July 3, l!lil
D-
GLUCOSEA N D GENTIOBIOSE IN HYDROI. : RADIOISOTOPIC DILUTION
4-( 1-Methyltetrahydropyridy1)-diphenylcarbinol(XI).To a11 ethereal solution of 0.4 mole of phenyl lithium, prep a r e l from 6.28 g. of bromobenzene and 0.56 g. of lithium, \vas added 3.17 g. of the mixture of methyl l-methyltetrahydroisonicotinate and methyl 1-methylisonipecotate ( I X ) from the low pressure hydrogenation of methyl isonicotinate methiodide. The mixture was heated under reflux for 3 hr. and, after cooling, poured into ice and water. The water and ether insoluble solid was removed by filtration giviiig 1.58 g. of X I , m.p. 179.0-179.8, after recrystallization from ethanol. A n d . Calcd. for CIOHSINO:C, 81.68; H, 7.58. Found: C, 80.93; H , 7 . 5 3 . The layers in the filtrate were separated and the water layer extracted with 50 ml. of ether. The dried extracts mere distilled leaving 1.72 g. of X , m.p. 130-132",asresidue. Catalytic Reduction of 4-( 1-Methyltetrahydropyridy1)-diphenylcarbinol (XI).-A solution of 2.0 g. of X I in 100 ml. of absolute methanol was shaken for one hour at room temperature with 0.2 g. of platinum oxide catalyst under 60 atm. pressure of hydrogen. Removal of the catalyst and solvent gave 1.95 g. (96.5%) of 1-methyl-4-piperidyldiphenylcarbinol (X), m.p. 125-130'. After recrystallization from methanol, the solid melted at 127.5-130.0' and showed no depression of melting point on mixing with an authentic sample of X. 1-Methyl-4-piperidylidenediphenylmethane (XU).-A solution of 4.0 g. of 1-methyl-4-piperidyldiphenylcarbinol (X) in 30 ml. of 1:l sulfuric acid-water was heated on a >team-bath for 1.5 hr., poured into water and neutralized. The basic solution was extracted with ether, and the combined, dried ether extracts were fractionally distilled. The fraction, b.p. 205-208' a t 12 mm., lit.8 b.p. 145-150' a t 1 mm., unlike the previously reported cases, crystallized on standing or seeding to give 3.09 g. (82.6%) of l-methyl-4piperidylidenediphenylmethane ( X I I ) , m.p. 55.0-56.3'. The hydrobromide of X I I , after recrystallization from ethanol, melted a t 264-266'. X I 1 hydrobromide is only sparingly soluble in water. Anal. Calcd. for C19H22BrS: Br, 23.2. Found: Br, 23.1. The Reaction of I-Methyl4piperidylidenediphenylmethane (XU) with Bromine Water. ( a ) At 100°.-To a suspension of 4 0 g of X I 1 hydrobromide in 50 ml. of water, heated on a steam-bath, was added in 5-ml. portions 82 ml. of saturated (0') bromine water. Heating was continued for 0.5 hr. or until all the solid was in solution and the reaction mixture was al!o\ved to stant! for 2 hr The rxeciuitated solit1 retlis.;olved on heating and the solution . . \vas iicntralizctl lrith potassium hydroxide. The amines
-
[ "OY
L RII3U rIoY FROM THE
3539
precipitated immediately and, after coagulation, were isolated by filtration to give 3.34 g. (air dried) of the mixture. Trituration of the amine mixture with petroleum ether (b.p. 30-60') gave, as the soluble component, 1.53 g. of 1methyl-4-ghenyl-4-piperidyl phenyl ketone (VII), m.p. 77.0-78.5 . The insoluble residue consisted of 1.44 g. of 4-hydroxy-1-methyl-4-piperidyldiphenylcarbinol(V), m.p. 158.0-160.8'. These products account for 89% of the starting material. ( b ) At 40-50°.-A suspension of 4.0 g. of l-methyl-4piperidylidenediphenylmethane ( X I I ) in 250 ml. of water was treated with 7 ml. of 48% hydrobromic acid a t 40-50" and 100 ml. of bromine water was added in portions. A large amount of undissolved solid remained even after the addition of 250 ml. of water. The mixture was cooled and the solid was removed by filtration and triturated with acetone. The acetone insoluble portion, 0.85 g., was found to be unreacted X I 1 hydrobromide, m.p. 263-265'. Concentration of the acetone solution gave 1.51 g. of a hydrobromide, m.p. 150" dec., which gave evidence of being 4bromo-I-methyl-4-piperidyldipheiiylcarbinol hydrobromide (XIII). Anal. Calcd. for C19H23BrpNO: l B r , 18.1; 2Br, 36.2. Found: Br (Volhard), 18.5; Br (Stepanow), 33.4.12 The acid filtrate from the removal of XI11 and X I 1 hydrobromide was neutralized with potassium hydroxide and the solid which separated was triturated with petroleum ether (b.p. 30-60'). From the petroleum ether 0.75 g. of 1methyl-4-phenyl-4-piperidyl phenyl ketone (VII), m.p. 75-77', was obtained. T h e petroleum ether-insoluble portion yielded only traces of 4-hydroxy-1-methyl-4-piperidyldiphenylcarbinol (V).
Acknowledgments.-The authors wish to express deep appreciation to Dr. S. M. McElvain for aid in initiating this problem and encouragement and suggestions which promoted the completion of the work. The authors also wish to express appreciation for a sample of 1-methyl-4-phenylisonipeconitrile furnished them by Sterling-Winthrop Research Institute of Rensselaer, N. Y. (12) T h e t o t a l halogen analysis of XI11 was consistently low a s compared with t h e theoretical value: however, i t is sufficiently high t o indicate t h e presence of two bromine residues. T h e low value for t h e total halogen analysis undoubtedly results from t h e relative ease of elimination of t h e bromine on t h e 4-position. One recrystallization of XI11 f r o m acetone lowered t h e bromine content t o 31.3%.
DURHAM, SEW HAMPSHIRE
RADIOCHEMISTRY LABORATORY, DEPARTMENT O F CHEMISTRY, \vASHINCTON UNIVERSITY]
Radioisotopic Dilution Analysis for D-Glucose and Gentiobiose in Hydrol BY JOHN C. SOWDEN AND ALFREDS. SPRIGGS' RECEIVED FEBRUARY 22, 1954 The D-glucose and gentiobiose contents of a representative sample of hydrol have been determined by radioisotopic dilution analysis. The D-glucose content by this method agrees well with the corresponding value obtained elsewhere by direct iLo!atioii methods. In contrast, the gentiobiose content as determined by the present method is coiisiderably greater than hitherto reported
Hydrol is the residual sirup obtained after the commercial crystallization of D-glucose from acid hydrolysates of corn starch. Since mineral acids catalyze not only the hydrolysis of oligo- and polysaccharides but also the condensation of monosaccharides,2hydrol is a product both of the acid hydrolysis of starch and of the acid reversion of D(1) Corn Industries Research Foundation Fellow.
( 2 ) A . Wohl, Ber., 23, 2084 (1890): E. Fischer, ibid., 113, 3687 (1890); H.F r a h m , A n n . , 6 1 6 , 187 (1944); E. Pacsu a n d P. T. M o r a , THISJ O U R N A L , 72, 1045 (1950): W. R. Fetzer, E. K. Crosby, C. E. E n g e l a n d L. C. Rirst, 2 n d . E n g . Chen.,46, 1075 (1953).
glucose. Oligosaccharides arising solely from the hydrolysis of starch are necessarily limited in structure to moieties of the original starch structure whereas no such limitations can be applied to the structures of those oligosaccharides resulting from acid reversion of D-glucose. Consequently, although D-glucose appears to be the only monosaccharide constituent of hydrol, the oligosaccharide fraction is complex and as yet has not been resolved completely into its component sugars. The first disaccharide to be identified as a con-
stituent of this "corii sugar molasses" was gentio- Thc octnncctatc (200 mg.) was deacetylated according t o thc b i o ~ eand , ~ compelling evidence that it arises solely directions of Thompson and iVo1fron1'~to yield anhydrous 8 gentiohiosc (52 rng., ni.17. 183--19k", cii. X 0 , O O O ct.;./min.: as a reversion product from D-glUCOSe has been re- tng .) . cently reported4 G-o-cr-D-Glucopyranosyl-D-gluStandard Radioactive Sugar Solutions.-The radionctivc cose (brachiose, isomaltose) also has been identified sugars were dissolved scpuratelp ill water t o give solutions in hydrol and can be considered partly as a product contailling 2-2.5 mg./ml. These solutions w r e stanciardusing 0.03-ml. aliquots, against known D-glucose :ind of starch hydrolysis and partly as a product of D- izcd, gentiobiose, respectively, in the manner described prcglucose reversion. 4,5 Other disaccharides that have viously.9 The result? of the stnnrlardizntioii~ :re .lion.ll been recognized positively as constituents of the i n Ta1)lcs I ; t i i d I I , hydrol mixture are a,a-trehalose5 and T A ~ LI E The former can be only a product of reversion S I ' A N D A R D I Z A T I O N OF STOCK l - C ' 4 - ~ ~ - SO1,7,i G ~ . 10s ~ ~ ~ ~ ~ ~ while the latter may arise both by the hydrolytic Specific Total and reversion routes. n-Glurnsr, radioactivity. radicmctivity Sample nig. cts 'min./mp cts./min Previous analyses of hydrol for specific sugars 1 19.97 375.9 7507 or classes of sugars have depended upon direct iso33. t58 220.8 741.1 lation of the sugar? or their d e r i v a t i ~ e s or ~ , upon ~,~ : 3 ,j5, 35 133. i i:l98 copper reducing methods.8 I n the present work, 4 61 8 4 1-21] 7 71 11.4 we have applied radioisotopic dilution analysis to 1 ii. 20 05 4 X.;i determine the amounts of u-glucose and gentiobiose, cllccl; 48 20 1,i4 2 7442 respectively, in a typical sample of hydrol. The adAvcrnge 7420 5 3.i vantage of this method in the analysis of complex sugar mixtures9 stems from the fact that it does TABLE I1 not rely on the efficiency of isolating the coinpoSTASDARDIZATIOS OF SI,OCK Cl"-GEsnnrirrmr SOLGI~ION nent being determined. Our analytical results for Specific T,,tal D-glucose agree well with the values obtained by Cxmtiolliwe, radioactivity, r:i 49 3 greater than values (5-5.77;) reported hitherto for 4 68.6 82 I1 tX23 this d i s a ~ c h a r i d e . ~It, ~is believed that the pres5 80.8 09.1 5383 cntly reported concentration of gentiobiose in our Chcck 5S.C 95 7 5 m ssniple of hydrol is accurate within the small limits of error attending the determinations of specific Avcrnge 55'76 i Ri radioactivity. i)
,
Radioisotopic Dilution Analysis for D-Glucose in Hydrol. -A sninple of 372.4 g. of hydrol was diluted t o 1 1. with clistillcd water. T o aliquots of 10 nil. cnch of this solution Hydro1.-The sample of hydrol was kindly furnished by M r . George T . Peckham, J r . , Research Director, Clinton were added aliquots of 0.50 mi. each of the stock r:itlioactivc Foods, Inc., Clinton, Iowa. The manufacturers analysis D-glucose solution. The solutions were deionized b y pasof this material showed moisture, 25.287,; ash, 5.857,: sage through Amberlite I.R.-100'6 nnd Duolitc .4--11Gion protein ( N X 0.25), 0.19%. The carbohydrate content, exchange resins, the effluents werc coticcntratctl a t retlucctl pressure arid the rcsulting sirups \rere dried by rcpcntctl culculatcd as the rcmaitidcr, n-as thus 5,i.G8%.,. l-C14-D-Glucose.-The lahelecl sugar ( ( a . 0.0; pc./tng.) concentrations a t reduced pressure with absolute cthnno!. 5 7 , was prepared from n-arabinose xmii thc iiitronicth:inc syn- The dried sirups irere dissolved in 20-nil. portions of 9 ethanol and seeded with minute traces of 0-glucosc. Aftcr thesis with C'l-nitronicthanc.'~ C14-Gentiobiose.-The Inlieled disacchnridc i v n s syiithc- crystallization was complctecl a t E O " , tlic superii:itatlt liqriitl cth sized by the Koenigs-Knorr method with the modifications !vas decanted and the sugar washed with cold %i70 of Reyiioltls 2nd Evans." 'I-niformly Inbclcd n-glucoscI2 For radioassay, the D-glucose was rccrystnllizctl i o con (9.50 nig., c a . 0.085 pc./iiig:) was converted to tcti-:L-O- specific radioactivity from 95% ethnnol with iiltct-nictlintc drying a t 110" i n high vacuum over phosplioruq I w l t o s i t l v . acetyl-D-glucopyraiios~-lbromide (1.13 g . ) and the latter was condensed with noli-radioactive 1,2,3,1-tetra-O-ncetyl-~- The samplcs of sugar tlius purifictl shoivctl i n . I ) . I 15.5- I liiO and [cz]*~D52.1°, equil. in r a t c r . ~ - g l t r c o p y r a i i o i e(0.905 ~~ g . ) t o give B-gcntioliiose octa:iceAs a. Glint check, thc analysis \vas rcpeatctl after llic ncltlit : i t e (0.821 g., n i . 1 7 . l R . i C , ~ N ] ~ -~t jI . lI c , c 2 in chlorofornl). -___ tion of a knowii ainouiit of D-glucose t o an aliquot of thc liy(:3) FI. Berlin, 'I'lIIS J O U R K A I . , 48, 1107, 2627 (1926). drol solution. The rrsults of t h c : u i n l v ~ c sfor r)-pIuco\c : ~ r < ' (4) A T h o m p s o n , 11.I,, K o l i r o n i a n d E. J . Q n i n n , ibi(l , 7 6 , 3002 qlionm in Table 111. ( I 9531. RadioisotoDic Dilution Analvsis for Gentiobiose in Hv (,i) E d n a lf.h f o n t s o m e r y a n d F. n. XVenkicy, J . Arcor. 0 . f i r . A j i . dro1.-To d q u o t s of 25 ml. each of L: solution cotitniiiiiig Chcniisis, 36, I0:K (1933). 332.3 g . of hydrol in 1 I . were ntlded :iliquots of 2.0(1 i n l . e x l i (C,) Celloliiose rerently hns been isolated in t h e form o i its crystalline of the stock radioactive gcntiobiose solution. T h octnacetate f r o m our sample of hydrol. Experiments are currently in then were chromatographed 011 columns (:300X progress t o determine whether this sugar is formed by reversion during n a r c o G-6O17-Celite-5:35I8 (1: 1 hy weight),19tr-ith water. t h e acid hydrolysis of starch or whether it is a n a r t i i a c t in this cnse, 57, ethanol and 1.57, ethanol as successive elution solverits.*" nrising f r o m cellulosic impurities i n t h e original starch. Gentiobiose was isolntcd from the lc5yocthaiiol cfflucitts-
Experimental
~
( 7 ) C . D. I-Inrd a n d S . 31. C a n t o r , THISJ O U R K A I , , 6 0 , 2G77 (1938). ( 3 ) 0 . T. Peckhain, J r . , nnd C. E . Rnael. . I , AT.TO(.0.fir. A g i . Ciieiifictc, 36, 437 ( 1 9 3 ) . (9) J. C. Sowden a n d R. Schafler, THISJOURSAL,74, 499 (1!I,j2)l ( I O ) J . C . Sowden, J . Biol. Cheiif., 180, 53 (1919). (11) D. D. Rcynolrl.: a n d W. I,. Rvnnq. Tlrrs J O I ' R F A I . , 6 0 , 233!1 (1928). (121 W e are indebted t o Professor \T. Z . IIasnirl, Unisersity of c:ilifornin, f o r generously supplying t h i s material. (13) Prepared according t o FI. A. T,nrdy and 11. 0.I.. Fisclirr, J . R i d C h ~ ; n .164, , 513 (1946).
(11) A. 'Thompson a n d I f . I.. TVolfrom, T i m J C ) I T R N * I . , 75, 3fX; iI !)i3) , (1-5) A product of R o h m a n d Haas Co., Philadclphi;~. I'cnnn. ( I C ) A product of Chemical Process Cn., Iled\v-ood City, C;ilii 117) A product of Atlas Poiviler C o . , h-ew York, h Y. (18) A product of Johns-hlanvillc Co., Kerv T o r k , h-.Y.
(19) R. I,. Whistler a n d 1).
1'. I l n r w , Tlr1.r J o r r ~ IL, s 7 2 , fi77
(1 950).
( 2 0 ) hI. I,. Wolfrom, A . T h o m p s o n , A . N. O'Seill nn,l T . '1' koivqki, ibiii., 74, I o n 2 ( 1 9 j 2 ) .
Gnl-
July 5, 1954
A CONVENIENT METHOD OF TABLE I11
D-GLUCOSE A N D GENTIOBIOSE CONTENT OF HYDROL Specific radioactivity of sugar, cts./min./mg.
46.9 46.6 46.4 4G.7 41 Oh
T o t a l weight of sugar, g.
Sugar in hydrol,a 72
D-Glucose 1.582 1,592 1.599 1.588 1.784
64.7 65 1 05.4 64.9 64 8“
Gentiobiose 451 0.495 9.07 8.88 460 ,485 31Fb .705 9.25’ a Based upon manufacturers assay of 65.68% ash-free, protein-free dry solids. Artificial hydrol sample made by adding 200 mg. of sugar to aliquot of hydrol solution. c After subtracting 200 mg. of known, added sugar. Sample
[COS I‘RIBUTIOS
FROM THE
PREPARING
2-DEOXY-D-RIBOSE
334 1
calculation: Weight of hydrol, 3.724 g.; D-glucose, g. = 7420 X __ X 0.001 = 1.582; u-glucose in hydrol, % = 46.9 1.582 X 100 = 64.7. 3.724 X 0.6568 after acetylation, as the p-octaacetate,?O m . p . after recrystallization from 95% ethanol, 194-195’; yields, 0.35-0.40 g. Deacetylation’P gave crystalline (3-gentiobiose. For radioassay, the sugar was recrystallized“ to constant specific radioactivity with intermediate drying a t 110” in high vacuum over phosphorus pentoxide. A check analysis was performed on a n aliquot of the hydrol to which known gentiobiose had been added. The results of the analyses for gentiobiose are shown in Table 111.
Acknowledgment.-We are pleased to acknowledge the generous support of this work by the Corn Industries Research Foundation, New York, N. U. ST.LOUIS,MISSOURI
DEPARTMENT O F CHEMISTRY, LVASHINGTON UNIVERSITY]
A Convenient Method of Preparing 2-Deoxy-D-ribose1 BY JOHN C. SOWDEN RECEIVEDMARCH1, 1954 The biochemically important sugar 2-deoxy-D-erythra-pentose (“2-deoxy-~-ribose,”“thyminose”) has been prepared in satisfactory yield from D-glucose in two simple steps. D-Glucose first was isomerized according t o the directions of Nef by hot, concentrated alkali t o give, among other products, a mixture of 3-deoxy-~-ribo-hexonicand 3-deoxy-~-arebo-hexon1c acids (the “D-dextro-weta-saccharinic acids”). T h e 3-deoxyhexonic acids then were degraded by the method of Ruff with hydrogen peroxide in the presence of ferric acetate to give the deoxypentose.
I n spite of intensive efforts,2 no simple and economical source has been found for 2-deoxy-~ribose since the discovery of this sugar as a constituent of the nucleic acids.3 The preparation of the deoxypentose, either from natural sources or through synthesis, has remained sufficiently difficult to hamper seriously any thorough study of its chemical or biochemical behavior. A preparation from D-glucose which makes 2-deoxy-~-ribosereadily accessible now has been accomplished. During their extensive studies of the saccharinic acids that are formed by the action of alkali on reducing sugars, Kiliani and co-workers isolated a crystalline 3-deoxyhexonic lactone through treatment of lactose4or galactose5with lime-water. The corresponding acid, when subjected to a Ruff6 degradation, produced a crystalline 2-deoxypentose7 (“metasaccharopentose”). Kiliani did not assign configuration to the deoxypentose, but it became apparent that the sugar was 2-deoxy-~-threo-pentose when Levene and Moris synthesized it from Dxylose by the glycal method. The respective constants recorded for the two preparations were : “metasaccharopento~e,”~ m.p. 95”, [ a ]0”~ in wa(1) A preliminary report of this work appeared in Abstracts Papers, Am. C h e m . Sot., 1 2 4 , l 5 D (1053). ( 2 ) W, 0.O v e r m d a n d A?. Stacey, A d v . Carbohydrate Chem., 8 , 45 (1953). (3) P. A. Levene a n d E. S. London, J. Bioi. Chem., 81, 711 (1929); 83, 793 (1029). ( 4 ) H. Kiliani, B e v . , 16, 2625 (1883). ( 5 ) H. Kiliani a n d H. S a n d a , ibid., 26, 1649 (1893). (6) 0. Ruff, ibid., 31, 1573 (1898); 34, 1362 (1901). (7) H. Kiliani a n d H. Naegeli, ibid., 36, 3528 (1902); H. Kiliani a n d P. LoeRler, ibid., 38, 2667 (1905). (8) P. A. Levene and T. hlori, J . Bioi. Chem., 83, 803 (1929).
ter; m.p. of benzylphenylhydrazone, 117-1 18” and “2-~ylodesose,~” m.p. 92-96’, [ a ] D -2” in water; m.p. of benzylphenylhydrazone, 116-115”. It is interesting that Levene and Mori apparently overlooked the preparation of Kiliani and Naegeli since they state, with reference to their own product, (‘xylodesose has now been prepared for the first time.” Beginning with D-glUCOSe, Nef isolated an epimeric pair of crystalline 3-deoxyhexonic lactones from the action of aqueous sodium hydroxide on the sugar.g He assigned to the acids the D-ribo and D-arabo configurations on the assumption that they were formed intramolecularly from D-glucose with preservation of the D-erythro configuration on carbons 4 and 5 . In addition, Nef made the observation that D-ghCOSe, when treated with hot, concentrated alkali, produces these two acids in a combined yield of about 20%. Thus, if the configurations assigned by Nef for these acids were correct, it was apparent that they should be, through application of the Ruff degradation, a fruitful source of 2-deoxy-~-ribose. Such, indeed, is the case. It has been found that, by combining Nef’s conditions for the alkaline isomerization of D-ghlCOSe with a subsequent Ruff degradation of the crude 3deoxyhexonic acids,lo 2-deoxy-~-ribosecan be obtained readily. Approximately 5 g. of the deoxypentose is produced in this way from 100 g. of Dglucose. (9) J. U. Nef, A n n . , 576, 1 (1910). (10) Shortly after t h e initial report of the present work,’ t h e degradation of “calcium dextro-meta-saccharinate“ b y t h e Ruff method to give 2 - d e o x y - ~ - r i b o s ewas reported by G . N. Richards, Chem. and Ind., 39, 1035 (1953).
