Dec., 1951
DEGRADATION OF AMYLOPECTIN TO PANOSE
5849
[CONTRIBUTION FROM TEE DEPARTMENT OF CHEMISTRY OB THE OHIO STATE UNIVERSITY]
Degradation of Amylopectin to Panose BY A. THOMPSON~ AND M. L. WOLFROM By the use of a combination of carbon and silicate chromatography a trisaccharide, 4-~-isomaltoppranosyl-~-glucosc or panose, has been isolated as its crystalline glycitol dodecaacetate from a reduced acid hydrolyzate of amylopectin. The hydrolysis was carried to 70% completion since calculations showed that a t this point a maximum yield of trisaccharide would be produced. The isolation of panose, which contains one a-l,&link, from this hydrolyzate constitutes further evidence for the conception that branching occurs a t the six position of D-glucose in the amylopectin molecule.
Considerable indirect evidence has accumulated saccharides were deacetylated and reduced to the that amylopectin is a branched polymer of D- corresponding glycitols with Raney nickel catalyst glucose with the point of branching at C-6of the D- and hydrogen under pressure. Upon reacetylaglucose molecule. This concept is further sup- tion with subsequent silicate chromatograph , one ported by the isolation of isomaltose (6-a-~-gluco- of these zones yielded crystalline material guclei pyranosyl-D-glucose), characterized as its crystal- being at hand from previous work') adequately line P-octaacetate12from the enzymic8 or acidq hy- identified as panitol dodecaacetate by melting drolyzate of amylopectin, evidence being offered point, rotation and X-ray powder diffraction patthat isomaltose was preformed in the molecule and tern. The yield of pure material was 0.1% in coma was not an artifact. We wish to report herein the parison with a calculated maximum of 0.3%. Maltotriose, as its crystalline 0-hendecaacetate, isolation of panoses (4-a-isomaltopyranosyl-~-gluCOS^^*^)^ characterized as its crystalline alditol do- has been isolated from enzymicaJOand acid'*' hydecaacetate, from the acid hydrolysis (with subse- drolyzates of amylose and amylopectin but panose quent reduction) of amylopectin. is the first trisaccharide containing the u-1,6-link In a previous publication*a statistical analysis of which has been identified, on a crystalline basis, in the hydrolytic degradation of branched chain mole- an acid hydrolyzate of amylopectin. This consticules of the amylopectin or glycogen type was made tutes further degradative evidence for branching at whereby the yields of those oligosaccharides which C-6 of D-glucose in the amylopectin molecule. contain one c~-1,6-glycosidic linkage could be calcu- Confirmatory to previous work,' small amounts of lated for any desired degree of hydrolysis. A de- maltose and isomaltose were isolated as their B-octailed treatment was recorded for the case of iso- taacetates from the reaction mixture. The fraction maltose. An exactly similar calculation can be normally expected to contain D-glucose was not inmade for the three possible trisaccharides from am- vestigated. Boissonnas" has pointed out the ylopectin containing one a-l16-link employing a chromatographic simplification effectable by reducbranching frequency of 1:25. This shows that a ing an aldose to the alditol (glycitol). It is hoped maximum yield of 0.91% trisaccharide will occur at that further application of the chromatographic 68.9% of complete hydrolysis. Since there are resolution of such reduced hydrolyzates may yield three possible trisaccharides containing one a- other crystalline derivatives of these difficultly l,6- and one cu-1,4-linkl the maximum yield of each crystallizable starch hydrolytic fragments. will be 0.91/3 or 0.3%. Experimental The amylopectin (waxy maize starch) hydrolysis (2y0carbohydrate in 0.05 N sulfuric acid a t 98') in Hydrolyds of Amylopectin.-Amylopectin in the form of the work herein reported was accordingly carried waxy maize starch (50 9.) was stirred vigorously in 2500 ml, of 0.05 N HISO, while heating in a boiling water-bath, out to ca. 70% completion (24 hr.), the course of the The course of the hydrolysis was followed by the copperreaction being followed by copper reduction. The reduction method of Somogyi.l* The reducing sugar was hydrolyzate material was subjected to a prelimi- calculated as glucose and the hydrolysis was continued until nary fractionatioa by the method of selective elution the reaction had reached 70% completion (24 hr.). The acid was removed by passing the solution through a from carbon as developed by Whistler and D u r s ~ . ~sulfuric 500 X 30 mm. (i.d.) column of Duolite A-4." The neuThe fractions were then acetylated (sodium acetate tral solution was then evaporated under reduced pressure to and acetic anhydride) and further separated by sili- 1250 ml. Fractionation of Amylopectin Hydrolyzate.-The above cate chromatography. The zones thus obtained waa placed on a 275 X 105 111111. (i.d.) column of which were considered most likely to contain tri- hydrolyzate activated carbon (Darco G601LCelite,U 1:l by wt.). The (1) Corn Industries Research Foundation Associate of The Ohio State University Research Foundation (Project 203). (2) M. L. Wolfrom,L. W. Georges and I. L. Miller, THIS JOIJBNAL, 69,473 (1947); ibid., T i , 126 (1949). (3) Edna M. Montgomery, F. B. Weakley and G. E. Hilbert, ibid., 69, 2249 (1947); ibid., 71, 1682 (1949). (4) M.L. Wolfrom, J. T.T p e e , T. T. Galkowski and A. N. O'NeiU, ibid., 79, 1427 (1950); ibid., 78, 4927 (1961). (S) S. C. Pan, A. A. Andreasen and P. Kolachov, Science, 119, 116 JOURNAL, (1960); S. C.Pan, L. W. Nicholson and P. Kolachov, THIS 73,2547 (1961). (6) D. French, Sciuncr, 118, 862 (1951). (7) M. L. Wolfrom,A. Thompson and T. T. Gallrowaki, Trim Jomn u , 18,rH)Qa(1961). (8) M. L. Wollmrn, B. N. b t t n and A. N. O'NeiU. Wd.,18, 6011 (1M1). (9) R,L. W h l N w nnd 0. P.DwW, W d , , 111,677 (1OWl.
D-glucose was removed from the column by washing with water (9 liters) until the eBuent became negative t o Benedict solution. This first portion of eiiluent was discarded. A second portion of sugar was obtained by washing the column with 570 ethanol (15liters) until the effluent again became negativdto Benedict solution. A third portion of material was obtained by washing the column with 15%ethanol
(10) M.L. Wolfrom,L. W. Georgea, A. Thompson and I. L. Miller, ibld., 71, 2873 (1949). (11) R. A. Boiaaonnaa, €I Ckim. & Acta, . SO, 1689 (1947). (12) M.Somogyi, J . B i d . Chum., 160, 61 (1946). (18) A pmdact of the Chemicnl R o c u Compnnt, Redwood City, Wifornia. (14) A prodact of bueo Dept., Atlas Powddlz &mp.ny, New Yorl, 8. Y . ( a m NO. a m prauut at Jahm.cld.n+lfl@L,, Raw Y a k , N.f.
VOl. 73
C. F. H. ALLENAND J. A. VANALLAN
5850
(8 liters). The 5% and 16% ethanol effluents were ewporated to sirups under reduced pressure and further dried by distillation with methanol under reduced pressure; yields 5 g. and 6 g., respectively. p-Isomaltose 0ctaacetate.-The 5 g. of amorphous material from the 5% ethanol effluent was acetylated with 2 g. of sodium acetate and 40 ml. of acetic anhydride a t a temperature just below the boiliig point of the mixture. The excew acetic anhydride was hydrolyzed by stirring with 200 g. af ice and water; yield 9 g, of acetylated sirup. This material was dissolved in Bo ml. of benzene and chromatographed on two 275 X 80 mm. (Ld.) columns of Magnesol'hCelites ( 5 :1 by wt.) and each was developed with 3000 ml. of benzene-t-butyl alcohol (5O:l by vol.). Four zones were located by streaking the extruded columns with permanganate indicator (1% KMnOI in 10% NaOH). The sectioned zones were eluted with acetone and the acetone was removed by evaporation under reduced pressure, The material from the three top zones failed to crystallize; that in the zone near the bottom crystallized from ethanol; yield 360 mg., m.p. 144-145" unchanged on admixture with authentic @-isomaltoseoctaacetate, [CZ]*~D+96" (c 2.9, chloroform). These values agree with those (143-141", -t97") accepted* for @-isomaltose octaacetate. Panitol Dodecaacetate.-The material (6 g.) from the 15% ethanol effluent from the carbon column was acetylated in the manner just described. The resulting acetylated material was chromatographed on Magnesol-Celite as described above except that 3000 ml. of benzene-t-butyl alcohol (35:l by vol.) was used as the developing agent. Four zones appeared on the column. The zone material was removed from the sectioned column by elution with acetone. After removal of the acetone the zone failed t o crystallize. The benzene-t-butyl alcohol effluent was evaporated to dryness to give @-maltoseoctaacetate; yield 0.5 g., n1.p. 155156", [ a I s 3 +63" ~ (accepted values: 159-160", +6:3').
-___
(16) A product of Westvaco Chloriue Products Corp., South Charleston,West Virginia
The material (0.9 g., fram the second zone from the top of the column was dissolved in 10 ml. of 0.05 N NaOCHa in methaaol and allowed to remain at 5" overnight. It was then diluted with 50 ml. of water and passed successively through ion exchange columns (150 x 20 mm. i.d.) of Amberlite 120'' and Duolite A-413. The solution and washings were evaporated to 50 ml. under reduced pressure. The sugar was then hydrogenated a t 1800 p.s.i. and 80' for 3 hr. in the presence of 2 g. of Raney nickel catalyst. After Gltration and removal of the solvent by evaporation under reduced pressure, fhe resultant amorphous material was again acetylated with sodium acetate 0.3 8.) and acetic anhydride (7 ml.) as described above. he resulting sirup crystallized from ethanol; yield 35 mg., m.p. 138-144'. The mother liquor was evaporated to a sirup and redissolved in 30 ml. of benzene. This solution was placed on a column (275 X So mm. i.d.) of Siene1kCelite" ( 5 : l by wt.) and developed with 4 liters of benzene-f-butyl alcohol (75: 1 by vol.). Three zones appeared on the column which were sectioned and eluted with acetone. Crystalline material was obtained from the zone near the column top; yield 90 mg., m.p. 140-145". The combined portions of crystalliie material (125 mg.) were further purified by three recr stallizations from ethanol; yield 70 mg. (O.l%), m.p. 14f-148" unchanged on admixture with authentic panitol dodecaacetate (m.p. 148.5-150°, [~C]=D 4-120' in chlorofom7), [~]SD +118" (c 2.6,chloroform). X-Ray powder drflraction data: 8.5119 -70,m 6.96-20, 5.73-5, 4.66-100, 4.0820, 3.38-40, 3.17-5, 2.97-10, 2.67-10,2.55-5. The X-ray diagram was identical with that of an authentic sample of panitol dodecaacetate.
(r
(17) A product of Rohm and Haas Co., Philadelphia, Pennsylvania. (18) A product of the Columbia Chemical Co., Batberton, Ohio. (18) Interplanar spacing, A. ; CuKn radiation. (20)Relative intensity a9 percentage strongcat line; estimated visually.
COLUMBUS 10, OHIO
R~CEIVED JUNE 7, 1951
[COMMUNICATION NO. 1403 FROM THE KODAKRESEARCH LABORATORIES]
Some 3,4-Diphenylcinnolhes and Related Compounds R Y C. F. H. ALLENAND J. A, VANALLAN 2-(Hydroxydesy1)-cyclohexanoneshave been converted into 5,6,7,&tetrahydrocinnolines by treatment with hydrazine. These are the first reduced cinnolines, other than 1,a-dihydro derivatives, to be described. A variety of subshncss mUlt on dehydrogenation in the presence of a palladium catalyst. The principal product is an indole, but a true cinnoline is also formed; the reaction is not a useful source of cinnolines. The cinndines described in this paper form salts with one equhrslent of an alkyl halide. Improved procedures are given for the preparation of benzil monophenylhydrazone and 3,4-diphenylcinnoline.
When 2-(hydroxydesyl)-4(or 5, or 6)-methylcyclohexanones (I)' are treated with hydrazine, water is eliminated as with other 1'4-diketones and reduced cinnolines (I ,2-diazanaphthalenes) (111) are formed. OH
I
r
lated unless phenylhydrazine or 2,bdinitrophenylhydrazine is used. These two derivatives have been previously described.2 While the monophenylhydrazone is readily cyclized, all attempts to cyclize the dinitro derivative failed. No attempt has been made to determine which of the two most likely structures is correct for the tetrahydrocinnoline (IVa, b) derived from phenylhydrazine. C6H6 )AqCaH1
I1
_-
IVa A
R I11
The intermediate hydrazone (If) cannot be isoe ( 1 ) 6.P.B.
Atla# and J. A. VonAllan, J. BPIr Chrm., 16, 716 (IDblIt
Cs"
IVb
The tetrahydrocinnolines (111) do not evolve methane when treated with methylmagnesium iodide; hence, it is unlikely that any hydrogen is attached to nitrogen. Since these substances da (2) C..
XI.
a.Allen,
Can, d . f i l l a r c h , 1,284 (lo'dl),
