May 5 , 1955
NOTES
2567
was extracted from the reaction solution with chloroform. The material remaining in the aqueous solution was treated as above seven times in all when the total material extracted by chloroform weighed 850 mg. This sirup was methylated 80 with methyl iodide and silver oxide four times when the vi product (800 mg.) had a methoxyl content of 55.9 (calcd. .e for octa-0-methyl methyl bionic ester “OMe” 57.6). k Hydrolysis of Methylated Bionic Acid and Identification of Products.-Disaccharide ester (750 mg.) was hydrolyzed 60 with 7y0 hydrochloric acid (20 cc.) for 3 hr. at 80’ during a which time the rotation had fallen from [ a ] ” D +86’ to a 8 constant value of +54O. The hydrolysate was neutralized 40 with barium carbonate, adjusted to pH 7.5 with barium RJ hydroxide and evaporated to dryness. The solid was t carefully dried and extracted with anhydrous ether t o colk The barium salt of lect the tetra-0-methyl-D-galactose. 20 the tetra-0-methyl-D-galactonic acid was extracted from the residue with chloroform.I2 The ether extracts were combined and evaporated t o dryness. The residual sirup (311 mg.) was converted t o the anilide which was recrystallized from ethyl acetate to give pure crystals (183 mg.) with m.p. 195-196’ (unde100 200 300 pressed by admixture with 2,3,4,6-tetra-0-methyl-~-galacTime, min. tose anilide). The chloroform extract was evaporated to a sirup (325 Fig. 3.-Hydrolysis of disaccharide A with an a-galactosimd.) which was dissolved in concentrated nitric acid (2.5 dase: 0, disaccharide A; 0 , melibiose; A, lactose. cc.). The solution was heated slowly to 100” on a waterbath and maintained a t that temperature for 5 hr. U’ater of an invertase preparation called “Brewer’s A.”4j16 The was added and the solution was distilled under reduced hydrolysis of the disaccharide a t 20’ was followed by depressure. Distillation and continuous addition of water termining the change in the rotation of the solution with a were carried on until all but a slight trace of nitric acid had polarimeter. The sugar solution was allowed to mutarotate been removed A little methanol was added and the to a constant rotation before treatment with the enzyme solution was concentrated t o a sirup (259 mg.) which solution. As references the hydrolyses of melibiose and was dried, dissolved in dry methanol and neutralized with lactose were carried out under the same conditions (Fig. 3). silver carbonate. Excess methyl iodide (0.2 cc.) was added Acetylation of Disaccharide A.-Anhydrous sodium aceand after the solution had stood for 1 hr. it was filtered and tate (50 mg.), disaccharide A (100 mg.) and acetic anhythe solid was washed with methanol. The washings and dride (1.0 cc.) were heated together a t i00’ for 30 min. filtrate were combined and evaporated to a sirup which crys- The product was isolated in the usual manner and crystals tallized. The product was recrystallized from an acetone, which had m.p. 223-227’ and [ ~ ] * O D4-186” (c, 0.5 chloroether and benzine mixture to give colorless crystals (156 mg.) form) were obtained in 10% yield from ethanol solution. which had m.p. 111-112° (undepressed by admixture with An amorphous material was obtained from the mother liquors authentic 2,3,4,5-tetra-O-methyl dimethyl mucate). in 80% yield and it had [ a ] m ~+111’ (c 0.5, chloroform). 2,3,4,5-Tetra-O-rnethyl Dimethyl Mutate.-The direct The same products in approximately the same yields were methylation of mucic acid with dimethyl sulfate and sodium obtained when the temperature of the above acetylation hydroxidela was found to be unsatisfactory due to the in- was increased and also when standard acetylation procesolubility of the sodium salt of mucic acid. A more indirect dures using zinc chloride or sulfuric acid as catalysts were approach was adopted using 2,3,4-tri-O-methyl-~-galactose employed. which had been prepared as a reference compound by the Anal. Calcd. for CaH38019 (crystalline octaacetate): method of Smith.14 C,49.51; H,5.65. Found: C,49.46; H,5.64. The 2,3,4-tri-0-methyl-~-galactose was oxidized to tri-0Hydrogenation of the A1dehydoacetate.-A sample of rnethylmucic acid which was esterified with methanol as the amorphous material from the previous experiment ( 150 described by Smith.” The 2,3,4-tri-O-methyl dimethyl mg.) was dissolved in glacial acetic acid (10 cc.) and shaken mucate (2.0 g., m.p. 101.5-102.5’) was dissolved in methyl with hydrogen at 4 atmospheres pressure for 24 hr. (platiiodide (20 cc.) and silver oxide (10 g.) was added. After num oxide catalyst, 50 mg.). The sirupy material obtained standing for 24 hr. the mixture was filtered, the solid was was non-reducing to Fehling solution and gave a negative washed with methyl iodide and the combined filtrate and Schiff test. washings were evaporated to a sirup. The sirup was methylated again as above. The product was distilled in high Acknowledgment.-The authors wish to express vacuum when the first fraction distilling over crystallized. their appreciation to E. I. du Pont de Nemours and The crystalline solid was sucked free of sirup and recrystallized from acetone to give a product (1.1 g.) with m.p. Co.,Wilmington, Del., for a research grant that made this investigation possible. 111..5-112.5° (depressed to 89-94’ by mixing with 2,3,4tri-0-methyl dimethyl mucate) and “OMe” 62.6, calcd. (1.5) T h e authors are indebted t o Dr. N. K Richtmyer of the Nafor CizHnOs 63.3. Anal. Calcd. for C12HnOa: C, 48.90; tional Institutes of Health, Bethesda, Md., for the xiit of a sample 0‘ H, 7 52. Found: C, 49.07; H , 7.61. this enzyme solution. Karrer and Peyer” reported m.p. 103’ for the product obtained from the direct methylation of mucic acid. FRICK CHEMICAL LABORATORY Periodate Oxidation of Disaccharide A.-A dry sample of PRINCETON UNIVERSITY disaccharide A (98.7 mg.) was dissolved in water and the PRINCETON, XEWJERSEY solution was diluted to exactly 50 cc. Aliquot portions ( 5 cc.) were taken and treated with 0.2 M periodic acid adjusted to pH 4.5-5.0 with sodium hydroxide. After Bisdehydro-carotenes suitable intervals the residual periodate was determined using 0.10 N sodium arsenite. “Blanks” were run siBY L. ZECHMEISTER A N D F. J. PETRACEK multaneously. Six moles of periodate per mole of disacRECEIVED NOVEMBER 22, 1954 charide were used up rapidly (Fig. 2). Hydrolysis of Disaccharide A by an a-Ga1actosidase.L Two years ago a deeply colored dehydrogenation A 0.870 solution of disaccharide A (10 cc.) in acetate buffer of PH 5.0 was treated with a highly active solution (0.1 cc.) product of &carotene, “bisdehydro-P-carotene,”
-
g
3
(12) Cf.W. N.Haworth and S. Peat, J. Chcm. Sac., 3094 (1922). (13) P.Karrer and J. Peyer, Hclu. Chim. Acta, 6,577 (1922). (14) F. Smith, J. Chcm. Soc., 1724 (1939)
C40Hb2,was prepared in our laboratory,’ and the (1) L. Zecbmeister a n d L. Wallcave, TEXIS J O U R N A L , ‘76, 4493
(1953).
2568
Vol. 77
NOTES
structural possibilities for it were narrowed down to a symmetrical (normal) I1 and a non-symmetrical (retro) formula I. Since recently, Inhoffen and RaspC2 have obtained 11, i.e., 3,4,3’,~’-bisdehydro&carotene by total synthesis and have found it different from our “bisdehydro-8-carotene,” the structure I should now be adopted for the latter.
propylidene derivatives of D-glucuronic acid and L-iduronic acid as well as their lactones. We failed to crystallize 1,2-0-isopropyIidene-D-xylo-dialdopentofuranose, but modified its purification. The alkaline hydrolysis of the cyanohydrins was conducted a t a low temperature according to the general conditions outlined by Isbell and co-workers, and the use of an ion exchange resin for generating the free acids was abandoned in favor of the method described by Mehltretter and associates4 which is simpler and enables the extraction of the products from a more concentrated solution. 1,3-0Isopropylidene-D-glucofuranuronic acid readily I 11, both end groups crystallizes from the evaporated extract, which also I t seems to be of some theoretical interest that, contains 1,2-O-isopropylidene-L-idurono-y-lactone in contrast to 11, compound I, now termed, retro- and some 1,2-O-isopropylidene-~-glucuronoy-lacbisdehydro-carotene, shows the following charac- tone; but the purification of the latter by recrystalliteristics : the retro structure increases the adsorba- zation is a wasteful process and efficient recovery of bility; it involves extensive fine structure in the the remaining material in the mother liquors as Dmain spectral band; and upon iodine catalysis it glucurono-y-lactone-ti-CIA, according to the method produces a stereoisomeric mixture that shows no of Sowden,’ requires isotopic dilution. To avoid marked cis peak, although cis forms are preponder- this it was found possible to by-pass the isolation ant in it. acid by of 1,2-O-isopropylidene-~-glucofuranuronic Inhoffen’s synthetic compound mentioned has, converting the mixture of the epimeric products to however, now been identified with a minor product the corresponding lactones, which could be sepxof the dehydrogenation of either a- or p - ~ a r o t e n e , ~rated by chromatography on clay according to the viz., “dehydrocarotene 111” (E::, 12.6 X l o 4 at general technique of Lew, Wolfrom and G ~ e p p . ~ A,, 471 mb, in hexane). Hence, both I and I1 I n this way a clear-cut separation of the two prodmay result from a direct dehydrogenating attack on ucts was achieved without isotopic dilution and the resulting pure 1,2-O-isopropylidene-D-glucofurancarotenes. urono-y-lactone-G-C14 was converted readily to D(2) H . H. I n h o f f r n a n d G . Rasp&,A i t i l . (in press; communicated t o glucose-6-C14by reduction with lithium aluniinuni u s b y Prof. Inhoffen who also kindly sent u s a sample of his synthetic preparation). hydride according to Roseman.6 (3) G. K a r m a k a r a n d I.. Zechmeister. THISJ O U R N A L , 77, 55 (1955). The identity of the second product as 1,2-0GATESA N D CRELLIN LABORATORIES OF isopropylidene-L-idofuranuronoy-lactone \Vas CHEMISTRY (No. 1598) proved by the reduction of this compound t o 1,2--0CALIFORNIA INSTITUTE OF TECHNOLOGY isopropylidene-L-idofuranose, the constants of PASADENA, CALIFORNIA which were in good agreement with those recorded by Meyer and Reichstein,’ who prepared this suhSynthesis’ of L-Iduronic Acid and an Improved stance by a different method. Xild acid hydrolysis of the isopropylidene group yielded crystalline L Production’ of ~ - G l u c o s e - 6 - C ~ ~ iduronic acid. To our knowledge, this is the first B Y F. SHAPIZADEH ASD M. L . ~ V O L F R O M recorded uronic acid in the idose series. RECEIVED DECEMBER 17, 1954 Isbell and co-workers3 have shown that thc In the synthesis of D-glUCOSe-6-C1Adescribed by proportion of the two epimeric aldonic acids formed Sowden,2 the main step is the introduction of the in the cyanohydrin synthesis involving the glycosiradioactive label by condensation of 1,2-O-isopro- dic group depends in part on the conditions under pylidene-D-xylo-dialdopentofuranose with CI4-la- which the reaction takes place. This is also true for beled sodium cyanide followed by the isolation of the formation of D-glucuronic and L-iduronic acids, 1,2-~-isopropy~idene-~-g~ucofuranuronic-G-~~~ acid since in the presence of sodium hydrogen carbonate from the reaction mixture by hydrolysis of the cy- and excess carbon dioxide a higher proportion of anohydrins, decationization with exchange resin the L-iduronic derivative could be isolated, whilc and extraction from the aqueous solution with the presence of sodium carbonate in the reaction ethyl acetate. \Ye experienced some difficulty in mixture reversed the proportion. However, in the isolating the above product in this manner and latter case, the over-all yield of the epimeric products was found to be lower. found it desirable to reinvestigate the reaction. These variations readily provide an iniprvved Preparation of 1,%O-isopropylidene-D-glucofuranyuronic acid is complicated by the possible impuri- yield of 1,2-0-isopropylidene-D-glucofuranuronoties of the sirupy starting material, the decompo- lactone-6-C14 as well as the corresponding L-idose sition of the products during hydrolysis, sensitivity (3) H. S. Isbell, J. V. Karabinos, H . I,. Frush, N. B. Holt, A . of the isopropylidene group to acid hydrolysis, and Schwebel a n d T. T.Galkowski, J . Research 5 a f 1 . Buy. S f a i z d a r d s , 48. finally by the formation of the two epimeric O-iso- 163 (1952). (4)
I ) This work W:I< carried o u t under c o n t r a c t (DA-33-019-or& I 17li. wi)ervisiiig agency, Ballistic Kesearch I,aburatories, Al~erdeen I’rovinp (>rotitid, Rlnryland) Ixtween t h e Ordnance Corps anS t a t e I‘niversity Research Frriindation (Project >!)I) ( 2 ) I C Snrv(lrri, ’1‘111s ~ < ~ I ~ R K 74, , ~ I 4177 . , (lc,a5!?) (
C. L. Mehltretter, B. H. Alexander, R.I.. Mellies and C . E. Rist,
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