Nov., 19-16
SYNTHESIS OF MALTOSE-~-PHOSPHATE AND D - X Y L ~ S E - ~ - P I ~ O S P ~ I ~ 2135 TE
[CONTRIBUTION FROM THE
DIVISIONO F
PLANT NUTRITION. COLLEGE O F AGRICULTURE, UNIVERSITY O F C)ALIFORNIA]
Synthesis of Maltose-1-phosphate and D-Xylose-1-phosphate BY W. R . MEACHER AND W. 2. HASSID The linkage between any pair of contiguous rnoies of periodate and gave rise to one mole of glucose units in the unbranched starch chain is formic acid. These data are consistent with the the same 'as in the disaccharide maltose. Starch, view that the structure for maltose-I-phospliate 1-phosphate which is a polymer of D-glucose, may therefore also is glucopyranosido-4-glucopyranosebe regarded as a polymer of maltose. Since and the structure of D-xylose-I-phosphate is Dstarch is formed as the result of "de-phosphory- xylopyranose- 1-phosphate. The maltose-1-phosphate and the xylose- l-plioslytic" condensation of a large number of D-glUCoSeI-phosphate units, it was thought that maltose- 1- phate when treated with potatwphosphorylase do phosphate might be similarly converted by phos- not undergo polysaccharide forination. phorylase into a polysaccharide. Experimental Inasmuch as D-xylose and D-glucose are strucPreparation of Maltose-1-(barium phosphate).-@turally identical, except that D-glucose has an Octaacetyl maltose was prepared by the method of Liiig additional carboil atom attached to the six-mem- and Baker8 and converted to the cu-broiiiolieptaacetylberet1 rinp, it was also of interest t o examine the maltose by thc geiieral proccrlurc of I%rauiis,Yslightly possibility of conversion of D-XybSe-1-phosphate niodified as follows: The reaction mixture, consisting of B-octaacetylnialtose, glacial acetic acid and hydrogen brointo a polysaccharide, xylan, by the same enzyme. mide a t 0 ° , wa5 poured into ice watcr and extracted with D-Glucose-I -phosphate produced throu'gh en- chloroform. The chloroform solutioti of the hroinodcctyl zymatic breakdown of glycogen or starch is iden- maltose \vas washed with icc water and dried with calciuin tical with the synthetically prepared e ~ t e r ' .its ~ , ~chloride, filtered and evaporated in 'i~ciciioto a thick +up. After thc additioii of pctroleuni ether and repcatcd stirritig, structure being a-D-glucopyranose-1-phosphate. the sirup was converted into a white, amorphous mass. When acted upon by animal or plant phosphoryl- Thc product was dried i i i a vacuum t l e x c a t o r over potasase this ester is converted by a reversible reaction sium hydroxide. A yiciltl of Y7('; of a-hronioheptaacetyl into polysaccharide. However, neither P-D-ghl- maltose was obtained. I t s specific rotation in chloroforin cose- 1 -phosphateJ nor the a-forms of D-mannose- (c, 2 ) was [.ID + 1T8",compared with BraunsIg value of [a]D 180.24". 1-phosphate and D-galactose-I-phosphate are Twcilty-five granis of the cu-brotiiohcptaacetylnialtose acted upon by phosphorylase to form polysacchar- was dismlvcd iri 150 nil. of dry benzene in which was suspended a slight escess of t h e theoretical amount of freshly i(is.5 6 7 1Ialtixe-1-phosphate and D-xylose-1-phosphate prepared trisilver phosphate ncedctl to react with the The misturc wai refluxed for oiie were prepnrei.1 by treating a-acetobromomaltose $romoacrtylinaltose, hour with itiwhaiiic:tl \tirring, w i t h a calciuiri chloride arid cr-aceiol-lronioxylose with freshly prepared tube attached to thc coridenscr. After cooliiig, the mixtrisilver phosphate arid by partially hydrolyzing ture was filtered with suction on a paper coated with the acetylated trisugar phosphate derivatives in diatoinaceous silica, "Hyflo-Siipcr-Cel" (Johns-Maiivillc), decolorized with charcoal alii1 again filtcrcd with suction. methanol solution with dilute acid. Since these The beiizciie was reiiiovetl by cvaporatioii uiider reduced esters were prepared through the reaction of the pressure, t h e residue stirred with petroleum ellicr and the corresponding a-halogen sugar derivatives and amorphous product thus obtaiiied dried in vacuo. The trisilver pl~osphate,~ the resulting maltose-l-phos- specific rotation of this product in chloroform (c, 2) was [ a ]+ ~ 120'. S o attempt was iiiaile to further purify phate and D-xylose-1-phosphateare most probably this iritermediate compound which is presumably trithe a-forms. (heptaacetyl-nialtose-l-)-phospliate.The yicld wai 915; When subjlxted to oxidation with sodium peri- of the theorctical. The iiiterniediatc coinpound was partially hydrolyzvtl otlate, the n:ialtose-l-phosphate consumed three (1.2 .V hydrochloric acid iii incthariol as described by moles of periodate and formed one mole cif formic with Cori, Colowick arid Coril arid the product isolated as a acid. The wxylose- 1-phosphate consumed two gclatiiioub bariuiil salt. Tlrc halt was dissolved in a mini-
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(1) C. I;. Cori, :3, 1'. Cnlowick a n d Gerty T. Cori, J . B i d . C h e w , 121, 465 (1937). (2) (a) C. S Hnnes, l'roc. Roy. Soc. (London), Bl28, 421 (l!jlO).; (b) Bl29, 17-1 (l!l:.O). (3) 31 I, \Volfliom a n d I). E. Pletclier. THISJ O U R N A L , 63, 1050 (lY41). (4) 51. F,. Wolfrom, C. S. S m i t h and A. E. Brown, i b i d . , 65, 2.52 (1943).
(5) S. P. Colowick, J Bioi. Chpm., 124,557 (1938). ( 0 ) 1:. J . Reithel. THISJ U U R N A L , 67, 103G (1945). ( i ) Inasmuch a s these galactose a n d miinnuse phosphale esters were pre1>3re(I from the corresponding a-acetobromo-sugar derivative.; xnd tri5ilvcr p h m p h a t e by a method similar t o t h a t used t o prepare u - o - ~ l u c r , ~ e - l - p h o ~ i J hthey ~ l e , are probably t h e a-forms. Reithe16 bas shown t h a t Ccilowick'ss D-galactose-l-phosiJhate prep . i w l in t h i , w:iy i s t h e a - f o r m a n d has pointed o u t t h a t when mono,ilver phsph:+tei s u-ed t h e @-isomers of aldose-1-phosphates are
, Ihl :,,n'YI
atnotint of watcr, precipitated by the addition of 1.5 vo1unic.q of taiitcd by plotting log (u - x) against timc, where a is equal to the total amount of phosphorus and z is equal to the amount of hydrolyzed phosphorus a t time t. The rate constant was calculated from the slope of the curve, K = 2.3n3 1 log (a - x ) / .l t , and was fouiid to be 3.21 X The hydrolysis rate constant K for dipotassium glucoseI-i.'hosphatc (Cori ester) determined under the same conditioils wafi 4.36 X as compared with Cori and coworkers'' value of 2.99 X lo determined a t 37" and 0.25 :Y hydrochloric acid. The first ant1 second dissociation constants of maltose1-phosphoric acid were determined by electrometric titration of the salt with 0.1 -V hydrochloric acid u4iig a glass electrode. The pIi; was calculated by incans of ran Slykc'.;" forlnu1;l.
VOl.
68
showed t h a t the barium salt of xylose-1-phosphoric acid contained 1.5 molecules of water of crystallization. Anal. Calcd. for C6H9O8PBal1/2H20: C, 15.30; P, 7.89; Ba, 35.01. Found: C, 15.25;. P,. 7.95; Ba, 35.32. Preparation of D-Xylose-1-(dipotassium phosphate).Two grams of the barium salt of xylose-1-phosphoric acid was dissolved in 30 ml. of warm water and treated with an equivalent amount of 105; potassium sulfate solution. The precipitated barium sulfate was removed through a precoated filter and absolute ethanol was added t o the filtrate to incipient turbidity. T h e solution was allowed t o remain at room temperature and absolute alcohol was added from time to time until a total of about 1.7 volumes of ethanol \vas reached. The mixture was then cooled, filtered, the crystals washed with dilute ethanol and recrystallized from watcr by addition of a n equal volume of cthaiiol. A yield of 1.9 g. was obtained. The xylose-l(tlipotassiuni phosphate) thus prepared is a white, nonhygroscopic crystalline product, shown t o contain two molecules of water of crystallization. Its specific rotation in water (c, 2) is [ a ] ~76". The xylose-1-ester is readily hydrolyzed with dilute acid, b u t stable t o alkali. I t shows no Fchling reduction on prolonged boiling. The estcr is completely hydrolyzed to xylose and inorganic phosphate when heated for ten minutes in 1 N sulfuric acid in a boiling water-bath. On hydrolysis of the solution, an osazoiie was obtaincd, identified as xylosazone. .4nal. Calcd. for C ~ H 9 0 8 P K ~ 2 H ~ OC:, 17.54; H , 3.83; P, 9.05. Found: C, 17.40; H,3.69; P, 8.90. Rate of Hydrolysis and Dissociation Constants of DXylose-1-phosphate.-The velocity constant for hydrolysis of the potassium salt of D-xylose-1-phosphoric acid in 0.376 &Yhydrochloric acid was determined a t 36' as previously described. The value for K of this ester under these conditions is 8.21 X The dissociation constants of the D-xylose-1-phosphate calculated by means of Van Slyke's formula and the The pK; was calculated by means of the HendcrsonHeiitlersori-Hasselhach equation are: pK,' = 1.25; p K : = Hasselbdch equation (1.15. = pH - log B / ( C - fj) Oxidation of D-Xylose-l-(dipotassium Phosphate) with Sodium Periodate.-Thc saitic procedure was employed The disiociation coilstants thus obtainctl for this estcr for oxidizing thc sylose-1-phosphate with sodium periodate arc pk'i = 1.52; pk" = 5 8 9 as in the osidatioii of the maltose-1-phosphate. I n the Oxidation of Maltose-1-phosphate with Sodium Perioositlatioii of one inole of dipotassium dihydrate xylose-ldate.-The technique for 'oxidizing carbohydrates with sodium periodate was descril~ctlby Hudson and co-work- phosphatc 2.05 iiiolcs of periodate were used with the proers.1* 111oxidizing the itialtosc-1-phosphate with sodium ductioii of t.O,j iiioles of formic acid, which closely agrees ivith the t heorctical requirements of two moles of periodate periodate Wolfroni and Plctcher'sJ procedurc for oxidation of glueosi,-l-phospliate wa5 iollowctl. Previous to osida- aiitl O I I C iiiolc of foriiiic acid. Action of Potato Phosphorylase on Maltose-1-phosphate tioii with periodate t h e riialtose-l-(bariuiii phosphate) and D-Xylose-1-phosphate.-Solutions of D-xylose-1-(disamples were treated with sodium sulfate to remove the potassium phosphate) and maltose-I-(barium phosphate), barium. The results showed t h a t 3.02 moles of periodate from which the barium was removed with potassium sulwere consumed, giving rise t o 1.1 moles of foritiic acid in fate, w r e adjusted with acetic acid to p H 6.0 and treated the osidal ion of one mole of barium pentahydrate maltosewith potato phosphorylasc. I n a control experiment DI-phosphntc. The theoretical requireinen t for osidatioii gluco~e-1-pho.pIiate ivas iiiiiilnrly treated. These inixof oiic tiiolc of glucopyraiiositlo--L-glurol,yr~i~ioi~~-l-phostiire.; \yere aiialyzctl for iiiiirgaiiie 1)hospliorus at . phate 15: eoiiiiiiiil)tioii of thrcc illole\ of I)criotln(e a t i t 1 1 lie t hirty-minute intervali. Ko hydrolysis of phobphate orproductioii of oiic iiiole O f foriiiic acitl. Preparation of Xylose-I-( barium phosphate). .Tetra- currctl under the iiillueiicc o f potato phosphorylase with iiialtoie- t-phosphate or o-sylose-1-I)hospIiatc. Iiiorgaiiic aCetyl-D-xybSe and a-broiiiotriacctyl xylosc were prepared phobpliorus appearctl i i i thc esperiiiient with 1)-glucose-1 by the method of Hudsoii and Twenty-five grains of a-broinotriacetyl-D-xylose was phosphate. The fortilatioil of starch was also observed when the solution was tested with iodine. I I I the case of treated with trisilver phosphate and the interniediatc itialtose-1-pliosphatc and n-sylov-1 -phosphate the starchproduct was partially hydrolyzed with 0.2 N hydrochloric iritliiic tcst \vas itt,g:itivc. acitl i i i ~ i i e t h n i i ~az i l tlc~crilieili i i tlic preparation of nialtosc-l-(l)ariuiii phuspliatc). A white seeiniiigly amurAcknowledgment: 'I'hc work rcpurtcd i l l this phous product wa.; ot)tainetl in yicld of 4 , 2 g., which had a paper was supported ill part by a grant from the specific rotation in watcr ((., 2) of [ a ] n 65,". Analysis
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(10) The value fur K relmrteil by Cori, cl al ,I is actually givetr as 1.30 X IO-:$, but it sloes not tcike into account t h e factor 9.303 necessary to c o n v e r t the logm t o loga. (11) D. I). Van >,lyke, J. B i d C h c m . , 64, 325 (1922). (12) E. I.. J n r k w m and C S . Hudson, T H I SJ O U R N A L , 59, 00.1 (1937); 69, 938 (13.10); II J o. h n s o n , ' I H I S J O U R N A L , 37, 2748 (1915).
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Corn Industries Research Foundation.
Summary The barium salts of maltose-1-phosphoric acid and D-xylose-1 -phosphoric acid have been synthesized and characterized. The D-xylose-1-phosphate has also been prepared as the crystalline potassium salt.
Nov., 1946
ULTRAVIOLET ABSORPTION SPECTRA OF THIOURACILS
2137
Data obtained from oxidation of these esters in 0.376 N hydrochloric acid at 36' and also their with sodium periodate show that the maltose ester dissociation constants were determined. is glucopyranosido-4-glucopyranose-1-phosphate Maltose-1-phosphate and D-xylose-1-phosphate and that the xylose ester is D-xylopyranose-1-phos- are not converted t o polysaccharide by potato phate. Both esters probably exist in the a-form. phosphorylase. The rate constants for hydrolysis of these esters BERKELEY, CALIFORNIA RECEIVED JUNE 5, 1946
[CONTRIBUTION FROM THE WELLCOME
RESEARCH LABORATORIES]
The Ultraviolet Absorption Spectra of Thiouracils1 BY GERTRUDE B. ELION, WALTERS. IDEAND GEORGEH. HITCHINGS The ultraviolet absorption spectra of pyriniidines have received a good deal of attention in recent years. However, except for the recent paper of Miller, Roblin and Astwood2 on 2thiouracil and its oxidation product, no thiopyrimidine spectra have been reported in the literature. During the present investigation of the absorption spectra of the mono- and dimercaptoanalogs of uracil (2,4dihydroxypyrimidine) and thymine (2,4-dihydroxy-5-methylpyrimidine)it was found that 2-hydroxy--Linercaptopyrimidine (4-thiouracil) and 2-hydroxy-4-mercapto-Zmethylpyrimidine (4-thiathymine) possess absorption bands so far removed from the usual absorption range of pyrimidines as t o be unrecognizable as members of that group. It is interesting that the anomalous spectra of these +thioderivatives are paralleled by their lack of antithyroid activity. Thus, Astwood3 has reported that whereas 2-thiouracil and 2,4-dithiouracil have approximately the same antithyroid activity, 4thiouracil is practically inactive. Results and Discussion The ultraviolet absorption spectra of 2-thiouracil , 2,4-dithiouracil, 4-thiouracil, 2-thiothymine, 2,4-dithiothymine and 4-thiothymine a t pH values of 1.0, 7.0 and 11.0 are reported here. A comparison of the spectra of these thiocompounds with the corresponding hydroxycompounds reveals soiiie expected as well as some unexpected differences. According to the data of Loofbourow, Stinison and Hart,4 uracil ;kt pH 7 shows an absorption maximum a t 25S0 A. with a mo:lecular extinction coeficient ( e ) of 10,600. As might be anticipated from the increased niass of sulftir, the spectrum of ?-thiouracil a t 1)H 7.0 (Fig. l ) shows ~i shift i i i the wave length of maximurn absorption to 2740 A., with a n E. value only slightly higher than for uracil. The replacement of both oxygens by sulfur, as in (1) Presented before the Division of Organic C.hetnistry a t the 109th meeting of the American Chemical Society, Atlantic City, S . J., April, 1946. (7) Miller, Koblin and Astwooil, THISJ U U R N A L , 67, 2201 (1'345). (3) Astwood, 13issell and Hughes, E i z d o c r i ~ i o l o g y37, , 456 (1945). (4) Loofbourow Stimson and Hart, Txrs J O U R X A I . , 65, 148 (10431.
2,4-dithiouracil (Fig. 2) results in a shift of the wave length of maximum absorption still further toward the visible range, and also the introduction of a second, weaker band a t 3600 8. The molecular extinction coefficient of this compound for the main band is much higher than for 2thiouracil. In contrast t o these two compounds,
c
2500 2900 3.730 \yaw leligtti i l l A. Fig. l.-.kbsorption spectra of %thiouracil: __ at pH 1.0; - - - a t p H 7 . 0 ; - . - . - a t p H 11.0.
the spectrum of 4-thiouracil (Fig. 3) is quite unusual. The pyrimidine band has been shifted so far toward the visible that in the usual range of pyrimidine absorption (2GOO-2800 A.) there occurs a minimum rather than a maximum in the spectral distribution curve. The peak occurs a t
