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. 3 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. I-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

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

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. 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 talosc [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.

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.

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.

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

to give additional product. The total yield from 100 g of melezitose is about 50 g.

Recrystallization.-One hundred grams of turanose is dissolved in 100 ml of hot water. After adding 5 g of a decolorizing carbon, the solution is filtered and then evaporated in vacuo to a sirup (n=1.490) containing about 80 percent of total solids. This sirup is dissolved in 100 ml of hot methyl alcohol. After the addition of 5 g of a decolorizing carbon, the hot solution is filtered. The filtrate, after cooling to room temperature, is seeded and preferably stirred while crystallization takes place. After several hours the crystals are collected on a filter and washed with methyl alcohol. The yield is about 75 g. The remaining sugar is reclaimed by concentrating the mother liquor, adding methyl alcohol, and separating the crystals which form.

In 4-percent aqueous solution, turanose gives [a]2+27.3° initially, changing in the course of an hour to an equilibrium value of +75.8°.

NOTES

The 3-glucosidofructose structure for turanose was first proposed by Isbell and Pigman [4].

2 The method for obtaining melezitose is described on page 472.

3 The nutrient solution is prepared by dissolving 2.5 g of NH4NO3, 0.3 g of KH2PO4, and 0.25 g of MgSÓ,.7H2O in 100 ml of water.

The seed crystals originally used at this Bureau were part of the product first discovered by Ď. H. Brauns.

REFERENCES

[1] C. S. Hudson and E. Pacsu, J. Am. Chem. Soc. 52, 2522 (1930).

[2] G. Tanret, Compt. rend. 142, 1424 (1906).

[3] M. Bridel and T. Aagaard, Bul. soc. chim. biol. 9, 884 (1927).

[4] H. S. Isbell and W. W. Pigman, J. Research NBS 20, 787 (1938) RP1104.

24. d-XYLOSE

Method.'-[1] One kilogram of shredded or broken corn cobs is boiled 2 for 3 hours with 6 liters of 7-percent sulfuric acid, after which the insoluble residue is separated on a filter and washed with water. The aqueous solution is neutralized with calcium carbonate, and after removal of the insoluble calcium sulfate, the liquor is treated with baker's yeast. When the ensuing fermentation is complete, about 100 g of decolorizing carbon is added and the solution is filtered.3 The filtrate is concentrated in vacuo to a sirup (n=1.442, 60 percent of total solids), which is then diluted with 3 volumes of methyl alcohol. After separation of the resulting precipitate, the alcoholic solution is evaporated in vacuo to a sirup (85 percent of total solids, n=1.503), which is mixed with 250 ml of methyl alcohol and seeded with crystalline d-xylose. After standing for several days, the crystalline product is separated and washed with 75 percent by volume of aqueous methyl alcohol. The yield is about 12 percent.*

Recrystallization.-One kilogram of xylose is dissolved with 400 ml of water containing a few drops of acetic acid. After the addition of 15 g of a decolorizing carbon, the hot solution is filtered and the filtrate is cooled and kept in gentle motion for several hours while crystallization takes place. The crystals which form are separated and washed with water or with aqucous alcohol. The yield is about 50 percent in the first crop. The sugar in the mother liquor crystallizes readily from sirups of about 75 percent of total solids.

a-d-Xylose melts at 145° C and in 4-percent solution gives [a]2+93.6° initially, changing in several hours to +18.8°.

NOTES

The commercial production of xylose from cottonseed hulls is described by Schreiber, Geib, Wingfield, and Acree [2].

The hydrolysis can be conducted in an Inchronel metal pot.

3 In the event that the solution at this point is dark colored, it should be clarified further by treatment with basic lead acetate followed by hydrogen sulfide [3].

4

By using the basic lead acetate clarification mentioned in note 3, the method can be used for the preparation of xylose from cottonseed hulls.

REFERENCES

[1] C. S. Hudson and T. S. Harding, J. Am. Chem. Soc. 40, 1601 (1918).

[2] W. T. Schreiber, N. V. Geib, B. Wingfield, and S. F. Acree, Ind. Eng. Chem. 22. 497 (1930).

[3] H. S. Isbell, J. Research NBS 13, 515 (1934) RP723.

XXXI. METHODS FOR THE PREPARATION OF CERTAIN SUGAR DERIVATIVES

In this chapter characteristic methods for the production of acetal and ketal derivatives, esters, ethers, glycosides, mercaptals, and oxidation and reduction products are given. A résumé of the characteristic properties of each group is followed by typical directions for the synthesis of specific compounds and a list of pertinent references. The methods are selected from those found in the literature as being of general use. No attempt has been made to give a complete bibliography or to report original work. Many of the original methods have been modified and improved in various ways. The physical constants of the products are selected from data in the literature and are not always to be found in the articles describing the method. Additional literature references are cited in the table of sugar derivatives (p. 704). Whenever possible the examples were chosen from the work of the members of this Bureau's staff. For this reason a general method applicable to any sugar may have been illustrated by a specific example for a rare and relatively inaccessible sugar.

1. ACETAL AND KETAL DERIVATIVES

General characteristics of the acetal and ketal derivatives.-The sugars and many of their derivatives condense with aldehydes and ketones to give compounds which are useful for the preparation of partially substituted sugars, and for the preparation and purification of derivatives and products of diverse types.

The configurational relationships of the hydroxyls determine in large measure whether or not a condensation will take place. The effect of configuration in relation to the particular ketone or aldehyde used has been reviewed [1, 2].

The condensation of acetone with a sugar is carried out by mixing the sugar with acetone and a dehydrating agent. Some of the dehydrating agents which have been used are hydrogen chloride [3], anhydrous copper sulfate [4], sulfuric acid [5, 6], zinc chloride [7], and phosphorus pentoxide [8]. The use of anhydrous copper sulfate rather than an acid is often desirable when glycosides are condensed