NOTES
Nov. 5, 1952 TABLE I Metal, % Calcd. ~~~~d
Procedure
Form and
(PhzP0z)zCu (BuzP0z)zCu [(CioHzi)zPOzIzCu
A A A
(PhzPOz)zBa
B
Blue needles Green needles Blue needles, m y . 940 Needles
Salt
m.p.
(BuzPOdzBa B [(P-CIC#H~)rPOzlzBa B [(C~oHzi)zPOz]zBa B
Needles Needles Needles, imp. under looo (PhzP0z)rMg B Needles B Needles (BuzP0z)nMg [ ( C I O H Z I ) Z P O ~ ] Z M ~C Needles, m.p. 82-88' [ ( P - C I C ~ H ~ ~ P O I IB ~ M ~Needles (PhrPOi),Ca B Needles (BurP0z)tCa B Needles [(C~oHzi)zPOz]rCa B Waxy needles [(P-CICaHn)zPOz]L!a B Needles (BuzP0z)zPb D Needles [(P-CIC~H~)aPO~lzPbD Needles D Needles [(CioHzi)zPOzlzPb (PhzP0z)zPb D Needles (PhzPOz)aPb A Powder
ii:i8
i!:
8.5
8.42
22.3 22'26 for 2.5Hz0 27'g3 27'85
::::: i:tl 5'3
5'4
6.42 3.4
:::5
4,07
4,3
i::: :::: i;:;; ~~:~~
36'89
36'75
32,2
42,,~
32.2
3 4 . l5
" T h e product obtained was substantially the basic salt. Procedure A gave a product that had an admixture of the basic salt, which could not be removed by repeated crystallizatioii.
5521
Procedure A.-Five grams of diphenylphosphinic acid, suspended in 50 nil. of water, was neutralized with N sodium hydroxide (phenolphthalein indicator). The filtered solution was treated with a slight excess (0.012 mole) of M cupric chloride. The product, in the form of light-blue tiny needles, was washed with water and ethanol, and dried in air. The procedure failed with the bis-p-chlorophenylphosphinic acid. The product consisted of two components neither of which had the properties of the desired salt. The free acid could not be regenerated from it by treatment with mineral acids, indicating a formation of a complex at the halogen atom. Procedure B .-Five grams of diphenylphosphinic acid, suspended in 50 ml. of water, was neutralized directly with A4 barium hydroxide. The filtered solution was evaporated to dryness and the residue was crystallized from 96% ethanol. The air-dried product contains 2.5 molecules of water which are lost by heating in vacuum a t 120'; the anhydrous product is extremely hygroscopic. Procedure C.-An intimate mixture of 5 g. of di-n-decylphosphinic acid (m.p. 87-88') with 1.5 g. of magnesium acetate tetrahydrate was heated under an infrared lamp until all odor of the eliminated acetic acid was removed. The product was washed with hot waterb followed by ethanol. I t forms tiny needles, m.p. 82-83 Procedure D.-A mixture of five grams of di-n-butylphosphinic acid and 3.4 g. of lead oxide was refluxed in 200 ml. of xylene with collection of the evolved water in a Starke-Dean moisture trap. The reaction was complete in 30 minutes. The hot solution was filtered rapidly and cooled. The product precipitated in the form of felted
.
TABLE I1
SOLUBILITY OF THE SALTS,IN GRAMS PER 100 ML. OF SOLUTION 450
250
96% Ethanol 350
0.0662 ,0216 .0016 .0430 ,0220 .0039
0.452 .068 .036 ,006 ,246 * 026 .112 13.38 0.430 .326 .290 .178 .400 .030 1.224 0.314 .636 .024
0.542 .084 ,042 .020 .250 .060 .114 14.05 0.538 .327 .288 ,266 ,286 .048 1.196 0.418 .760 .038
Water Salt
25'
(BuzP0z)zPb 0.0654 [(ClCeH4)zPOaIzPb .0076 * 0008 [(CioHzi)zP0zlzPb (PhzPOzIzCu .0400 (BuzPOz)zCu ' .0172 [( CioHii )zPOzIaCu .0034 (PhzPOzhBa .112 (BuzPOd2Ba Over 188 [( ClCsH&P0zl~Ba 1.128 [( CIOHZI)IPOZ 1~Ba 0.0036 (PhzPOz)sMg 3.554 (BuzPOzhMg 6.570 [(C1CsH&POz]zMg 0.382 [( CioHz~)rPOz IzMg .0012 (PhzP0z)zCa ,704 (BuzP0z)ZCa 4.636 [(C1C7H&P02]zCa 0.114 [ ( C I O H Z ~ I P O Z I Z C ~0.0029
350
0.066 .017g .0012 .0424 .OB6 .0038 .111 Not detd. 1.056 0.0016 3.350 4.714 0.342 .0010 .698 4.362 0.114 0.0036
.110 1.018 0.001 2.616 3.168 0.302 .0008 .694 4.318 0.114 0.0036
Benzene
450
25O
354
450
0.588 ,112 ,056
0.032 .094 .034
0.030 .092 .052
0.158 ,090 .572 000 ,816 25.418 0.240
.ooo
.ooo
.324 .log ,152 21.24 0.916 0.334
.os0 .110 ,138
.ooo
.018 .OOo
....
.ooo
0.728 .I96 .076 1.199 0.712 2.970 0.062
.168 .OO6 .144 .OOo ,002 .I18 .O28
.ooo .248 2.758 .19g
.ooo
.022 4.208 0.000 2.438
0.002 ,200 .004 .002
.Bo
.066
.
.000
.024 4.366 0.000 Gel. Gel. 15.774 0.004 .002 .334 0%
bilities in water of the magnesium, barium and calcium salts of dibutylphosphinic acid and of the magnesium salt of diphenylphosphinic acid. The high solubility of barium dibutylphosphinate in 96% ethanol, and that of copper di-n-decylphosphinate, barium di-n-decylphosphinate, magnesium di-n-butyl- and di-n-decylphosphinates in warm benzene should be noted. The tendency of the salts of barium, calcium and magnesium to have higher solubility in cold water than in hot water is also of interest. The inverse solubility-temperature relation is also seen in alcoholic solutions of magnesium and calcium diphenylphosphinates and magnesium bis-p-chlorophenylphosphinate. Experimental Part
In studies concerned with the acid-catalyzed cleavage of a terminal phosphate group from the
Preparation of the Salts.-The dures were used.
(1) Presented in part before the Second International Congress of Biochemistry, Paris, France, July 21-27, 1952.
following typical proce-
needles which were recrystallized from water. The product was anhydrous after air-drying. This procedure when used with diphenylphosphinic acid gave a product that was largely the basic salt, containing 42.74% of lead. The procedures used in the preparation and the properties of the products are listed in Table I. AUBURN, ALA.
Rates of Hydrolysis of Fructose-6-phosphoric Acid1 BY S. L. FRIESS RECEIVED APRIL30, 1952
552%
SOTES
adenosine triphosphate molecule, i t was essential to have some estimate of the magnitude of the rate for the competing, non-terminal hydrolysis reaction a t the ribose-phosphorus linkage. For this purpose, fructose-6-phosphoric acid (FPX) was selected as an available model compound to yield order of magnitude information on the hydrolysis rate of the primary sugar hydroxyl-phosphorus grouping. Previous report^^,^ on the hydrolysis of fructose mono- and diphosphates have indicated a greater lability toward aqueous hydrochloric acid of the 1-phosphate linkage as compared to the G-grouping,? and a rate constant3 for the relatively rapid hydrolysis of fructose-1-phosphate (in 0.1 N HCl a t 100') amounting to 1 X sec.-'. In the present work, the 6-phosphate ester hydrolysis was carried out under somewhat milder conditions, closely approximating those used in previous kinetic work" on the acid-catalyzed hydrolysis of triphosphoric and pyrophosphoric acids ; reaction teinpcratures of 40 and 30' were employed, and a t each temperature hydrolysis rates were determined a t two levels of acidity. N o additional inert salt was present in these rate runs beyond that introduced in the sample preparation procedure. The stoichiometric hydrolysis reaction
-+
+
Vol. i 4
phosphate under equivalent conditions will proceed largely by cleavage a t the terminal phosphate grouping, without a significant contribution from slower hydrolysis a t the internal sugar-phosphorus bond. In this connection, it is also of considerable interest that both the acid-catalyzed4*6and enzymatically-catalyzed7 hydrolysis of the related polyphosphate, triphosphoric acid, involve cleavage of a terminal phosphate residue. Further, the data of Table I permit a rough comparison of the temperature coefficients for the hydrolysis of FPA a t relatively low (0.025 N ) and high (0.09 Nj levels of added acid. For the temperature interval employed, both low acidity (runs 1 and 3) and high acidity (runs 2 and 4) determinations lead to virtually the same temperature coeficient for kl,i.e., a factor of about 3.7 for 10.3'. This would imply that both the catalyzed and uticatalyzed contributions to observed kl values have the same temperature dependence. As a final observation, the results of run 5 employing HC1 as catalyst (compared to 1 and 2 using IIzS04) point to the relative insensitivity of the hydrolysis to the nature of the strong acid used as catalyst.
Experimental The barium salt of fructose-&phosphoric acid was used was followed by colorimetric evaluation of inorganic direct!y as supplied by the Nutritional Biochemicals Corphosphate evolved, using the method of Lowry poration, Analyses for total hydrolyzable phosphate and barium ion indicated a maximum purity of 95.5% with and Lopeze5 First-order rate constants were for respect to barium fructose phosphate. Triply distilled calculated for the exceedingly slow hydrolyses over water was used in all rate determinations. roughly the first several per cent. of each reaction, In a given rate run, a weighed portion of barium salt was corresponding to a total reaction time of about 10 dissolved completely in about 25 ml. of water, giving a pale solution, To this was added the equivalent quandays. Values for these constants and their ob- yellow tity of sodium sulfate, in small portions with continual served deviations are sumniarized in Tablc I . stirring. The required amount of a standard solution of acid catalyst was then added by pipet, with stirring. After 'LABLE I standing in the cold for about five minutes, the mixture was directly into the 50-ml. volumetric flask used as the L ~ C I D - C ~ Y ~ A L Y Z EHYDROLY-SIS D OF F R U c . l - O S ~ - 6 - P ~ i u S P ~ ~ o R Ifiltered c rcaction vessel, with about 13 i d . of water being used in the Ac1u (FP.1) transfer and subsequent washing. The solution was then Reactiun placed in the constant temperature bath, allowed t o come temperaturc,'l Initial concentrations hi, to temperature, and made to volume with preheated water. Run oc. Acid catalyst, S FP.4, .\I sec.-I X 10' Aliquots (2 ml.) of reaction mixture were withdrawn a t 1 38.93 H2S04, 0.0247 0,0210 1 , 0 9 i.0 . 06 intervals of roughly 24 hours, delivered into about 40 ml. of ,0210 1 . 7 3 i .07 water containing 0.4 millimole of sodium acetate, made to 50 HzSOd, ,0914 2 39.!13 .0210 4 . 11 i .10 nil., and finally analyzed colorimetrically6 for inorganic phosH2S04, ,0247 ;; 50.33 phate. Concentrations of FPA were calculated from initial .(I210 6 . 5 2 f . 4 i values and the subsequent analytical values for phosphate. HzS04, .(1
