[CONTRIBUTION FROM THE C H I N I S T R Y
LABORATORY, NATIONALINSTITUTE
u. s. PUBLIC HEALTHSERVICE]
OF
HEALTH,
MELEZITOSE MONOHYDRATE AND ITS OXIDATION BY PERIODATE' NELSON K. RICHTMYER
AND
C. S. HUDSON
Received April $6, 1946
The composition of the large, clear crystals of melezitose (1) which crystallize from its aqueous solutions at room temperature was established definitely b y Georges Tanret (2) as CleHtr?Ola.2H20. Thesc crystals lose their transparency as a result of efflorescence, and on complete drying by heat the loss of both molecules of water of crystallization can be demonstrated. The specific rotation has been reported by most investigators to be [& f88" to f89" for dried, "anhydrous" melezitose, but similar values have also been quoted for melezitose dihydrate. As a preliminary step in the present investigation, molezitose dihydrate was prepared by crystallization from water. The large, clear crystals, upon standing in the air at room temperature, became completely white within a few days, and their weight became constant after one molecule of water of crystallization had been lost. The second molecule of water of crystallization was removed readily by heating the powdered monohydrate at 110" in vacuo. However, the anhydrous melezitose absorbed moisture very rapidly until it had regained one molecule of water of crystallization. Melezitose monohydrate, C~.H32016"rrO, thus appears to be the stable form of the sugar under normal atmospheric conditions; the use of this monohydrated form is recommended for the preparation of melezitose solutions of accurate composition. The specific rotation of melezitose monohydrate was determined as [alto$88.5' in water (c, 2), which corresponds to [cr]t0 +85.6" for melezitose dihydrate, and to [a]:' f91.7' for anhydrous melezitose. From this experience we may conclude that most samples of "dried" melezitose whose rotations were measured by previous investigators consisted essentially of melezitose monohydrate. The generally accepted formulation of melezitose (I) as [3-(cr-~-glucopyranosy~)-/?-~-fructofuranosy~]-a-~-glucopyranos~de has been developed in the course of many researches since the isolation of the sugar over a century ago by Bonastre (1). All points in this formula have been established with reasonable certainty except that the /?-D-fructofuranosyllinkage is written only by analogy with the corresponding linkages in the other naturally occurring sugars-sucrose, gentianose, raffinose, and probably stachyose (3) and verbascose (4). Unlike those sugarR, melezitose cannot be hydrolyzed by invertase (6-D-fructofuranosidase) ( 5 ) , presumably because of the nearness of the fructofuranoside linkage to the glucosyl radical which is attached through oxygen to carbon 3 of the fructose moiety; hence definite proof that this linkage is of the 8- rather than of the a-type is lacking. 1 Presented in part before the Washington Section of the American Chemical Society, May 10, 1945.
610
OXIDATION OF MELEZITOSE
611
The pyranoid-ring structures of the two glucosyl groups were established by Zerripl6n and Braun (6), and by Leitch (7), through the methylation of melezitose and the subsequent hydrolysis of the hendecamethylmelezitose. Although their conclusions concerning the sirupy trimethylfructose portions were found later to be incorrect, the fact that Miss Leitch was able to convert her trimethylfructose to a sirupy tetramethyl derivative which agreed in its index of refraction and in its rotation in several solvents with the standard values for 1,3,4,6-tetramethylD-fructose is regarded as strong evidence for a fructofuranoid ring in melezitose. The presence of the fructofuranoid ring has now been proved conclusively, and the presence of two glucopyranoid rings has been confirmed, by the oxidation of melezitose (I) with sodium metaperiodate and with periodic acid by the procedures developed in this Laboratory by Jackson and Hudson (8). When one molt: of the sugar was allowed to react with an excess of sodium periodate, four moles of oxidant were consumed, and two moles of formic acid were liberated; no fomtaldehyde could be detected in the reaction mixtures.* The absence of formaldehyde shows that the ring structures must be limited to 2,5 or 2,6 in the fructose unit and to 1,5 and 1,6 in the glucose units. A 2,6-ring in fructose would require one mole of periodate; 1,5- and 1,&rings in glucose would require two and three moles of periodate, respectively, for each glucose unit; since the totall consumption of periodate was only four moles, a 2,6-ringed fructose cannot be present in melezitose because the two glucose units alone must consume at least four moles of periodate. Therefore the fructose unit must have a 2,5 (furanoid)ring and each glucose unit must have a 1,5 (pyranoid)-ring in order to account for tlhe periodate consumed and for the two moles of formic acid liberated. Very similar results were obtained by the oxidation of melezitose with periodic acid. The evidence from the analytical data was supplemented through the isolation and identification of formic acid (111) as the crystalline barium salt. The structure of the tetraaldehyde (11) was confirmed by its further oxidation with bromine water to the corresponding tetrabasic acid, and subsequent hydrolysis of the latter to three products: glyoxylic acid (IV), which was converted to crystalline oxalic acid (V) for identification; D-glyceric acid (VI), which yielded crystalline calcium D-glycerate ; and D-fructose (VII), which was levorotatory in solution, formed D-glucose phenylosazone when heated with phenylhydrazine, and could be identified conclusively by its conversion t o the characteristic D-fructose p nitropheny Ihydrazone. All1 these results are in complete accord with those predicted by theory for the oxidation of melezitose of the structure shown by formula I. EXPERIMENTAL PART
Melezitose monohydrate. Sixty grams of purified melezitose, prepared from honey-dew honey (9), was dissolved in an equal weight of warm water. The solution was filtered into a
* 111 a study of the determination of free primary hydroxyl groups in methylated sugars, Jeanloe [Helv. Chim. Acta, 27, 1517 (1944)] showed that no formaldehyde was liberated in the reaction between melezitose and potassium periodate; he did not determine the amount of reagent consumed.
612
N. K. RICRTMYER AND
C . S. HUDSON I
I
probably B
HCZl\-O
C-CH2 OH
1
I
'-0-CH
1
HO CH
I
I
HCOH HCO
2
HCOH
I
HCO
CH, OH
I
CHzOH I
CH2 OH Melezitose (I)
i
1
CHO
1
HLO-
CHO
I
CH2 OH
I
1
I
CH2 OH
HCO
I
CHz OH (11) [Brr; HBr
CHs OH CHO 2 1 COOH
(Iv) Br,
COOH 2 1 COOH
(VI
COOH
+2
I HCOH I
CH2 OH
(VI)
+
I co I I
HOCH HCOH
I
HCOH
I
CHZ OH
(VW
crystallizing dish and allowed t o atand, loosely covered, for several weeks undisturbed in a room kept a t 20'. The crystals separated as clusters of large, clear prisms; these were removed, wiped carefully with filter paper t o free them from adhering mother liquor, and left
OXIDATION OF MELEZITOSE
A13
overnight in the air. There was no change in their appearance. An 18-g.portion of the clear crystals was powdered and weighed quickly. Efflorescence began at once with the loss in weight becoming constant a t 3.3574 within three days in the air a t room temperature; the calculated value for the loss of one molecule of water from melezitose dihydrate, C I ~ H , & . ~ H Z Ois , 3.33%. The remainder of the large, clear crystals, also 18 g., after standing an additional twentyfour hours in the air, began to show pinpoints of white spots which grew in size until the crystals became entirely white. The crystals retained their original form without becoming crumbly, and their faces had a shiny luster. The loss in weight after about two weeks was constant a t 3.5%, corresponding again to the loss of one molecule of water of crystallization by efflorescence and the formation of melezitose monohydrate. A smaller sample of powdered melezitose monohydrate was dried for six hours at 110' in vacuo. The water of crystallization was removed completely, but was regained with such rapidity under atmospheric conditions that considerable difficulty was experienced a t first in securing a n accurate weight of the anhydrous material. The return from the anhydrous to the monohydrated stage became complete in about two days in the air at room temperature; the weight remained constant thereafter except for slight increases during periodri of humid weather. Anal. Calc'd for C l a I I J 2 O 1 ~ . HC,41.38;H,6.56;HzO, ~0: 3.45. Found: C, 41.30; H , 6.58; H20, 3.45. Dr. W. T. Haskins of this Laboratory has recrystallized melezitose by dissolving i t in a n equal m-eight of water at 60" and then adding four volumes of warm 95% alcohol. The granular product thus obtained was filtered, washed, and dried in the air overnight. It also had the composition of a melezitose monohydrate. Anal. Calc'd for ClsHs201a.HzO: HzO, 3.45. Found (4 hours a t 110" in vacuo) : H,O, 3.44. Speci$c rotation of melezitose monohydrate. The purified melezitose which had been recrystallized from water as described above was identical in rotation with a sample which had been twice recrystallized from water and alcohol by Dr. Haskins. This rotation, [a]; +88.5" in water (c, 2 to 4), characterizes melezitose monohydrate; from i t may be calculated the values [a]: $85.6" for melezitose dihydrate and [a]: $91.7" for anhydrous melezitose. The rotations +88' to +89" reported for "dried melezitose" by many earlier investigators undoubtedly referred to samples which at the time of weighing consisted principally of melezitose monohydrate. Oxidation of melezitose with sodium metaperiodate. T o 2.6122 g. of melezitose monohydrate in 175 ml. of watcr was added 60 mi. of 0.4365 M aqueous sodium periodate (5.22 molecular equivalents), and the volume was adjusted exactly to 250 ml. The rotation, observed in a 4-dm. tube, dropped from +3.70° (circular degrees, calc'd) to +1.19" during the first hour, to f1.04" in two hours, and to $0.90" in four hours, then rose slowly to +1.01" by the end of twenty-four hours and remained constant for several days. This final rotation corresponds to [a];+29.5" for the expected tetraaldehyde (11). The titration of aliquots showed the consumption of 3.57, 3.68, 3.96, 4.W, and 4.04 equivalents of periodate after two, five, twenty-four, forty-eight, and seventy-two hours, respectively. The production of formic acid seemed to approach the theoretical value of two equivalents more slowly, the titrations indicating 1.67,1.77, and 1.85 equivalents after twenty-four, forty-eight, and seventytwo hours, respectively. These values increased very slop, ly thereafter, presumably due to secondary oxidation reactions. h'o formaldehyde could be detected with dimethyldihydroresorcinol. Oxidation of melezitose with periodic acid. A solution of 26.12 g. of melezitose monohydrate in 1600 ml. of water &-ascooled t o 4", and to i t was added 413 ml. of cold 0.5445 M periodic acid (4.5 molecular equivalents). The mixture was kept in the refrigeretor at 4" because the reaction proceeded too rapidly at room temperature. The volume was adjusted to Zoo0 ml. After twenty-four and forty-five hours the titration of aliquots shoned that 3.87 and 4.03 equivalents of periodic acid had been consumed, and that 1.78 and 1.98 equiva-
614
N. K. RICHTMYER AND C. 8. HUDSON
lents of periodic acid had been consumed, and that 1.78 and 1.98 equivalents of formic acid had been liberated, respectively. No formaldehyde could be detected. The oxidation was stopped after forty-six hours, t o avoid secondary reactions, by adding aqueous barium hydroxide to the ice-cold reaction mixture until i t was faintly alkaline to phenolphthalein. The insoluble barium iodate and barium periodate were removed by filtration. The rotation of the filtrate corresponded to [a]: +29.6" for the expected tetraaldehyde (11), in excellent agreement with the value +29.5" which had been obtained by the oxidation of melezitose with sodium periodate. At this point in one experiment the filtrate was concentrated in vacuo to a dry sirup and 200 ml. of methyl alcohol was added to extract the expected tetraaldehyde. The undissolved residue consisted of 9.2 g. of elongated prisms which were recrystallized from water and identified as barium formate; the theoretical yield was 10.2 g. Anal. Calc'd for C2H:Ba04: C, 10.56; H, 0.89; Ba, 60.40. Found: C, 10.60; H , 1.04; Ba, 60.39. I n the principal experiment the filtrate was acidified and oxidation of the tetraaldehyde effected by the addition of 25 ml. of bromine. The rotation changed from positive to weakly negative, becoming constant within four days; after four more days the excess bromine was removed by aeration. T o hydrolyze the expected tetrabasic acid, the solution was heated a t 85' for twenty-three hours, the rotation reaching a constant and somewhat higher rotation than before. Next, the expected glyoxylic acid (IV) was oxidized to oxalic acid (V) by adding 5 ml. of bromine and allowing the mixture to stand in the dark for two days; excess bromine was removed by aeration. Aqueous barium hydroxide was then added until the solution was barely alkaline to phenolphthalein. The precipitated barium oxalate weighed 4.1 g., representing only a 37% yield, although in a nearly parallel experiment a 71% yield mas obtained. This product was dissolved in hot dilute hydrochloric acid, and the barium ions were precipitated with sulfuric acid; upon concentration of the filtrate, oxalic acid dihydrate was obtained and identified, after two recrystallizations from water, by its melting point and mixed melting point, and by titrations with alkali and with potassium permanganate. The filtrate from the barium oxalate precipitate was freed from barium ions with sulfuric acid, and from bromide ions with silver carbonate; the excess silver ions were precipitated with hydrogen sulfide, and the solution was aerated to expel the dissolved hydrogen sulfide. The solution, which presumably contained D-fructose and D-glyceric acid, was concentrated, neutralized barely to phenolphthalein with limewater, and the calcium salts were Precipitated, in several fractions, with methyl and ethyl alcohols. The final filtrate was concentrated to a sirup which was extracted with absolute ethyl alcohol. The crude calcium salts were dissolved in water and the solution was treated with decolorizing carbon and concentrated. The crystalline product which separated as a hard cake of prisms neighed 7 . 8 g. It was recrystallized from water to a constant rotation of [a]: +15.5" in water (c, 0.7), and melting point about 142'with decomposition; these values are in agreement with those reported by Jackson and Hudson (lo), and, with the analyses belon, identify the product as calcium D-glycerate dihydrate. Anal. Calc'd for C~HloCaOs.2H20: Ca, 14.00; H20, 12.59. Found: Ca, 13.90; H20 (at 110" in vacuo), 12.42. The absolute alcohol extract had a levorotation equivalent to 3.3 g. of fructose; the theoretical yield LTas 8.9 g., but undoubtedly a portion of the sugar was lost through the destructive action of the warm acid solution which was used t o hydrolyze the tetrabasic acid. One-third of this solution was concentrated t o remove the alcohol, and the residue was dissolved in water. Phenylhydrazine and a small amount of acetic acid were added, and the mixture, after being marmed on the steam-bath for two hours, yielded 1.5 g. of D-glucose phenylosazone, m.p. 210" with decomposition. The product was identified further by converting 1 g. of i t to 0.44 g. of D-glucose phenylosotriazole according t o Hann and Hudson (11); the rotation, [a]: -81.7" in pyridine ( c , 0.8), and melting point, 195-196',
OXIDATION OF MELEZITOSE
615
are in agreement with the values reported by those authors. A mixed melting point with an authentic sample of the osotriazole showed no depression. Another third of the absolute alcohol extract was concentrated to 75 ml. and boiled gently for two minutes with 1.3 g. of p-nitrophenylhydrazine. The solution, upon concentration in vacuo to a small volume, yielded 1.5 g. of reddish, pellet-like crystals which melted at 174-175’ with decomposition. The product was recrystallized once from 95y0 alcohol, once from water, and then twice from 95% alcohol; the melting point of 182Owith decomposition, the analysis, and the habit of crystallizing first in yellow, prismatic needles which changed to yellow prisms (see the following paragraph), identified the substance as D-frUCtose p-nitrophenylhydrazone. Crystalline modifications of D-fructose p-nitrophenylhydrazone. The reaction between n-fructose and p-nitrophenylhydrazine according to the directions of van der Haar (12) yielded yellow, prismatic needles melting at 182’ with decomposition. However, the first attempts to isolate this compound from the absolute alcohol extract which was presumed to contriin fructose from the melezitose oxidation and degradation products resulted in the separation of small, yellow plates. Further recrystallizations of the “known” fructose p-nitrophenylhydrazone from 95% alcohol then revealed that in our Laboratory this compound iiow usually crystallizes from concentrated solutions, upon cooling, as fine, yellow, prismatic needles which hegin to change spontaneously within two or three hours at room temperature to clusters of darker yellow, plate-like prisms; the change is complete within one or two days; from more dilute solutions only the latter prismatic form may appear. I n the experiments just performed, all samples of D-fructose p-nitrophenylhydrazone, whether prepared from pure fructose or from the fructose solution derived from melezitose, crystallized subsequently from 95% alcohol in this manner. There appears to be no difference in melting: point or composition between the two modifications. Anad. Calc’d for C12H17S~0,:C, 45.71; H , 5.44; N, 13.33. Found (needles, from pure fructose) : C, 45.71; H , 5.27; N(Kjeldahla), 13.08. (prisms, from pure fructose): C, 45.93; H , 5.32; N , 13.13. (prisms, from “melezitose” fructose): C, 45.70; H, 5.46; N , 13.23.
The authors wish to thank Dr. Arthur T. Ness and Mr. Charles A. Kinser of this Institute for carrying out the microchemical analyses. SUMMARY
The stable form of crystalline melezitose under normal conditions is the monohydrate, ClsHs201s.Hz0,with [a]:’$88.5” in water. A study of the oxidation of melezitose with sodium metaperiodate and with periodic acid has proved conclusively the presence of the fructofuranoid ring in this sugar. BETIIESDA, MD. REFERENCES (1) A review, “Melezitose and Turanose,” by C. S. Hudson, will appear in “Advances in Carbohydrate Chemistry,” Academic Press, Inc., New York, Vol. I1 (in press). (2) TANRET, Bull. soc. chim., [3] S6, 816 (1906). (3) OifuKI, Sci. Papers Inst. Phys. Chem. Research (Tokyo), 20, 201 (1933).
* Du.mas nitrogen determinations were unsatisfactory with the samples of this p-nitrophenylhydrazone; c f . h’iederl and Niederl, “Micromethods of Quantitative Organic Analysis,” Second Edition, John Wiley and Sons, Inc., New York, 1942, p. 98.
616
N. K. RICHTMYER AND C. S. HUDSON
(4) MURAKAMI, Proc. Imp. Acad. (Tokyo), 16, 12 (1940);Chem. Abstr., 94,3694 (1940). ( 5 ) See ADAMS,RICHTMYER, AND HUDSON, J . A m . Chem. Soc., 66, 1373 (1943). (6) ZEMPL&NAND BRAUN, Ber., 69,2230 (1926). (7) LEITCH,J. Chem. SOC.,688 (1927). (8) JACKSONAND HUDSON, J . A m . Chem. SOC.,(a)69, 994 (1937); (b)61, 1532, footnote 8 (1939). (9) HUDSON, J. 070. Chem., 9,470 (1944). (10) JACKSON AND HUDSON, J . A m . Chem. Soc., 62,960 (1940). (11) HANNAND HUDSON, J . Am. Chem. SOC.,66, 735 (1944). (12) VAN DER HAAR,“Anleitung zum Nachweis, zur Trennung und Bestimmung der Monosaccharide und Aldehydsiiuren,” Verlag von Gebrtider Borntraeger, Berlin, 1920, p. 191.
