purified by recrystallization from a 4-percent solution in water. Two and one-half liters of distilled water is heated to boiling and to this is added 100 g of crude guanosine. The mixture is stirred vigorously to hasten solution and when dissolved it is filtered immediately through a little decolorizing carbon on a large Büchner funnel. The filtrate is cooled and allowed to stand overnight, whereupon crystalline guanosine separates. The product is collected on a filter, washed with water and then with alcohol, and dried at a low temperature (about 35° C). Usually one recrystallization provides sufficient purification. However, if the crystals in the magma do not show clean-cut end faces, or if the magma appears somewhat gelatinous, a second recrystallization after filtration through carbon may be desirable.

Separation of adenosine. From the filtrate obtained from the crude guanosine, adenosine may be precipitated without further purification by the addition of picric acid, or if desired, a preliminary treatment may be given to remove the small excess of magnesia as follows:

To the filtrate obtained from the crude guanosine is added a solution of about 2 g of ammonium phosphate, followed by dilute barium hydroxide to about pH 9, or as long as a precipitate continues to be formed. The solution is filtered. The filtrate is immediately treated with dilute sulfuric acid in sufficient quantity to precipitate any barium salts, which are then separated by filtration. The addition of dilute sulfuric acid to the filtrate is continued until the solution remains just acid to congo red. At this point a nearly saturated solution of picric acid in hot 90-percent alcohol is added until no more immediate precipitate is formed. Approximately 50 g of picric acid is required. The precipitate is filtered, washed with water slightly acidified with picric acid, then with alcohol, and finally is air-dried. About 100 to 115 g of air-dried crude adenosine picrate containing some cytidine picrate is obtained.

Purification of adenosine picrate. The crude adenosine picrate may be sufficiently purified by one or two recrystallizations from hot water using a little decolorizing carbon in the filtration. The crude wet adenosine picrate is taken from the filter, and without drying, is added immediately to boiling water until the solution is nearly saturated, whereupon it is filtered at once through washed carbon on a large Büchner funnel. The solution is allowed to crystallize overnight, and the crystals are filtered, washed with water and alcohol, and air-dried. Adenosine picrate may be hydrolyzed directly without removing the picric acid.

Hydrolysis of guanosine. One hundred grams of recrystallized guanosine is dissolved in 10 liters of approximately 0.1 N sulfuric acid at the boiling temperature, and hydrolysis at boiling temperature is allowed to proceed for 1 hour, after which the solution is treated with an excess of silver sulfate dissolved or suspended in hot water.

323414°-42---32

The

solution is allowed to cool and stand overnight at room temperature (or preferably in the icebox at as low a temperature as is practicable), and the insoluble guanidine-silver sulfate compound is separated by filtration.

The filtrate is neutralized with barium hydroxide to a pH of about 6.4 to 7.0, in two stages; the bulk of the barium sulfate is filtered off while the solution is decidedly acid, since the barium sulfate will be found to filter much more readily in acid than in neutral solution. The neutralization of the small remaining amount of acid is completed to a pH of 6.4 to 7.0, using bromthymol blue, a glass electrode, or some other convenient pH indicator. The solution is filtered through a Büchner funnel, using enough decolorizing carbon to prevent the clogging of the filter by the barium sulfate. The neutral filtrate is concentrated under reduced pressure at a low temperature to a thick sirup, which is taken up with about 500 ml of warm absolute alcohol. A small quantity of decolorizing carbon is added and the solution filtered. The filtered solution is evaporated again in vacuo to a thick sirup, which is diluted with a little warm absolute alcohol and seeded with d-ribose. Crystallization usually starts within a few minutes and is completed overnight. The crystals are collected on a filter, washed with alcohol, and dried.

Ribose may be purified by dissolving in a very small amount of water, adding alcohol, filtering through carbon, and allowing to stand until crystallization is complete, preferably in the icebox.

The mother liquors, both from the crude and from the recrystallized ribose, may be retreated to yield further crops of crystals.

Hydrolysis of adenosine picrate.-One hundred and fifty grams of recrystallized adenosine picrate is dissolved in 10 liters of boiling water. When solution is complete, 70 g of sulfuric acid is added and hydrolysis is allowed to proceed for 1 hour at 100° C. The solution is allowed to stand overnight at room temperature, or preferably in the refrigerator, in order that the insoluble adenine picrate may completely crystallize. After separation of the adenine picrate by filtration, the sulfuric acid in the filtrate is neutralized in two steps, as described for guanosine, and the filtrations in each case are made through a matt of decolorizing carbon on a large Büchner funnel. From this point on, the procedure is identical with that described above for guanosine.

The yield from 500 g of nucleic acid assaying 83 percent has been found to be about 35 g of ribose from the guanosine and 25 g from the adenosine picrate, or a total of 60 g of crystalline d-ribose.

NOTES

1 Nucleic acid purchased from several chemical firms has been found satisfactory. 2 If preferred, the nucleic acid may first be dissolved in water containing a little ammonia and the magnesia then added, although this procedure is not necessary.

The sizes of vessels and the quantities of materials specified are based in part upon the capacity of the autoclave available. If the reaction is conducted in a flask, more vigorous stirring may be used without splashing.

The yield is materially reduced if stirring is not used. This probably results from the formation of relatively insoluble magnesium nucleotides which, if not kept in suspension by stirring, cake upon the bottom and cause the hydrolysis to proceed less smoothly.

At this stage the reaction of the filtrate should preferably be slightly alkaline, about pH 7.5 to 8.0. An acid reaction indicates that insufficient magnesia was used. It is important that the solution not be allowed to become acid during the hydrolysis, because at the high temperature employed there would be danger of hydrolyzing some of the riboside and so losing ribose in the reaction mixture.

Avoid heating longer than is necessary as the guanosine is easily hydrolyzed. The same is true of adenosine.

'The decolorizing carbon should be thoroughly washed with hot water.

To avoid bumping, a drying oven which will accomodate a 12-liter flask is excellent for carrying out the hydrolysis, both of guanosine and of adenosine picrate.

REFERENCES

[1] F. P. Phelps, U. S. Patent 2,152,662.

[2] F. P. Phelps and F. J. Bates, Publication pending.

[3] P. A. Levene and E. P. Clark, J. Biol. Chem. 46, 19 (1921).

[4] H. Bredereck, Ber. deut. chem. Ges. 71, 408 (1938).

[5] W. Jones and H. C. Germann, J. Biol. Chem. 25, 93 (1916).

21. -SORBOSE

Biological source. The biological synthesis of l-sorbose from d-sorbitol by bacterial fermentation has been developed by the Industrial Farm Products Research Division of the Bureau of Agricultural Chemistry and Engineering, and yields of more than 90 percent are obtained [1].

Crystallization.-Three parts of sorbose are dissolved with 2 parts of water containing a few drops of acetic acid. After the addition of a small quantity of a decolorizing carbon the hot solution is filtered. The clear filtrate is allowed to cool slowly while it is stirred. Crystallization takes place as the solution cools. After standing for several hours at room temperature or below, the crystals are separated and washed, first with a mixture of methyl alcohol (2 volumes) and water (1 volume) and then with undiluted methyl alcohol. About 50 percent of the crude sugar separates in the first crop of crystals. By evaporating the mother liquor in vacuo to a sirup of about 70 percent of total solids, additional sorbose crystallizes. In 11-percent aqueous solution, l-sorbose gives [a]=-43.7° initially, changing in several hours to 43.4.°

REFERENCES

[1] P. A. Wells, J. J. Stubbs, L. B. Lockwood, and E. I. Roe, Ind. Eng. Chem. 29, 1385 (1937).

22. d-TALOSE

1

Method.-[1, 2, 3] d-Talose may be prepared from galactose through the intermediate preparation of pentaacetylgalactose (see page 488), 1-bromoacetylgalactose (see page 500), triacetylgalactal (see page 532), and galactal. An aqueous solution of galactal (150 g in 1,500 ml of water) is cooled to 0°C and treated with a solution of 174 g of perbenzoic acid in 1 liter of ether. The mixture is stirred at 0°C for 4 hours and for several additional hours while the temperature is allowed to rise slowly to room temperature. The aqueous phase is separated and extracted three times with ether and three times with chloroform. The purified aqueous solution is then concentrated in vacuo to a thin sirup (about 70 percent) and allowed to stand overnight. About 5 g of difficulty soluble monobenzoyltalose crystallizes and is separated by filtration and washed with water.2

The mother liquors are evaporated to a thin sirup which is taken up with methanol and allowed to crystallize. The crystals are separated and the mother liquor is again evaporated and crystallization allowed to take place. This process is repeated until no further crystallization occurs, The various crops are then combined and

3

extracted for an hour with eight times their weight of gently boiling methanol. The undissolved material is removed by filtration and the filtrate is evaporated to a thin sirup which crystallizes to give crude d-talose.5 The sugar is recrystallized by dissolving it in water and concentrating the solution to a sirup which is diluted with methyl alcohol and seeded. Pure d-talose may be obtained by recrystallizing once more the material obtained. From 150 g of galactal a total of about 50 g of d-talose may be obtained. This corresponds to a yield of about 80 g of d-talose from 500 g of galactose.

The form of d-talose obtained under ordinary conditions melts at 133° to 134°C and in 4-percent aqueous solution exhibits a rapid complex mutarotation from an initial value of [a]=+68.0° to an equilibrium value of [a]2+20.8°.

The pyridine rearrangement of galactonic acid and the reduction of the talonic acid formed has also been used for the preparation of talose [4, 5].

NOTES

1 Preparation of perbenzoic acid. Thirty grams of finely ground sodium peroxide is added with vigorous stirring to 400 ml of ice and water and after the addition of 200 ml of cold ethyl alcohol (-5°C), 25 ml of benzoyl chloride dissolved in 100 ml of cold ether is added. The mixture is stirred for several minutes and is then filtered through a large Büchner funnel. The filtrate, after acidification with 700 ml of cold (0°C) normal sulfuric acid, is extracted with four 150-ml portions of ether. The solution should be kept as cold as possible until the final acidification; about 20 g of perbenzoic acid is obtained.

2 Alcohol should not be used for washing this product.

3 Additional material may be obtained from the mother liquor by heating it near the boiling point with 0.05 N sulfuric acid for several hours. The cold solution is extracted with ether, neutralized with barium carbonate, filtered, and evaporated as before. This treatment hydrolyzes the benzoyl derivatives of galactose and talose which may be present.

4 Usually this material is nearly pure galactose with an optical rotation between [a] +76° and +80°. If the rotation is below this, another extraction is advantageous.

5 The optical rotation of this material is between [a] = +25° and +30°.

REFERENCES

[1] W. W. Pigman and H. S. Isbell, J. Research NBS 19, 189 (1937) RP1021. [2] P. A. Levene and R. S. Tipson, J. Biol. Chem. 93, 631 (1931).

[3] M. Bergmann and H. Schotte, Ber. deut. chem. Ges. 54, 440 (1921).

[4] W. Bosshard, Helv. Chim. Acta 18, 482 (1935).

[5] C. Glatthaar and T. Reichstein, Helv. Chim. Acta 21, 3 (1938)

1

23. TURANOSE (3-(a-d-GLUCOPYRANOSIDO)-d-FRUCTOSE) 1 Method. [1, 2, 3] One hundred grams of pure melezitose 2 is dissolved in 1 liter of boiling water containing 4 ml of concentrated sulfuric acid. The solution is boiled gently for 15 minutes and is then neutralized with an excess (10 g) of calcium carbonate and cooled to 37° C. A cake of compressed baker's yeast and 20 ml of nutrient solution 3 are added, and fermentation is allowed to take place at 37° C for about 4 days, after which about 10 g of a decolorizing carbon is added and the solution is filtered. The filtrate is evaporated in vacuo to a sirup of about 88 percent of total solids (n=1.511). The sirup is taken up with 150 ml of methyl alcohol, and after the addition of 5 g of a decolorizing carbon the solution is filtered. The filtrate is seeded with crystals of turanose and allowed to stand. After several days the crystals are separated and the mother liquor is concentrated again.