PHYTIN AND PHOSPHORIC ACID ESTERS OF INOSITE.

BY R. J. ANDERSON.

(From the Chemical Laboratory of the New York Agricultural Experiment Station, Geneva, N. Y.)

(Received for publication, April 2, 1912.)

In continuation of the physiological investigation concerning the metabolism of the organic-phosphorus compound known as phytin, which has been and is being carried out at this institution by Dr. Jordan, a closer study of the chemical properties of this substance, phytin, became necessary. Much work has already been done and reported on this subject by various investigators. Definite information, however, concerning different kinds of salts formed by the free phytic acid or inosite phosphoric acid is seldom met with in the literature. Frequently impure salts have been analyzed.

Posternak, who first successfully prepared phytin in pure form,l also studied its chemical’properties. Among the salts mentioned2 is one, calcium-magnesium, as well as one crystalline, calcium- sodium, double salt, for which he gives the formula, 2C2H4Pz09Na4 +C2H4PzCaz + 8H20. Winterstein describes a calcium-magne-

sium compound which, after removing the calcium with oxalic acid and precipitating with alcohol, contained 42.24 ‘per cent PZOs and 12.97 per cent MgO. Patten and Hart,4 working in this laboratory, isolated from wheat bran an impure magnesium- calcium-potassium compound. Levene5 describes a semi-crystal- line barium salt which corresponds to a tetra-barium phytate. Vorbrodt! mentions a crystalline barium salt obtained by partially

1 Rev. gkn. de bot., xii, p. 5; Compt. rend. acad. des xi., cxxxvii, p. 202. 2 Compt. rend. acad. des xi., cxxxvii, pp. 337 and 439. 8 Ber. d. d. them. Gesellsch., xxx, p. 2299. 4 Amer. Chem. Journ., xxxi, p. 566. 6 Biochem. Zeitschr., xvi, p. 399. 6 Anzeiger Akad. Wiss. Krakau, 1910, Series A, p. 414.

471

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472 Phytin and Esters of Inosite

neutralizing phytic acid with barium hydroxide and evaporating in vacuum, to which he assigns the formula, C12H26046Ba7Pll. Although crystalline, this compound was undoubtedly impure. By neutralizing the mother-liquor from the above with barium hydroxide he obtained an amorphous precipitate of the composi- tion C, 5.75; H, 0.77; Ba, 52.97; P, 11.60 per cent. This corre- sponds approximately with a hexa-barium phytate.

Of the several salts mentioned in this paper some were obtained from commerbial phytin and from an organic-phosphorus-mag- nesium compound by precipitating with barium chloride and barium hydroxide; others were prepared from previously purified phytic acid. These products will be more fully described in the experimental part.

The tri-barium phytate, CsH1209[(P0~H)z Ba18, is obtained pure as an amorphous white powder by repeatedly precipitating barium phytate in 0.5 per cent hydrochloric acid with a like volume of alcohol. It may also be obtained in crystalline form by dissolving the amorphous salt in a 10 per cent solution of phytic acid in which it is very soluble and from which it again slowly crystallizes out on standing at ordinary temperature.

A penta-barium phytate, C6H14027PsBai, is obtained when a solution of the tri-barium phytate in 0.5 per cent hydrochloric acid is neutralized with barium hydroxide and then made faintly acid with acetic acid.

The penta-barium ammonium phytate, CsH12027P6Ba5(NH&, is obtained when the above mentioned amorphous tri-barium salt is digested with dilute ammonia.

Thepenta-magnesiumammoniumphytate,CBH,2027PsMgS(NH1)2, is thrown down as a white amorphous precipitate when excess of magnesia mixture is added to an aqueous solution of phytic acid, or when ammonium phytate is precipitated with magnesia mixture.

A tetra-cupric di-calcium phytate, CsHlzOzrPsCu&az, in nearly pure form is obtained when a slightly acid solution of calcium ammonium phytate is precipitated with excess of copper acetate. If the magnesium ammonium phytate is precipitated under the same conditions an impure compound is obtained which contains about 1 per cent Mg, 0.6 per cent N, 34 per cent Cu and 15.6 per cent P. No effort was made to obtain these salts pure. It was

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R. J. Anderson 473

only desired to find out to what extent other bases were removed when precipitating with copper acetate.

Starkenstein’ claims that commercial phytin always contains free inosite together with inorganic phosphates and that merely drying the substance at 100°C. causes nearly complete decomposi- tion into inorganic phosphate and free inosite.

That phytin is so easily decomposed seemed very improbable as several months’ work on the substance has shown that it is

relatively stable when pure and when no miheral acids are present, Moreover Contardi8 reports that when phytin is heated in an auto- clave with pure water for several hours to a temperature of 200°C. only very small quantities of inosite could be isolated.

In order to determine if inosite is present in determinable quantity 100 grams of commercial phytin in the form of the acid calcium salt, imported from Europe and which had been kept, in the laboratory for several years, was shaken up with 1 liter of water, filtered at once and washed with water. The filtrate was precipitated with barium hydroxide, again filtered and the excess of barium precipitated with carbon dioxide and the filtrate from the latter evaporated on the water-bath. In the very slight residue which remained, consisting mostly of barium carbonate with a trace of barium chloride, no trace of inosite could be detected by the most painstaking method of isolation. Of the same phytin, lQ0 grams were dried to constant weight at 115’C. and was then treated in the same manner. Even here no trace of inosite could be obtained. Subjecting to the same treatment 50 grams of the same phytin, after previously mixing with 0.5 gram ihosite, resulted in the recovery of 0.4 gram inosite.

This proves that phytin is by no means so easily split as Starken- stein claims. The results in his case may have been due to other causes besides mere drying at 100°C.

The same author (Zoc. cit.) also states that when phytic acid is precipitated with ammoniacal magnesia mixture it is not the mag- nesium ammonium compound which is formed but only the diffi- cultly soluble magnesium phytate. This is an error. Under these conditions the previously mentioned penta-magnesium ammonium phytate, CsH12027PaMg,(NH&, is formed.

For the free phytic acid Pbsternakg proposed the empirical for- mula, CzHs09Pz, which he considered to have the following con- stitution:

7 Biochem. Zeitschr.. xxx, p. 59. 8 Atti R. Accad. dei Lincei Roma (5), xviii, i, p. 64. 9 Compt. rend. acad. des sci., cxxxvii, p. 439.

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474 Phytin and Esters of Inosite

H I OjCHO. po (OHI

‘$THO. PO (OH)3

and which finds expression in the name “anhydro-oxymethylen di-phosphoric acid.

As is well known the free acid, as well as its salts, is ‘easily split under the influence of dilute mineral acids into inosite and ortho- phosphoric acid. This fact and the discovery by NeuberglO that both inosite and phytin yield furfurol when distilled ‘with phos- phorus pentoxide and phosphoric acid, respectively, lead him to believe that the inosite ring exists already formed in phytin. In accordance with this view he proposed the following structural formula fop the acid:

/O\ / \ (OH)aP $(OHJ3

CH-CH

(OH>3 1 1 (OH)3

P- 0-CH CH-O-P

O< II > (OH), P-0-CH-CH-O-P (OH)3

This is just treble the molecular weight of the anhydro-oxymeth- ylen di-phosphoric acid of Posternak.

Suzuki and Yoshim;rall considered that phytic acid was the hexa-phosphoric acid est.er of inosite.

Starkenstein12 believes that phytin represents a cqmplex pyro- phosphoric acid compound with inosite and he proposes the follow- ing constitutional formula:

lo Biochem. Zeitschr., ix, pp. 551 and 557. I1 Bull. Coil. of Agric. Tokyo, vii, p. 495.

I2 Biochem. Zeitschr., xxx, p. 56.

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R. J. Anderson 475

(OH)2 (OHIs P=O.HO.HC-CH.OH.O=P

I 10 /

(0% P=O.HO.HC CH.OH.O=P

I I

(OH)2

(OH)2Pq0.H0.HC-CH.OH.O=P (OH)2

\

\

\

/

0

Vorbrodt (Zoc. tit.) proposes still another formula. It is impossible at the present time to decide definitely beiweeri

any of the above constitutional formulas, as the substance has not, yet been synthesized in the laboratory.

As represented by the empirical formula, CsH2402,Pe, phytic acid corresponds to a hexa-phosphoric acid ester of inosite plus 3H20, CsHsOti PO(OH)zle, + 3H20.

At present it is impossible to say whether the compound repre- sents a pyrophosphate or if the water is linked in some other way. That the acid contains twelve acid (OH) groups as expressed in the formula of Starkenstein, which wouldalso be the case if it were a hexa-phosphoric acid ester of inosite, and not eighteen (OH) groups as in the formula of Neuberg, seems certain, for in no case have we been able to prepare any salt in which more than twelve H- valences were replaced by bases.

As observed by Starkenstein only one-half of the twelve (OH) groups are particularly reactive. This finds expression in the fact that the barium salt obtained from .acid solutions contains only 3Ba to 6P. As suggested by the above author, it is probable that these reactive hydroxyls are adjacent but linked to different phosphoric-acid residues. The salts with binary bases would then be represented by the following:

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476 Phytin and Esters of Inosite

-OH

-P=O -0

> M

-0 -P=O

-OH

A further confirmation of this is found in the fact that the tri- barium phytate as well as other similar salts of phytic acid with binary bases are strongly acid in reaction.

The presence of only eight acid (OH) groups, however, can be shown by titrating an aqueous solution of the acid with decinor- ma1 sodium hydroxide using phenolphthalein as indicator. Patten and Hart (Zoc. cit.) who titrated with decinormal barium hydroxide using phenolphthalein as indicator obtained results agreeing with a hexa-barium salt.

Of special interest in connection with the constitution of phytin are the phosphoric acid esters of inosite.

Neuberg and Kretschne9 report obtaining a poly-phosphoric acid ester of inosite by their method of preparing phosphoric acid esters of the carbohydrates and glycerine, that is, by the action of phosphorus oxychloride. The product however, could not be obtained pure as it was found impossible to separate it from the inorganic phosphates.

Contardi’J claims to have prepared the hexa-phosphoric acid ester of inosite by heating inosite with an excess of phosphoric acid in a stream of carbon dioxide to 160° to 165°C. The product was purified as the barium salt and after decomposing the latter with sulphuric acid the free ester was obtained, which he describes as identical with phytic acid. The same authorI claims to have prepared poly-phosphoric acid esters of mannite, quercite and glucose by the same method. .

Carre,‘6 however, repeating these experiments found that the products described by Contardi were merely mixtures of free phos- phoric acid and the polyhydric alcohols in question together with

I3 Biochem. Zeitschr., xxxvi, p. 5.

I4 Atti. R. Acead. dei Lincei Roma, (5), xix, i, p. 23. 15bid., p. 823. I6 Bull. sot. chim. de France, (4), ix, p. 195.

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their decomposition products mixed with some monobarium phos- phate.

Many fruitless efforts have been made in this laboratory to syn- thesize phytic acid and the hexa-phosphoric acid ester of inosite. All experiments in this direction lead only to the tetra-phosphoric acid ester of inosite, CsH,(OH)204 [PO(OH)&.

The method of Contardi was modified to the extent that inosite, either dry or with water of crystallization;was heated with phos- phoric acid, previously dried at 100°C. to constant weight, in vacuum to a temperature of 140’ to 160°C. for about two hours.

The same product, viz., the tetraphosphoric ester was obtained whether the phosphoric acid was present in large or small excesss above six molecules of H3P04 to one molecule of inosite. When it was present in less quantity than this, however, for instance one molecule of inosite to three molecules of H3P04, then a mixture of esterswas formed. It was found impossible to separate these prod- ucts completely owing to the fact that they possess about the same solubility.

The tetraphosphoric ester is most conveniently isolated by means of its barium salt. The separation of the ester from the excess of the phosphoric acid or barium phosphate succeeded because its barium salt is much less soluble in dilute alcohol acidified with hydrochloric acid than is barium phosphate.

The new ester is a well characterized compound, very similar in appearance and reactions to phytic acid. By heating with acids, inosite and phosphoric acid are regenerated. It gives a white precipitate with the ordinary molybdate solution, and with excess .of silver nitrate a white precipitate is also produced. These reac- tions are identical with those of phytic acid.

The inosite used in these experiments was prepared from the crude magnesium compound previously mentioned and carefully purified by recrystallization.

The reason why phytic acid could not be obtained by the action of phosphoric acid on inosite is no doubt to be found in that it is not a simple ester but a complex compound as suggested by’starkenstein. It is, however, difficult to understand why the hexa-phosphoric ester was not obtained by’this method. The only explanation that can be offered is that under the conditions of these experiments it is not stable.

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One reason alleged by Starkenstein for considering phytin a pyrophosphate is based upon its giving a white precipitate with silver nitrate. This is certainly a characteristic reaction of pyro- phosphates. Yet the tetraphosphoric ester gives a pure white precipitate with the same reagent. As the ester cannot be in. the form of a pyrophosphate the fact that phytic acid gives the same colored silver compound is not necessarily an indication that it pepresents a pyrophosphate compound.

The phytic acid used in these experiments was prepared from products obtained from two different sources. The starting mate- rial in one case was a calcium phytate imported from Europe; the other was a crude natural magnesium organic phosphorus compound extracted in this country and kindly supplied us by Dr. Carl S. Miner of Chicago.

As shown by the analyses of the carefully purified salts and of the free acid, these two preparations were identical and they were also identical with the product described as phytic acid by Poster- nak and other investigators.

EXPERIMENTAL PART.

Tri-barium phytate.

The commercial phytin was purified for analysis by means of the barium salt. Thirty grams calcium phytate were dissolved in a small quantity of 0.5 per cent, hydrochloric acid, diluted to about 2 liters with’ water and a concentrated solution of 30 grams barium chloride was added. The precipitate was dissolved w&hout fil- tering by the addition of just sufficient dilute hydrochloric acid. It was then precipitated by adding barium hydroxide to faintly alkaline reaction. The mixture was then acidified with acetic acid and after standing over night was filtered and well washed in water. It was re-precipitated in the same manner three times. After finally filtering and washing in water the substance was dis- solved in about 1 liter of 0.5 per cent hydrochloric acid, filtered and the filtrate precipitated by adding a like volume of alcohol. After repeating this operation the substhnce was filtered, washed free of chlorides with 50 per cent alcohol and finally washed in alcohol and ether and dried in vacuum over sulphuric acid.

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The product so obtained was a light, perfectly white semi-crys- talline or amorphous powder. Placed on moist litmus paper, it showed a strong acid reaction. It is very slightly soluble in water, slightly soluble in acetic acid and readily soluble in mineral facids.

For analysis the substance was dried at 130°C.

0.2728 gram substance gave 0.0352 gram Hz0 and 0.0643 gramCO*. 0.2763 gram substance gave 0.1749 gram BaSOd and 0.1675 gram Mg,PIO,. 0.1909 gram substance gave 0.1206 gram BaSOa and 0.1154 gram Mg,P20,. For C6H120J(P03H)2Ba]a = 1120. Calculated: C = 6:42; H = 1.60; P = 16.60; Ba = 36.78 per cent. Found : C = 6.42; H = 1.44; P = 16.89; Ba = 37.25 per cent.

P = 16.85; Ba = 37.17 per cent.

The barium salt prepared in the same manner from a natural crudemagnesium organic phosphorus compound gave the following result on analysis:

0.2057 gram substance gave 0.0273 gram Hz0 and 0.0480 gram COs. 0.1422 gram substance gave 0.0886 gram BaSOl atid 0.0841 gram. Mg,P1O,. Found: C = 6.36; H = 1.48; P = 16.48; Ba = 36.66 per cent. ’

The two salts are therefore identical.

Crystallized tri-barium phytate.

One gram purified phytic acid was dissolved in 10 cc. water and 4 grams of the above mentioned tri-barium phytate added. It was filtered from traces of undissolved particles and allowed to stand for two days at room temperature. The substance has then sepa- rated as a heavy crystalline powder of irregular form. From less concentrated solutions the substance separates in small, needlc-

shaped crystals. The substance was filtered, washed well in water and finally in

alcohol and ether and dried in the air. For analysis it was dried at 120%.

0.1972 gram substance lost 0.0153’gram HzO. 0.2028 gram substance gave 0.1251 gram BaSO( and0.1216 gram Mg2PZ07. Found: P = 16.71; Ba = 36.30 per cent. Calculated for 5 HzO: 7.44; Found: 7.75 per cent.,

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Penta-barium phytate.

This salt is obtained on neutralizing a solution of the tri-barium phytate in 0.5 per cent hydrochloric acid with barium hydroxide and then acidifying with acetic acid. The precipitate was filtered, washed thoroughly in water, alcohol and ether and dried in vacuum over sulphuric acid.

The product was a white amorphous powder. For analysis the substance was dried at 130°C.

0.2970 gram substance gave 0.0307 gram H20 and 0.0500 gram CO,. 0.2507 gram substance gave0.2080 gram BaSOl and 0.1207 gram Mg2P207.

0.1856 gram substance gave 0.1543 gram BaSOl and 0.0899 gram MglPzOi For C6H1402rP6 Bas = 1391. Calculated: C = 5.17; H = 1.00; P = 13.37; Ba = 49.37 per cent.

Found: C = 4.59; H = 1.15; P = 13.42; Ba = 48.82 per cent.

P = 13.50; Ba = 48.92 per cent.

Penta-barium ammonium phytate.

When the tri-barium phytate is digested in dilute ammonia it is transformed into the penta-barium ammonium salt and ammo- nium phytate. The latter product, however, was found to contain some barium.

Two grams of the analyzed tri-barium phytate were digested for two hours in 25 cc. of 2.5 per cent ammonia, filtered and washed in dilute ammonia and finally in alcohol and dried in vacuum over sulphuric acid. The product was a heavy white amorphous pow- der. On moist litmus paper it showed a neutral reaction.

For analysis the substance was dried at 130°C.

0.1509gramsubstancegave0.1~5gramBaSO~and0.~762gramMg2P207. 0.1747 gram substance gave 0.0026 gram N (Kjeldahl).r>

For CsHlzOzrPsBas (NHJ2 = 1425. Calculated: P = 13.05;Ba = 48.19; N = 1.96percent. Found : P = 14.07;Ba = 46.99; N = 1.48 per cent.

By evaporating the filtrate from the above to dryness on the water-bath an amber-colored mass remained which after drying at 13O’C. gave the following result on analysis:

Found: P = 20.51; Ba = 6.65; N = 10.48 per cent.

17 This and subsequent nitrogen determinations were made by Mr. M. P. Sweeney.

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R. J. Anderson 481

Penta-magnesium ammonium phytate.

Two grams phytic acid were dissolved in 400 cc. water and then precipitated by adding excess of magnesia mixture slowly and under constant shaking. After the precipitate had settled the super- natant liquid was decanted, the residue filtered and washed with water until free from chlorides and finally washed in alcohol and ether and dried in vacuum over sulphuric acid.

The product was a fine white amorphous powder and weighed 2.7 grams. It reacts neutral on moist litmus paper. For analysis

it was dried at 130°C.

0.1089 gram substance gave 0.0832 gram Mg2P20T for P. 0.1089 gram,substance gave 0.0705 gram Mg,PrOr for Mg. 0.1248 gram substance gave 0.0039 gram N \ 0.0893 gram substance gave 0.0028 gram N ( (Kjeldahl). For CsHlzOa,PcMga (N&)2 = 859.5. Calculated: P = 21.e; Mg = 14.13; N = 3.25 per cent. Found : P = 21.29; Mg = 14.13; N = 3.12 - 3.13 per cent.

If the phytic acid is first neutralized with ammonia and then precipitated with magnesia mixture the same product is obtained.

Two grams phytic acid in 400 cc. water were neutralized with ammonia, precipitated with excess of magnesia mixture, filtered, washed free of chlorides with dilute ammonia and then in alcohol and dried in vacuum over sulphuric acid. For analysis the sub- stance was dried at 130°C.

, Found: P = 21.49; Mg = 13.96; N = 3.47; 3.48 per cent.

Tetra-cupric c&calcium phytate.

To a solution of 2 grams phytic acid in 200 cc. water excess of .calcium chloride was added and the solution then neutralized with ammonia. The precipitate was just dissolved in dilute hydro- . chloric acid and the solution precipitated with copper acetate. The bluish-green colored copper compound was filtered off, washed with water until free from chlorides and then in alcohol and dried in vacuum over sulphuric acid.

The dry substance was a light-blue amorphous powder. It is very slightly soluble in water or in very dilute acids, readily soluble

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in the ordinary dilute mineral acids. It is readily soluble in 2.5 per cent ammonia with a deep-blue color. In this solution con- centrated ammonia or alcohol produces a light-blue colored pre- cipitate.

The compound represents a nearly pure tetra-cupric di-calcium phytate. It contained 0.17 per cent N.

For CaH1z09 (PO&I&. (PO&a)2 = 1036. Calculated: Cu = 24.51; Ca = 7.72; P = 17.95 per cent. Found : Cu = 25.58; Ca = 7.69; P = 16.85 per cent.

If a slightly acid. solution of magnesium ammonium phytate is precipitated with copper acetate a light blue colored copper com- pound is obtained. After washing and drying it gave the follow- ing result on analysis:

Mg = 1.11; Cu = 34.27; N = 0.64 and 0.52; P = 15.66 per cent.

This -compound is exceedingly soluble in dilute and concentrated ammonia. By the careful addition of.alcohol to the ammoniacal solution a substance separates in light blue colored crystals on standing. This is evidently a complex copper-ammonium. salt but it was not further examined.

Phytic acid.

This was prepared after the method of Patten and Hart (Zoc. cit.). The analyzed tri-barium salt was decomposed with the cal- culated quantity of decinormal sulphuric acid. After removing the barium sulphate, the solution was precipitated with copper acetate. The copper compound was decomposed with hydrogen sulphide, the copper sulphide filtered off, the filtrate concentrated in vacuum and finally dried in vacuum over sulphuric acid. The products obtained from both the calcium phytate and the magne- sium compound were light amber colored, very thick liquids and

. corresponded in all respects with the body described by other inves- tigators as phytic acid.

For analysis the substance was dried at 13O’C.

a. From calcium phytate.

0.3193 grim substance gave 0.0917 gram Hz0 and 0.1238 gram CO*. 0.1505 gram substance gave 0.1424 gram Mg,P20,.

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b. From the magnesium compound.

0.2789 gram substance gave 0.0804 gram Hz0 and 0.1101 gram CO,. 0.1236 gram substance gave,0.1169 gram MgzPzOr. For CsH24 OerPs = 714.

calculated: Found: Found: \ I

c. . . . . . . . . . . . . . . . . ..10.03 10.57 1Or;S H . . . . . . . . _ . . . . . . . . . . . . . . . . . 3.36 - 3.21 3.22 P . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.05 26.37 26.16

Titrated against decinormal sodium hydroxide using phenolphthalein as indicator the following results were obtained:

0.2648 gram acid required 30.7 cc. s NaOH. Calculated for 8NaOH: 29.65 cc. 0.1593 gram<acid required 18.60 cc. $ NaOH. Calculated for 8NaOH: 17.60 cc.

Inosite from the crude magnesium compound.

Twenty-five grams of the air-dried substance, containing 20 per cent of moisture, was heated with 100 cc. of 30 per cent sul- phuric acid in a sealed tube for about three hours at a temperature of 140°C. Two tubes equally charged were heated at the same time. After cooling the reaction mixture was of dark brown color and a considerable quantity of magnesium salts had crystallized out.

The contents was washed into a beaker, filtered and diluted with water to about 1500 cc. The sulphuric and phosphoric acids and the magnesium were then precipitated by barium hydroxide, filtered and well washed in hot water. The filtrate was evaporated to about 350 cc. and the excess of barium removed by carbon dioxide, filtered, the filtrate decolorized with animal charcoal and then evap- orated on the water bath to a syrupy consistency. This was taken up in a small quantity of hot water, filtered and alcohol added to the filtrate until a cloudiness was produced. By scratching with a glass rod crystallization began; more alcohol was then added and the mixture placed in the ice-chest over night. After filtering and washing in alcohol and ether and drying in the air the product weighed from 5.1 to 5.4 grams. From the mother liquor a further quantity of crystals from 0.4 to 0.6 gram could be obtained on the addition of ether and allowing to stand for twenty-four hours in the cold.

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484 Phytin and Esters of Inositk

For purification the raw product was dissolved in six parts of water and again brought to crystallization by the addition of alco- hol as before. ,It was then obtained in large, thin, colorless plates.

It gave the reaction of Scherer. The dried substance melted at 220°C. (uncorrected).

Dried at lOO”C., 0.4136 gram substance lost 0.0669 gram HZ0 and 0.1600 gram lost 0.0258 gram HZO.

The dried substance was analyzed.

0.1342 gram substance gave 0.0791 gram H20 and 0.1981 gram CO,. For C&He (OH)8 = 180. Calculated: C = 40.00; H = 6.66; 2HzO = 16.66 per cent. Found : C = 40.26; H = 6.59; 2HzO = 16.17 - 16.12 per cent.

This substance was used in subsequent experiments with phos- phoric acid. Some 40 grams of inosite were prepared in this way.

Tetra-phosphoric acid ester of inosite.

Crystallized inosite (4.32 grams, 2 molecules) was powdered and mixed in a distillation flask with 24 grams phosphoric acid (about 24 molecules or double the quantity required to form the hexa- phosphoric ester). The acid had been previously dried at 100°C. to constant weight. The flask was connected with the vacuum pump and heated in an oil bath to 140” to 160°C. for about two hours. By 120” water began to come over and the reaction was practically complete at the end of one hour. After cooling the reaction mixture was a thick, reddish-brown colored, nearly solid mass. This was dissolved in about 1 liter of water and a solution of 40 grams of barium chloride in 400 cc. of water was added. The barium salt of the ester was then precipitated by the addition of about 1 liter of al’cohol.

A solution containing phosphoric acid and barium chloride in the same dilution as above remains perfectly soluble on the addi- tion of a like volume of alcohol.

.The voluminous flaky precipitate was filtered off at once and thoroughly wagbed in 333 per cent alcohol.

For purification the substance was dissolved in 700 cc. of 0.5 per cent hydrochloric acid, filtered from slight insoluble residue, the filtrate diluted with 500 cc. of water, some barium chloride added and then precipitated by the addition of a like volume of alcohol. This was repeated a second time. The substance was

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then dissolved in 500 cc. of 0.5 per cent hydrochloric acid, precipi- tated by adding barium hydroxide to slightly alkaline reaction, then acidifying with hydrochloric acid and adding 500 cc. alcohol. After filtering and washing as before the substance was again twice precipitated from 0.5 per cent hydrochloric acid solution with alcohol and finally washed in 50 per c&t alcohol, alcohol and ether and dried in vacuum over sulphuric acid. The product weighed 8.9 grams. It was a white voluminous amorphous powder. On moist litmus paper it showed a strong acid reaction. The solu- bility of the product was practically the same as forthe tri-barium phytate.

For analysis it was dried at 100” and 13O’C.

0.3252 gram substance lost 0.0281 gram H20.

0.2697 gram substance gave0.0442 gram Hz0 and0.0878 gram CO,. 0.2038 gram substance gave0.0300 gram Hz0 and0.0685 gram COS. 0.2482 gram substance gave 0.1505 gram BaSOa and 0.1434 gram MgZP20?. 0 1833 gram substance gave 0.1108 gram BaSO* and 0.1075 gram Mg,P*O,.

0.1776 gram substance gave 0.1074 gram BaS04 and 0.1038 gram Mg2P20,. For G,Hs{OH)zOa[(POaH)2 Ba]t = 770.7. Calculated: C = 9.34; H = 1.55; P = 16.08; Ba = 35.64 per cent.

Found : C = 8.87; H = 1.83; P = 16.10; Ba = 35.68 per cent. C = 9.16; H = 1.64; P = 16.34; Ba = 35.57 per cent.

P = 16.29; Ba = 35.58 per cent. calculated for 4 HzO: 8.55; Found: 8.64 per cent.

Another lot prepared by heating 1.80 grams dry inosite (1 mole- cule) with 7.9 grams dry phosphoric acid (about 8 molecules) and isolated in the same manner gave the following results on analysis :

0.2879 gram substance lost 0.0240 gram HzO. The dried substance was analyzed.

,0.2639 gram substance gave 0.0452 gram Hz0 and 0.0936 gram CO%. 0.1480 gram substance gave 0.0866 gram BaSOn and0.0846 gram Mg2PsOT. 0.1632 gram substance gave 0.0959 gram BaS04 and 0.0933 gram MgtP20,.

Found: C = 9.67; H = 1.91; P = 15.93; Ba = 34.43 per cent. Hz0 = 8.33; P = 15.93; Ba = 34.58 per cent.

A third lot prepared by heating 1.80 grams dry inosite (1 mole- cule) with 5.88 grams dry phosphoric acid (6 molecules) and isolat- ing in the same manner as before gave the following:

C = 9.69; H = 1.75; P = 16.06; Ba = 36.33 per cent.

It is apparent therefore that in each of the above experiments the same compound was produced.

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486 Phytin and Esters of Inosite

The free tetra-phosphoric ester.

About 5 grams of the purified barium salt was decomposed by digesting it with the calculated quantity of decinormal sulphuric acid. After removing the barium sulphate the solution was pre- cipitated with excess of copper acetate. The copper precipitate was filtered, thoroughly washed with water, suspended in water and decomposed with hydrogen sulphide. The copper sulphide was removed by filtration, the filtrate concentrated in vacuum and finally dried in vacuum over sulphuric acid until it was of a thick, syrupy consistency.

For analysis the substance was dried at 130°C.

0.3020 gram substance gave 0.0933 gram Ha0 and 0.1577 gram COz. 0.1605 gram substance gave 0.01387 gram Mg,P20,. For C~HG(OH)~ 0, [PO(OH)& = 500. Calculated: C = 14.40; H = 3.20; P = 24.80 per cent. Found : ’ C = 14.24; H = 3.45; P = 24.09 per cent. 0.1663 gram substance required 16.5 cc. decinormal sodium hydroxide

using phenolphthalein as indicator. This corresponds to five acid (OH) groups.

Calculated for 5NaOH: 16.63 cc.

Properties of the free ester.

The concentrated aqueous solution of the ester is very similar to phytic acid. It is a very thick amber-colored liquid of sharp acid, slightly astringent taste and strong acid reaction. On longer keeping in the desiccator over sulphuric acid it becomes hard and brittle and may be powdered. It is then very hygroscopic.

The dry substance is slowly but completely soluble in alcohol, readily soluble in water.

The concentrated aqueous solution gives a white precipitate with silver nitrate in excess which dissolves on largely diluting with water. The precipitate is readily soluble in ammonia, dilute nitric, sulphuric and acetic acids, insoluble in glacial acetic acid.

With ferric chloride it gives a white or faintly yellowish precipi- tate which is very sparingly soluble in acids..

With lead acetate a white precipitate is produced, readily soluble in dilute nitric acid.but sparingly soluble in acetic acid.

With barium chloride it gives a white precipitate slightly soluble in acetic acid but readily soluble in hydrochloric and nitric acids.

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R. J. Anderson 487

Calcium chloride does not give a precipitate but on heating the calcium salt is thrown down as a white precipitate which redissolves on cooling.

Magnesium salts do not cause a precipitate and on heating the solut.ion merely turns cloudy; on cooling it clears up again.

With the ordinary molybdate solution it gives in the cold a white voluminous flaky precipitate which slowly turns yellowish in color. Phytic acid under the same conditions gives a white precipitate which remains unchanged in the cold. On drying at 110’ or 130’ the substance turns very dark in color.

The ester, like phytic acid, fails to give directly the Scherer reaction for inosite.

Inosite from the tetra-phosphoric ester.

Ten grams of the purified barium salt was heated with 25 cc. 30 per cent sulphuric acid in a sealed tube to about 150°C. for three hours. After precipitating the sulphurii: and phosphoric acids with barium hydroxide the inosite was isolated by the usual method and recrystallized from hot dilute alcohol. It was filtered and washed in alcohol and ether and dried in the air. Yield, 1.52 grams. It was obtained in the form of small colorless six-sided plates, free from water of crystallization.

The air-dried, water-free substance melted at 221’C. (uncor- rected.)

0.2094 gram substance gave 0.1259 gram HtO and 0.3033 gram COS. 0.1360 gram substance gave 0.0827 gram Hz0 and 0.1991 gram CO,. For CBHIZO~ = 180.

Calculated: C = 40.00; H = 6.66 per cent. Found : C = 39.50; H = 6.72 per cent.

C = 39.93: H = 6.80 per cent.

As already mentioned, if a mixture of inosite and phosphoric acid is he&ted when less than six molecules &PO4 are present to one molecule inosite, a mixture of esters is obtained. It was found impossible to separate these bodies as barium salts and obtain pure compounds since their solubilities are acparently nearly alike.

Dry inosite (3.60 grams, 2 molecules) and 5.88 grams dry phos- choric acid (6 molecules) was heated in a distillation flask as before to 180’ to 190’ for about two hours, until water ceased coming over. The reaction mixture was in the form of a very bulky, thin

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488 Phytin and Esters of Inosite

flaky mass, very brittle and of yellowish-brown color, mixed with some very dark-colored substance. It was broken up with a glass rod and removed from the flask and treated with water in which the dark-colored portion was readily soluble, but the lighter-colored substance was insoluble in this medium. It was powdered in a mortar and thoroughly washed in water and alcohol and dried in vacuum over sulphuric acid.

The substance was apparently insoluble in boiling water, in boiling dilute acids and in glacial acetic acid; also insoluble in alco- hol, ether and other organic solver&. After drying at 130” the substance was analyzed.

0.2500 gram substance gave 0.0838 gramHz0.and0.2085 gramCOt. 0.1500 gram substance gave 0.1145 gram Mg,PzO,. 0.1542 gram substance gave 0.1178 gram MgzP20,.

Found: C = 22.74; H = 3.75; P = 21.28; 21.29 per cent.

This agrees approximately with a mono-pyro-phosphoric ester of inosite but the phosphorus is too high.

It was decided to purify it by means of the barium salt. The substance was dissolved by boiling in dilute sodium hydroxide in which it gave a dark amber colored solution. After filtering, it was precipitated with barium chloride, the barium precipitate filtered and washed free of alkali. It was then dissolved in 500 cc. 0.5 per cent hydrochloric acid and precipitated by barium hydrox- ide. After filtering and washing it was repeatedly precipitated with alcohol from 0.5 per cent hydrochloric acid solution until finally a small amount of a white amorphous powder was obtained. After drying at 130” this was analyzed.

0.2028 gram substance gave 0.0412 gram Hz0 and 0.0979 gram CO*.

0.2207 gram substance gave 0.0413 gram Hz0 and 0.1042 gram CO,. 0.1982 gram substance gave 0.0996 gram BaSOl and 0.1103 gram Mg2Pz07. Found: C = 13.16; H = 2.27; P = 15.51; Ba = 29.57 per cent.

C = 12.88; H = 2.09.

In this compound the relation between the carbon and phos- phorus is nearly 6C to 3P which would indicate a tri-phosphoric ester. The substance was, however, far from pure and lack of material prevented any further investigation of this body, which is apparently a mixture of various esters.

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R. J. AndersonESTERS OF INOSITE

PHYTIN AND PHOSPHORIC ACID

1912, 11:471-488.J. Biol. Chem. 

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