Nature of the Phospho-organic Compound formed by Yeast-Juice and Zymin from the Hexoses and Phosphate.

The formation and properties of the compound produced from phosphates in the manner just described have been investigated by Harden and Young [[1905, 2]; [1908, 1]; [1909]; [1911, 2]], Young [[1909]; [1911]], Iwanoff [[1907]; [1909, 1]], Lebedeff [[1909]; [1910]; [1911, 5], [6]; [1912, 3]; [1913, 1]]; and Euler [[1912, 1]; Euler and Fodor, [1911]; Euler and Kullberg, [1911, 3]; Euler and Ohlsén, [1911]; [1912]; Euler and Johansson, [1912, 4]; Euler and Bäckström, [1912]], but its exact constitution cannot as yet be regarded as definitely known. [p048]

Phosphates undergo this characteristic change when the sugar undergoing fermentation is glucose, mannose, or fructose, and it may be said at once that no distinction can be established between the products formed from these various hexoses; they all appear to be identical. The compound produced is, as already mentioned, not precipitated by ammoniacal magnesium citrate mixture, nor by uranium acetate solution. It can, however, be precipitated by copper acetate (Iwanoff) and by lead acetate (Young). The preparation of the pure lead salt from the liquid obtained by fermenting a sugar with yeast-juice or zymin in presence of phosphate is commenced by boiling and filtering the liquid. Magnesium nitrate solution and a small quantity of caustic soda solution are then added to precipitate any free phosphate, and the liquid well stirred and allowed to stand over night. To the neutralised filtrate lead acetate is then added together with sufficient caustic soda solution to maintain the reaction neutral to litmus, until no further precipitate is formed. The liquid is then filtered or, better, centrifugalised, and the precipitate repeatedly washed with water until a portion of the clear filtrate gives no reduction when boiled with Fehling's solution. It is essential that this washing should be thorough as evidence has recently been obtained of the formation under certain conditions of a hexosephosphate, the lead salt of which is not so sparingly soluble as that of the hexosediphosphate [Harden and Robison, [1914]]. The lead precipitate is then suspended in water, decomposed by a current of sulphuretted hydrogen, the clear filtrate freed from sulphuretted hydrogen by a current of air, and finally neutralised with caustic soda. The removal of phosphate and conversion into lead salt are repeated twice, and the resulting lead salt is then found to be free from nitrogen and to have a composition represented by the formula C₆H₁₀O₄(PO₄Pb)₂. Lebedeff carries out the preparation in a somewhat different manner. The fermentation is effected by means of air-dried yeast (150 grams to 1 litre of water, 210 grams cane-sugar and 105 grams of a mixture of 2 parts Na₂HPO₄ and 1 part NaH₂PO₄) and the liquid (about 700 c.c.) after boiling and filtering, is treated with an equal volume of acetone. About 300 c.c. of a thick liquid is precipitated and this is redissolved in water and precipitated by an equal volume of acetone two or three times. The final liquid is then precipitated with warm lead acetate solution and filtered and washed with dilute lead acetate solution until the filtrate is clear and no longer reduces Fehling's solution after removal of the lead [[1910]]. Euler and Fodor [[1911]] on the other hand precipitate the free phosphate with magnesia mixture and then add acetone, dissolve the syrup thus precipitated in water and add copper [p049] acetate solution. A blue copper salt is precipitated which is thoroughly washed with water and used for the preparation of solutions of the acid. A solution of the free acid can readily be prepared by the action of sulphuretted hydrogen on the lead salt suspended in water. It forms a strongly acid liquid, which requires exactly two equivalents of base for each atom of phosphorus present to render it neutral to phenolphthalein. It decomposes when evaporated, leaving a charred mass containing free phosphoric acid. The acid is slightly optically active, and has [a_D] = + 3·4°. A number of amorphous salts have been prepared by precipitation from a solution of the sodium salt, and of these the silver, barium, and calcium salts have been analysed with results agreeing with the general formula C₆H₁₀O₄(PO₄R′₂)₂. The magnesium, calcium, barium, and manganese salts, which are only sparingly soluble, are all precipitated when their solutions are boiled but re-dissolve on cooling, and this property can be utilised for their purification. The alkali salts have only been obtained as viscid residues.

A difference of opinion exists as to the molecular weight and constitution of this substance. Iwanoff [[1909, 1]] regards it as a triosephosphoric acid, C₃H₅O₂(PO₄H₂), basing this view on the preparation of an osazone which melted at 142°, but when recrystallised from benzene gave a product melting at 127°–8°, which had the same appearance, melting-point, and nitrogen content as the triosazone formed by the action of phenylhydrazine on the oxidation products of glycerol. Neither Lebedeff [[1909]] nor Young could obtain Iwanoff's osazone, and all attempts to reduce the acid with formation of glycerol either by sodium amalgam or hydriodic acid were unsuccessful (Young). There is therefore practically no serious experimental evidence in favour of Iwanoff's view.

On the other hand, Harden and Young regard the acid as a diphosphoric ester of a hexose. This view is based on the fact that when the acid is boiled with water, or an acid, free phosphoric acid is produced along with a levo-rotatory solution containing fructose and possibly a small proportion of some other sugar or sugars. (Euler and Fodor however did not obtain a hexose in this way [[1911]].) The acid itself only reduces Fehling's solution after some hours in the cold, rapidly when boiled, whereas when its solution is first boiled, and then treated with Fehling's solution in the cold, the products of decomposition bring about reduction in a few minutes. The reduction brought about when the acid is boiled with Fehling's solution is considerably less (33 per cent.) than that produced by an equivalent amount of glucose. The behaviour of the compound towards phenylhydrazine is also in complete agreement [p050] with this view. Lebedeff found [[1909], [1910]] that the acid or its salts heated with phenylhydrazine in presence of acetic acid gave an insoluble compound which was ultimately found to be the phenylhydrazine salt of hexosemonophosphoric acid osazone

C₆H₅NH·NH₂·H₂PO₄·C₄H₅(OH)₃·C(N₂HC₆H₅)CH(N₂HC₆H₅)

[Lebedeff, [1910]; [1911, 6]; Young, [1911]]. After recrystallisation from alcohol this compound forms yellow needles, melting at 151°–152°. It is decomposed by caustic soda yielding a sodium salt

Na₂PO₄·C₄H₅(OH)₃·(CN₂HC₆H₅)·CH(N₂HC₆H₅)

and on boiling with caustic soda decomposes giving a hexosazone (free from phosphorus) which is probably glucosazone, and in addition glyoxalosazone, probably as the result of a secondary decomposition. Towards acids it is remarkably stable yielding with hydrochloric acid a hexosonephosphoric ester from which the original osazone can be regenerated (Lebedeff). Lebedeff at first [[1910]] argued from the formation of this osazone that the original hexosephosphate contained only one phosphoric acid group per molecule of hexose. It was however shown by Young [[1911]] and subsequently confirmed by Lebedeff [[1911, 6]] that one molecule of phosphoric acid is split off during the formation of the osazone, even in neutral solution. Moreover it has been found that in the cold hexosediphosphoric acid reacts with 3 molecules of phenylhydrazine forming the diphenylhydrazine salt of hexosediphosphoric acid phenylhydrazone

(C₆H₅NH·NH₂·H₂PO₄)₂·C₆H₇(OH)₃·N₂HC₆H₅.

This compound crystallises out when 1 volume of alcohol is added to a solution of 3 molecules of phenylhydrazine in one of the acid and forms colourless needles melting at 115°–117°. p-Bromophenylhydrazine yields an analogous compound melting at 127°–128°.

Precisely the same products are given with phenylhydrazine by the hexosephosphoric acid prepared from glucose, mannose, and fructose, proving that all these sugars yield the same hexosediphosphoric acid, a point of fundamental importance.

Direct measurements of the molecular weight of the acid by the freezing-point method, combined with the determination of the degree of dissociation by the rate of cane-sugar inversion, are indecisive, but indicate that the acid has a molecular weight considerably higher than that required for a triosephosphoric acid.

A similar uncertainty attaches to the determination of the molecular weight from the freezing-point depression and conductivity of the acid potassium salt [Euler and Fodor, [1911]]. Euler however concludes [p051] that both a hexosediphosphoric acid and a triosemonophosphoric acid are formed, but has not prepared any derivatives of the latter.

As regards the constitution of the hexosephosphoric ester several suggestions have been made by Young, but no decisive evidence at present exists. The identity of the products from glucose, mannose, and fructose may be explained by regarding the acid as a derivative of the enolic form common to these three sugars (p. [97]), or by supposing that portions of two sugar molecules may be concerned in its production. The formation and composition of the hydrazone and osazone are of great importance as they indicate that in all probability one of the phosphoric acid residues is united with the carbon atom adjacent to the carbonyl group of the hexose. They moreover render it certain that the original phosphoric ester is a hexosediphosphoric ester and not a triosemonophosphoric ester.

Hexosediphosphoric acid has not as yet been discovered in the animal body. The action of a number of enzymes upon it has been examined [Euler, [1912, 2]; Euler and Funke, [1912]; Harding, [1912]; Plimmer, [1913]] with the following results.

The lipase of castor oil seeds, a glycerol extract of the intestinal mucous membrane of the rabbit and pig, and an aqueous extract of bran have a slow hydrolytic action, whereas pepsin and trypsin are without effect. Feeding experiments with rabbits and dogs indicate that the ester is capable of hydrolysis in the animal body, a large proportion of the phosphorus being excreted as inorganic phosphate. The ester is also decomposed by Bacillus coli communis.

It is remarkable that the hexosephosphate is not fermented nor hydrolysed by living yeast, a fact observed by Iwanoff, Harden and Young, and Euler, although, according to the experiments of Paine [[1911]], the yeast cell is at all events partially permeable to the sodium salt.