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P.ANDERSON, E. S . X ~ X WAKD E LR.LR1. BURTON
ratio of dichloropyridaziiie and the sodium thiophenolatc. The procedure was otherwise much as describpd in the case of thiosalicylic acid. X waxy, pale yellow solid was obtained as the crude product which separated during the reflux period. It was triturated well with ether and the creamy solid collected ( A ) . The aqueous material and ether were retained (B). Solid A (9.4 g. from 7.5 g. of 3,fi-dichloropyridazine; m.p. 138-140') was crystallized from cyclohexane (800 cc.) t o give feathery needles, m.p. 150-150.5' ( 5 . 6 g.). The liquors were retained for work-up. iliialyses indicated t h a t A was the bis compound yield, based on 4-chlorothiophenol consumed, as evaluated). Anal. Calcd. for C1cHIOC1zN?SA: C1, 19.41; S, 7.67. Found: C1, 19.51; llj,7.41. The aqueous liquors ( B ) from the crude product were extracted well with ether, and these combined with the ether washings. Extraction of the ether (retained as C) with 10% caustic ultimately led t o recovery of ca. 47, of 4-chlorothiophenol; C was washed with sodium chloride solution, dried and stripped of solvent, leaving a white solid which melted a t 94-96' (6.0g. from 7.5 g. of dichloropyridazine). Evaporation of the cyclohexane mother liquors from A left a white residue, m.p. 91.5-96' (3.6 g. from above run). Tlie crude materials were combined and crystallized from pentane to yield fine white needles, m.p. 96.5-97.5'. This was
Vol.
s1
3-chloro-G-(l-cliloroplicnyltliio)-pyrid~ziii~, a yield bcina obtained. Anal. Calcd. for CloHcClaN2S: N, 10.90; S, 12.47. Found: N, 10.73; S, 12.6:3. When two equivalents of sodium 4-chlorothiophenolate were employed, the bis type was formed in 997c yield. 3,6-Bis-(3-diethylaminopropylthio)-pyridazine Bis-(4-nitrobenzobromide).-This quaternary salt was made by mixing the requisite baselc with 2.1 equivalents of 4-nitrobenzyl bromide and heating on the steam-bath (without solvent) for an hour. The crude product was pulverized, triturated with acetone, boiled in acetone-ethanol, and then crystallized twice from ethanol. A 62% yield of powder having a n orange cast was obtained, m.p. 205-206.5° dec. Anal. Calcd. for C Z ~ H ~ ~ B ~ ? N F , O Br, ~ S19.91; ?: S, 7.99. Found: Br, 19.89; S, 8.20. I
Acknowledgments.-It is a pleasure for the authors to make recognition of the friendly interest which Dr. C. 11. Suter and (the late) Dr. J. S. Buck have shown in these researches, in addition to the generous support, given graciously. RESSSELAER, N. Y .
[COTTRIBUTIOS PRO11 THE S A r I O S A L INSTITUTE O F -1RTHRITIS A S D hlETABOLIC DISEASESAND 1HE XATIONAL INSTITUTE O F S E U R O L O C I C A L DISEASES ASD BLIKDNESS,XaTIONAL INSTITUTES O F HEALTH, PCBLIC HEALTH SERVICE, DEPARTMEKT O F HEALTH, EDUCATION A S D IYELFARE]
u. s.
Enzymatic Syntheses of C1'-Labeled Uridine Diphosphoglucose, Galactose 1-Phosphate, and Uridine Diphosphogalactosel BY E. P. A N D E R S O N , ~ELIZABETH S . MAXWELL AND ROBERT MAINBURTON RECEIVED MAY1, 1959 Methods are described in detail for the enzymatic syntheses of CI4-labeled uridine diphosplioglucose from glucose-C14,and of labeled galactose 1-phosphate and uridine diphosphogalactose from g a 1 a ~ t o s e - C ~T ~ h. e syntheses are feasible on a preparative scale, and essentially pure samples of the labeled uridine diphosphoglycosyl compounds can b e isolated in good yield. I n the synthesis from galactose, the intermediate galactose 1-phosphate can be isolated as the barium salt, or, in a somewhat different procedure, the over-all synthesis of uridine diphosphogalactose from free galactose can be carried out in a single incubation. Methods are also defined for the enzymatic synthesis, on a preparative scale, of C'4-labeled uridine diphosphoglucuronic acid.
Uridine diphosphoglucose (UDPG3), first discovered by Leloir and co-workers4 as a coenzyme for the transformation of Gal-1-P to G - l - P , 3has been shown to act as a glucosyl donor in a variety of enzymatic reactions for the biosynthesis of diand polysac~harides.~-~ UDPG also undergoes enzymatic conversions to UDPGal'O , I 1 and to (1) A preliminary report on part of this work has already ai,peared; E. P. Anderson and €I If.Kalckar, Absts. Am. Chem. Soc. 5C, Minneapolis, September, 19.55. (2) Fellow in Cancer Research of the American Cancer Society. Present address: liational Cancer Institute, ATational Institutes of Health, Bethesda, Maryland. (3) T h e following abbreviations are used: U D P G , uridine diphosphoglucose; UDPGal, uridine diphosphogalactose; UDPG.4, uridine diphosphoglucuronic acid; Gal-1-P, a-D-galactose 1-phosphate; G - l P, a-D-glucose 1-phosphate; G-6-P, glucose 6-phosphate; 6-PG, 6-phosphogluconate; A T P , adenosine triphosphate; A D P , adenosine diphosphate: U T P , uridine tiiphosphate; C T P , cytidine triphosphate; PP, inorganic pyrophosphate; P,, inorganic orthophosphate; D P N , diphosphopyridine nucleotide; T P N , triphosphopyridine nucleotide; *, C'd-labeled. (4) R. Caputto, I,. F. Leloir, C. E. Cardini and A . C. Paladini, J . Bioi. Cizrm., 184, 333 (1950). ( 3 ) E. Cabib and I,. F. Leloir, ibid., 231, 239 (19%58). ( 0 ) C. E. Cardini, I,. IT. Leloir a n d J. Chiriboga, ;bid., 214, I1!1 (19.55). ( 7 ) I,. F. Leloir and C. E. Cardini, ibid., 214, 1;17 (1952). ( 8 ) L. F. Leloir and C. E. Cardini, THISJ O U R N A L , 79, ti3-$0 ( l < l 5 7 ) , (9) L. Glaser, J . B i d . C h r m . , 232, (i27 (1938). ( l o ) L. F. Leloir. i l r c h . Biochein B i o p h y s . , 33, 180 (l!iZl),
UDPGA,I2 both of which have likewise been iniplicated as glycosyl donors in biosynthetic reactions. l3-I6 Because of the interest in having radioisotopelabeled uridine diphosphoglycosyl compounds for use in the exploration of such pathways of glycosyl transfer and as tools to assay for interconversions of the nucleotide compounds themselves, enzymatic syntheses were undertaken to label these compounds with C14 in the carbohydrate portion of the molecule. Such syntheses have been developed for UDPG and UDPGal, starting in each case with a readily available radioactive substrate, and achieving synthesis on a preparative scale in good yield and with a high degree of purity. In (11) H. A i . Kalckar, B. Braganca and A. Munch-Petersen, S a l i w e , 172, 1039 (1953).
(12) J. L. Strominger, E. S.hIaxwell, J. Axelrod and H. 31. Kalckar, J . Diol. Chein.. 224, 79 (1957). (13) J. E. Gander, W. E. Petersen and P. I). Boyer, Aici:. Biochcrii. R i o g h y s . , 60, 259 (195fi). (14) R. 31.Burton, 31.A. Sodd and R . 0. Brady, J . B i d C h e w . , 233, 1053 (1958). (16) I,. Glaser and I). H. Brown, P r o c . . Y d . .Acrid. Sei,, 41, 2.73 (1965). (16) A . RIarkovitz, J. A. Cifonelli and A. D o r f m a n , Biocliim. et Bioplzys. Acle, 28, 453 (1458). (17) H. 51. Kalckar, E. P. Anderson and I;. J . Isselbncher, ibid., 20, 2ti2 (19%).
Dec. 20, 1931)
ENZYMATIC SYNTHESIS OF C’I-LABELED URIDINE DIPHOSPHOGLUCOSE
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addition, methods are available for the enzymatic synthesis, on a preparative scale, of UDPGA-C.I4
the equilibrium is distinctly unfavorable for the forniation of G-1-P, and inorganic pyrophosphatase was used to pull the over-all reaction in the direction of UDPG synthesis by cleavage of the PP formed simultaneously (reaction 4). Experimental Glucose-C14, labeled either uniformly or specifically in carbon atom 6, was incubated together with hexokinase, Materials .-Uniformly labeled glucose-Ci4 (0.4 73 pc. / mg.) was obtained from Tracerlab, Inc. Glucose-6-C14 ATP, an ATP-regenerating system consisting of phosphopyruvate and pyruvic phosphokinase, phosphoglucomutase (2.22 pc./mg.) and galactose-l-C14 (2.13 pc./mg.) were prepared by the Xational Bureau of Standards. Crystalline and its cofactors, UTP, UDPG pyrophosphorylase and inurganic pyrophosphatase. U D P G formation was measured A T P (disodium salt), D P N , T P N and UDPG were obtained from either Sigma Chemical Company or Pabst Labora- by assaying D P N reduction in the presence of UDPG dehytories. Crystalline hexokinase was also purchased from drogenase,28a highly specific and essentially quantitative method; yields of 80-95% U D P G from glucose could be Fabst Laboratories. Crystalline phosphoglucomutase1* was achievetl. generously provided by Dr. Victor Najjar. PhosphopyruThe UDPG-C14 formed was isolated by conventional vate was a gift of M r . LVilliam Pricer. Pyruvic phosphokinaselg was kindly donated by Dr. ,Jerard Hurwitz and inor- methods, involving separation from non-nucleotide contaminant< by adsorption on and elution from charcoal, f d ganic pyrophosphatase20by Dr. Russel Hilmoe. Yeast U D P G pyrophosphorylase was prepared by the lowed by further purification with paper chromatography. method of Munch-Peterson, et U Z . , ~ ~ ; the cut representing The product so isolated was then assayed for radioactivity 0.45-0.6 saturation in the second ammonium sulfate frac- and, enzymatically, for UDPG content. The glucose-Ci* was found t o be incorporated without isotopic dilution. tionation was stored in 0.6 saturated ammonium sulfate, Yields up to 80% could be obtained with this isolation Drowhich improved the stability of the enzyme. UDPG cedure (giving a very satisfactory over-all yield from gludehydrogenase was purified according to the procedure of Stroniinger, et al. , 1 2 and glucose-6-phosphate dehydrogenase cose-C14of 65-i5 T,). This rnethod h i s been convenient for laborator)- synaccording to the method of Kornberg and Horecker.22 thesis and has also proved feasible for the commercial prepGalactokinase was prepared by the method of Leloir and aration of UDPG.z9 Other satisfactory procedures for the TruccoZ3 from galactose-adapted Saccharonzyces fragilis. biosynthesis of C D P G have also been described. The T h e organism was adapted to galactose by several transfers , ~G ~ a n g ~ l iof, ~Murthy ~ through galactose-containing liquid media.24s25 The galac- methods of Burma and I l l ~ r t i m e rof ~ ~ of Glaserg all :iIso depend upon UDPG tose-adapted yeast was then grown in 10-liter batches using and H a n s e r ~and pyrophosphorylase; the first two utilize leaf homogenates 2% galactose medium, a 105%inoculum, and constant vigorous aeration. The cells were harvested, after two days of plant tissues (sugar beet and Impatiens lzolstii, respecgrowth a t 30’, with a Sharples continuous flow centrifuge, tively), while the last two procedures, like ours, use the yeast enzyme. I n addition, an excellent chemical method33 washed with water and air dried. Liver Gal-1-P uridyl transferase was purified by the pro- and an isolative procedure34are also available. Of these ~ *a n g ~ l i , ~ Murtlly 1 cedure of Kurahashi and Andersonz6; preparation of the various workers, Burma and M ~ r t i m e r , G and H a n ~ e and n ~ ~Glaserg have also prepared the C14-labeled yeast fraction used in the alternative synt.hesis of VDPGal compound. A mixture of UDPG-C14 and U I ) P G X ~ - C ~ ~ is described below. UDPG and UDPGA.-UDPG-C1* was synthesized from has also been prepared enzymatically by crude extracts of Saccharomyces fvagilis . 3 5 free g1uc0se-C~~ by the following series of enzymatic reacIn a separate reaction UDPG could be converted nearly tionsi quantitatively to UDPGX with the UDPG dehydrogenase. Glucose* A T P +G*-6-P ADP hexokinase Details of this procedure have been described previouslyLz for the preparative sj-nthesis of unlabrled material and are G*-6-P G*-l-P phosphoglucomutase fully applicable to the preparation of labeled UDPGX from U DPG-Ci4. G*-1-P U T P f-+Procedure for the Preparation of UDPG-C14.-Various UDPG* PP U D P G pyrophosphorylase procedures were tried for carrying out the different erizymatic steps of the synthesis separately, but the simpler pyrophosphatase method of using a single incubation gave highly satisfactory results and was routinely used. Incubation of the reagents and enzymes on a small scale gave best yields; the reac2P, tion mixtures from several sinal1 vessels could then be pooled Free glucose was phosphorylated by A T P in the presence of for product isolation on a preparative scale. X total volume hexokinase (reaction I), the G-6-P formed was converted to of 10 ml. incubation mixture contained 5 pmolcs of glucoscG-1-P by phosphoglucomutase (reaction 2), and G-1-P was CI4 (labeled uniformly or in carbon atom 6), 7.5 pmoles of reacted with U T P in the presence of U D P G pyrophosphor- ATP, 30 pmoles of phosphopyruvate and 10 pmoles of U T P . ylase to form U D P G and inorganic pyrophosphate (reaction The reaction tris-(hydroxymethy1)-aminowas run in 0.1 3z7). The limiting step was apparently reaction 2 , in which methane buffer, pH 7 . 5 . Trace amounts of MgCl? ( 4 pmoles) and of glucose 1,6-diphosphate (0.008 p ~ n o l e were ~~) (18) V. 8 . Najjar, in S . P. Colowick and N. 0. Kaplan. “Methods in added, together with an excess of cysteine (275 pmoles) and Enzymology,” Vol. I, Academic Press, Inc., hTew York, N. Y., 1955, of each of the necessary enzymes, hexokinast, pyruvic phosp. 294.
+
+
+
+
.1
(19) A. Kornberg a n d W. E. Pricer, J . B i d . Chem., 193, 481 (1951). (20) L . A. Heppel a n d R. J. Hilmoe, ibid., 192, 87 (1951). (21) A. Munch-Petersen, H . M. Kalckar and E. E. B. Smith, Kgl. D a m k e V i d e n s k a b . Selskab. B i d . M e d . , 22, 3 (1955). (22) A. Kornberg and B. L. Horecker, in S. P. Colowick and N. 0. Kaplan, “Methods in Enzymology,” Vol. I, Academic Press, Inc., New York, N. Y., 1955, p. 323. (23) L. F. Leloir and R. E. Trucco, in S. P. Colowick a n d N. 0. Kaplan, “Methods in Enzymology,” Vol. I, Academic Frees, Inc., h-ew York, N. Y., 1955, p. 290. (24) R. E. Trucco, R. Caputto, L. F. Leloir a n d S. Mittelman, Arch. Biochem., 18, 137 (1918). (25) J. F. Wilkinson, Biochenz. J., 44, 460 (1949). (26) K. Kurahashi and E . P. Anderson, B i o c h i m . el B i u p h y s . A c f a , 29, 498 (1958). (27) This pyrophosphorolytic cleavage of U D P G has been described and certain characteristics of t h e enzyme studiedzl; use of t h e reaction for t h e formation of U D P G from G-1-P has also been reported (E. P. Anderson, H. M. Kalckar and A. Munch-Petersen, Pubbl. Staz. ZooI. N a p o l i , 29, 119 (1957)). T h e reaction is specific for U T P , a s distinguished from A T P or C T P , ‘ and can also be used for t h e assay of
U T P (H. M . Kalckar and E. P. Anderson, in S. P. Colowick and N. 0 . Kaplan, “Methods in Enzymology,” Vol. 111, ilcademic Press, Inc., New York, h’. Y., 1957, p. 976). ( 2 8 ) J. L. Strominger, E . S. Maxwell and H . M . Kalckar, in S . P. Colowick and N . 0. Kaplan, “Methods in Enzymology,” Vol. 111, Academic Fress, Inc., Xew Yolk, N. Y . , 1957, p. 974. (29) B y P a b s t Laboratories, Milwaukee, Wisconsin. (30) D.1P. Burma and I). C. Mortimer, A r c h . Biocliriii. B i o p h y s . , 62, 16 (1956). (31) N. C. Ganguli, J . B i d . Chem., 233, 337 (1958). (32) S. K . M u r t h y and R. G. Hansen, J . D a i r y Sci., 39, 925 (1956). (33) J. G. Moffatt and H. G. Khorana, THISJ O U R N A L , 80, 3750 (1958). (34) H . G. Pontis, E. Cabili a n d L. I?. I A u i r , Ijirichirn. cl B i o p h y s . A c t a , 26, 116 (1957). (35) R. E. Trucco, S a l u v e . 174, 1103 (1934). (30) T h e diphosphate in particular is needed in unly minute amounls and m a y not be a necessary addition if larger quantities of the phosphoglucomutase are used.18 T h e diphosphate used here was a generous gift from Dr. Hans Klenow.
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E. P.ANDERSON,E. S. AIAXWVELLAND I
