1596
SHAMGAR AND LEIBOWITZ
milder isomerization of galactitol at 4.0% nickel for three hours (at 170" and 1900 p.s.i.g. hydrogen) resulted in the formation of considerably more DLtalitol (14.1%) than m-glucitol (2.7%). This indicates that isomerization is initiated a t the ends of the molecule (1,2-enediol) and progresses toward the center (2,3-enediol) as isomerization conditions become more severe.
Aclcnowledgment. The authors wish to express their appreciation to Drs. J. D. Brandner, J. W.
VOL.
26
Le Maistre, Leo Kasehagen, L. k'. Gleysteen, Sidney Cantor, and Paul Becher for many helpful suggestions. Optical rotation and infrared data were supplied by Mr. Richard Smith and elemental analysis by Dr. Fred Cheng. Column chromatographic data were supplied, .in part, by Mr. Stanford P. Herron and Mr. Clarence Hall. We are grateful to Dr. N. K. Richtmyer for a sample of D-talitol. WILMINGTON,
DEL.
[ C O N T R I B U T I O N FROM T H E D E P A R T M E N T OF B I O L O G I C h L CHEMISTRY, T l I E
HEBREW UNIVERSITY]
On the Nitration of D-Fructose. I1 AIIARON H. SHAMGAR
AND
JESHAYAHU LEIBOWITZ
Received July 14, 1960 Among the reduction products of a mixture of nitrates formed by nitration of D-fructose with nitroniuin sulfate, a comcould be isolated. This proved to be a new type of sugar derivative: a compound of an anhydride pound of formula CDH,~O, of *fructose with pyruvaldehyde, probably having an acetal-like linkage between the two components.
In our previous communications on this subject1V2 to a formula of C9HI4O7,i.e., a C6-C3 compound. This compound is easily hydrolyzed. The C3by the action of different nitrating agents on D- moiety could be split off by brief heating with fructose. The nature of these products depends dilute acids and proved to be volatile, so that it largely upon the acidity of the reaction mixture. could be separated from the hydrolyzate of I by Whereas nitration with diiiitrogen pentoxide yields distillation. The distillate reduced Fehling's solua monomeric nitrated derivative of ~-fructose,~tion rapidly, was optically inactive, and gave a in strongly acid media, the formation of di-D- 2,4-dinitrophenylosazoneof melting point 145'. The paper chromatogram of the hydrolyzate fructose dianhydrides is predominant. Only one of these dianhydrides could be obtained as a nitrate revealed two spots: one identical with that for Dester in the form of crystals; the others were iso- fructose, and another, moving faster than trioses, lated after reductive denitration. These anhydrides giving a reaction with resorcinol. This second spot gave a positive anthrone test, but did not reduce could be detected only if the developed chromatoFehling's solution. When hydrolyzed they yielded gram were dried without elevation of temperature. All of these properties of the Ca-component of 1 n-fructose quantitatively, with one exception. This exceptional compound (I) exhibits a strik- indicated its identity with pyruvaldehyde. This ing similarity to the compound described as di-D- component was either present in 1 or was derived it during hydrolysis. It is well known that ~ fructose dianhydride I1 by Jackson and G ~ r g e n , from but it proved to be a sugar derivative of quitc a trioses afford pyruvaldehyde when they are treated new type: When hydrolyzed, the change in its with acids.6-8 Compound I could, therefore, be rotatory power indicated the formation of D- either a compound of (a) u-fructose with a triose fructose in a yield of about the hydrolyzate or (b) u-fructose with pyruvaldehyde; neither the reduced an amount of Benedict's solution cquiva- analytical data nor the molecular weight, dctcrlent to 15Oy0 of fructose, and it also reduced sodium mined allow a clearcut distinction. In order to dehypoiodite equivalent to an aldose content of cide between them, control experiments were about 3070. The molecular weight and analytical performed on the formation of pyruvaldehyde from data for I, (mol. wt. 260, C, 45.7; H, 6.2) correspond 1,3dihydroxy-2-propanone and glycerose by the action of acids. I$ was found that far higher con(1) A. H. Shanigar and J. Leibowits, J. Org. C h . ,25, centrations of acid, or much more prolonged heat430 (1960). ing, was necessary in order to produce traces of (2) A. Shamgar-Schwager and J. Leibowits, Bull. Res. pyruvaldehyde from the trioses than from I. Counc. of Israel, 7A, 84 (1958). ( 3 ) M. Sarel and J. Leibowits, J. Org. Chem., 24, 141 We also made sure that no pyruvaldehyde is pro-
we described the large variety of products obtained
(1959).
(4) R. F. Jackson and S. Goergen, - . Bur. Standards J. Research, 3 , 27 (1929). ( 5 ) R. F. Jackson and E. McDonald. Bur. Standards J. Research, 6 , 709 (1931).
(6) 0. Pincuu, Ber., 31, 31 (1898). (7) W. Lloyd Evans and Brook, Am. Chem. J., 3 5 , 115 (1906). (8) C. Eiidcrs, Biochem. Z., 312 (1942).
MAY
1961
NITRATION OF D-FRUCTOSE.I1
duced from D-fructose under the conditions of our experiment. Therefore, unless it is presumed that a triose, at the moment of the cleavage of its linkage' to another-sugar, behaves differently from free trioxcs, it must be concluded that I is a compound formed by union of an anhydride of D-fructose with pyruvaldehyde; these could be joined together by an acetal-like linkage. Such a compound could be produced during the nitration of D-fructose in one of two ways: (1) The cleavage of C6-chains to Cechains, with subsequent dehydration of the latter and their condensation with intact C6-chains. The occurrence of anhydride formation and polymerization reactions during acid nitration has been demonstrated in our preceding communication.' (2) Some of the di-Pfructose dianhydrides produced .during acid nitration could be partially degraded to a C r Csproduct. Compound I could not be detected among the products of nonacid nitration of D-fructose. EXPERIMENTAL
Reagents. Absolute nitric acid (dl6 1.52) wa8 prepared by vacuum distillationQof a mixture consisting of one part of 70% nitric acid and two parts of 98% sulfuric acid (v/v) a t temperatures not exceeding 20" (0.4 mrti.), and the distillate was collected in liquid air. D-Fructose (N.B.C.) was ground and sieved (mesh 100 and up) and dried over phosphorus pentoxide to constant 'weight. Dry ethanol, used for crystallizations, was prepared from commercial absolute ethanol by means of magnesium and iodine.lO Chloroform waa purified by means of aqueous sulfuric acid and washed with water until neutral, dried over potassium carbonate, and distilled shortly before use. Catalytic hydrogenolysis WM carried out in a Parr apparatus at a hydrogen pressure of 60 lb./sq. in. (approx. 4 atm.) using twicc as much palladium catalyst11 (10% on charcoal) aa nitrates. After 20 min., the reactions with diphenylamine and Nessler's reagent were negative. Paper-chromatographic separations (descending) were performed on Whatman No. 1filter paper (for chromatography). The developing mixture consisted of 3 parts of butanol, 2 parts of pyridine, and 1 part of water." The spots were detected by means of a resorcinol phosphate solution." Nitration of D-fructose by nibonium sulfate. Twenty grams of &fructose was dissolved, with rapid stirring, in 200 ml. of absolute nitric acid at 5", and this temperature waa maintained during the entire procedure; 400 ml. of cold, concd. sulfuric acid waa added over a period of 10 min., followed by 300 ml. of dry chloroform. After stirring for 5 min., the (upper) chloroform layer was siphoned off, 200 ml. of fresh chloroform was added, and the mixture was stirred for 3 min. This procedure was repeated once more, and the combined chloroform extracts were repeatedly washed with 500-ml. portions of ice cold, distilled water, until the aqueous layers were neutral towards litmus. After (9) An air-blasted, "Speedivac" rotatory pump, singlestage model 1SC30, Edwards & Co. Ltd., London, was used. (10) A. J. Vogel, Practical Organic Chemistry, Longmans, Green & Co., Ltd., Imdon, 1951, p. 166. (11) L. P. Kuhn, Anal. Chem.,20, 276 (1950). (12) E. J. McDonald and B. K. Goss, Anal. Chem., 24, 422 (1952). (13) L. Sattler and P. W. Zerban, Ind. Eng. Chem., 41, 1401 (1949).
1597
the first two washings, the chloroform layer was rapidly removed from the acid aqueous phase, disregarding turbidity. To the faintly yellow chloroform solution, a small amount of p-benzoquinone was added. The solution was dried twice for 2 hr. over anhydrous sodium sulfate and then concentrated i n vucuo at 25-30'. The resulting thick sirup was dissolved in 150 ml. of dioxane. This solution was concentr&ed i n vacuo to about half its volume, 120 ml. of 95y0 ethanol was added, and the solution was reduced in the presence of 20 g. of palladium catalyst. After solvent removal in vacuo, about 15 g. of a glassy residue was obtained which reduced Fehling's solution readily and exhibited a slightly negative optical rotation. This solid was dissolved in 200 ml. of water, 5 g. of baker's yeast (prewashed with a dilute D-fructose solution) was added, and this suspension was kept for 2 days a t 30". The yeast was removed by filtration with a small amount of Celite "Hiflo,"l4 the alcohol formed during the fermentation was removed in W O , and the resulting solution w11s reincubated with 3 g. of yeast for 2 days. Upon final removal of the yeast by filtration, and of the solvent by distillation in vacuo, about 6 g. of a sirup was obtained. This sirup was dissolved in ca. 30 ml. of absolute ethanol, and this solvent was removed in v u m . The opaque residue was treated with 10 ml. of rigorously anhydrous ethanol and set aside for crystallization. After 3 to 4 weeks, small platelets a p peared which soon grew into little squares (I).I n order to separate them from the sirupy mother liquor, 5 ml. of abs.bS0lute ethanol waa added, the sirup was liquefied by shaking, and the crystals were removed by rapid filtration. From the mother liquor, two further crops of these crystals could be obtained. After recrystallization from absolute ethanol, [ a ]+15.6' ~ ( c 6.2, water); m.p. 203-205" (uncorr.). Anal. Calcd. for CsH1407: C, 46.2; H, 5.98. Found: C, 45.7; H, 6.24. Molecular-weight determinations were carried out in water (AK 18.5): 185.5 mg. of I in 3.000 g. water, A T = 0.425", mol. wt. = 268. 185.5 mg. of I in 6.000 g. water, A T = 0.225', mol. wt. = 252. 185.5 mg. of I in 10.000 g. water, A T = 0.130", mol. wt. = 264. Calcd. for CQH~~O~, mol. wt. = 234; calcd. for CoHI4O7.H20,mol. wt. = 252. On a paper chromatogram, I had the R, value 1.25, as compared to &fructose, R j = 1.00. Acetylation of I. Compound I (37.9 mg.) was dissolved in 2 ml. of pyridine and 3 ml. acetic anhydride was added. This mixture was kept for 36 hr. at room temperature, after which the acetylating mixture was removed by a stream of dry air. The resulting residue (11) was moistened with toluene and redried aa above. This procedure was repeated four times, until the odor of acetic acid had disappeared. Compound I1 was dried thoroughly by dissolving i t repeatedly in absolute ethanol and evaporating i n vacuo; I1 remained as a white sirup which could not be crystallized. [ a ] -10.7' ~ (c 2.55, benzene). The molecular weight determination waa performed in benzene: 67.5 ma. of I1 in 2.64 benzene; -AF = 0.31', mol. wt. = 400. Calcd. for COHIIO~OCOCHI~ 360. Hydrolysis of .'Compound I (185 mg.) was dissolved in 10 ml. of water, a D = $0.29" ( c 1.85). To 8 ml. of this solution, 2 ml. of 5N sulfuric acid waa added, and the resulting solution was transferred to a Rmall ampoule which was sealed and kept immersed in boiling water for 1 hr. After cooling the ampoule, its contents reduced Fehling's solution readily at room temperature and had B rotation of about -1.30'. Prolonging the hydrolysis by 1 hr. caused no change in rotation. Amberlite IR-4 was added to this acid solution to neutrality, and a few drops were taken for paper chromatography. Care was taken not to dry the developed paper with II stream of hot air. With resorcinol, two spots could be detected, one identical with that for D(14) Celite (Hiflo), a siliceous filter-aid produced by Johns Manville Co., New York, N. Y.
1598
VOL. 26
YUEN AND SUGIHARA
fructose, the second a t R3 0.92 (or, relative to D-fructose, RJ 3.18). On the same chromatogram, lI3-dihydroxy2-propanone gave a relative R f 2.20. Quantitative ozidation o j the hydrolyzate of I. Total reducing sugars were determined according to Benedict15; 1 ml. of Benedict’s reagent corresponded to 1 par of D-fructose. Calculations: 15 ml. of Benedict’s reagent = 1.85 ml. of hydrolyzate soln. 1.48%. 10 ml. of Benedict’s reagent = 1.22 ml. of hydrolyzate soln. 1.48y0. Hence, 1.48 mg. of hydrolyzate is equivalent in reducing power to 22.6 mg. of D-fructose (or 153% of a hexose). For aldose, an iodimetric estimation was uscd.16 Standardizations: 3.00 ml. of approx. 0.1N iodine soln. = 2.30 ml. of 0.1N sodium thiosulfate. 3.00 ml. of this iodine soln. 1 ml. of a 1.37% D-fructose soln. = 2.27 ml. of 0.1N sodium thiosulfate. Calculations: 3.00 ml. of this 0.1N iodine solution 1 ml. of hydrolyzate 1.48y0 = 1.65 ml. of 0.1N sodium thiosulfate. This equals 5.6 mg. aldohexose, or 2.8 mg. of aldotriose. Correspondingly 1 ml. of the hydrolyzate 1.48Yo contains 0.076 p~ reducing sugar, including 0.031 p~ aldose. The ratio aldose/total reducing sugar = 1:2.4. Distillation of the hydrolyzate of I. Compound I (50 mg.) was dissolved in 5 ml. of 4N sulfuric acid and kept in a closed ampoule at 100’ for 1hr. The cooled hydrolyzate was trans-
ferred to a small distillation unit, and the distillate was collected in Dry Ice. It reduced Fehling’s solution upon gentJlewarming. To 2 ml. of the distillate, 5 drops of phenylhydrazine, 0.1 g. of sodium acet,ate, and 10 drops of acetic acid were added, and the mixture was kept for 30 min. A crystalline mass settled out; i t was dissolved in hot ethanol and concentrated in a stream of dry air. Crystallization set in (probably of sodium acetate) and the crystals were washed with little ethanol and removed by filtration. After several repetitions of this procedure, a small amount of yellow crystals appeared, m.p. 145’ (pyruvaldehydeosazone, m.p. 148’). The low yield did not permit recrystallization. This hydrolysis was repeated, using 0.2N sulfuric acid and reducing the time of hydrolysis to 30 min. The first drops of the distillate reduced Fehling’s solution readily. In a similar manner, 1,3-dihydroxy-2-propanone, Dfructose, and di-D-fructose dianhydride I were subjected to hydrolysis for 30 min. with 0.2N sulfuric acid and with 4N sulfuric acid. With 0.2N acid, none of these distillates exhibited any reducing action with Fehling’s solution. With 4N acid, the reduction was negligible.” Using 6N sulfuric acid and the same conditions of hydrolysis, 1,3-dihydroxy2-propanone yielded an appreciable amount of a volatile, reducing product.
(15) S. R. Benedict, J . A m . Med. Assoc., 57, 1193 (1911). (16) F. A. Cajori, J. Biol. Chem., 54, 617 (1922).
(17) However, a paper chromatogram of these hydrolyzates revealed faint spots, identical with those for pyruvaldehyde.
+
+
JERUSALEM, ISRAEL ~~
[CONTRIBUTION FROM THE
DEPARTMENTS O F CHEMISTRY, UNIVERSITY O F UTAH AND ARIZONA STATE UNIVERSITY]
Crystalline ~ , ~ - x y ~ o - 3 - H e x u ~Ao sNew e , Hexose’ GEORGE U. W E N * AND JAMES M. SUGIHARA Received August 5 , 1960 Monobenaoylation of 1,2 :4,5-di-O-isopropylidene-~,~-galactitol followed by oxidation with chromium trioxide in pyridine yielded crystalline GO-benzoyl-l,Z :4,5-di-O-isopropy~idene-~,~-zylo-3-hexulose. Debenzoylation and deacetonation provided a crystalline hexose, pres11Tned to be ~,~,-zyb-3-hexulose. This series of reactions constitute the first synthesis of an unsubstituted 3-ketose.
An alternative path circumventing the formaI n an earlier communication from this laboratory, the preparation of crystalline 4-0-benzoyl- tion of an enediol during the debenzoylation step 1,2 :5,6-di-0-isopropylidene-~-arabo-3-hexulose was was devised using 1,2 :4,5-di-O-isopropylidene(I)(formula of D-isomer only given), reported. The compound was obtained by oxida- ~,~-galactitol* a-diacetone dulcitoII6 as the starting compound. tion of 3-0-benzoyl-1,2:5,6-di-O-isopropyIidene-~mannitol with chromium trioxide in anhydrous Because this substance contains a primary and a pyridine. Deacetonation and debenzoylation pro- secondary hydroxyl group, selective benzoylation cedures applied to the substituted 3-hexulose failed of the former would be a distinct possibility.6 to yield crystalline products and thus deterred an Under controlled conditions, 6-0-benzoyl-l,2 :4,5unequivocal synthesis of the parent 3-ketose. di-O-isopropylidene-D,cgalactitol(11) was obtained The failure of the debenzoylation reaction to pro- in a crystalline form. Oxidation of this compound ceed in a desired fashion was attributed to the in an anhydrous system with chromium trioxide formation of an enediol when the ester linkage was in pyridine afforded crystalline 6-0-benzoyl-1,2 : cleaved. This enediol would provide a mixture of 4,5-di-0- isopropylidene - D,L- rylo - 3 -hexulose (111). four isomers. (1) This rePearch has been supported by National Science
Foundation Grant No. G7302. (2) A member of the “Research Participation for College Teachers Program” at the University of Utah, Summer 1959, sponsored by the National Science Foundation. (3) J. M. Sugihara and G. U. Yuen, J. A m . Chem, Soc., ?9,5780 (1057).
(4) This name is used in preference to 2,3:5,6-di-O-isopropylidene-n,L-galactitol suggested by Hann. Maclay, and Hudson (Ref. 5), as the name of the 3-hexulose would logically follow without inversion of the carbon chain. ( 5 ) R. M. Hann, W. D. Maclay, and C. S. Hudson, J . A m . Chem. SOC.,61,2432 (1939). ( 6 ) J. RI. Sugihara, Advances in Carbohydrate Chem., 8 , 35 ( 1953).
