1975Phosphorylated Sugars. Part XV.' Syntheses of 3-Deoxy-~-erythro-and 3-Deoxy-~-threo-hexu~osonic Acid 6 4 Dihydrogen Phosphates)By Francois Trigalo, Witold Jachymczyk, John C. Young, and Ladislas Szab6,' Equipe No. 55 du CentreThe title compounds were obtained by oxidation of glucometasaccharinic acid and 3-deoxy- D-xylo-hexonicacid 6-phosphates with vanadium(v) oxide-potassium chlorate and isolated by ion-exchange chromatographyAlthough fairly stable in N-hydrochloric acid a t 50'. 3-deoxy- D-eryrhro-hexulosonic acid 6-phosphate is rapidlydestroyed by 0.1 N-hydrochloric acid a t 95'. When treated with bases of various strengths the same compoundundergoes aldol cleavage between C-3 and -4 and gives pyruvic acid and D-glyceraldehyde phosphate; thelatter is immediately transformed into DL-lactic acid and phosphate ion.Concomitant with this reaction, alkali-stable phosphate esters of unknown structure are also formed.National de la Recherche Scientifique, lnstitut de Biochimie, Universitd de Paris-Sud, 91 405 Orsay, France3-DEOXY-D-eYythtrO-HEXULOSONIC ACID 6-PHOSPHATE (11)and the D-threo-isomer (XVIII) have been identified byDoudoroff and his colleagues as intermediates of D-glucose and D-galactose metabolism in Pseudomonads.Both are cleaved by distinct and specific aldolasesbetween C-3 and -4 to yield equimolar amounts ofpyruvate and D-glyceraldehyde phosphate. The 6-phosphate of 3-deoxy-~-erythro-hexulosonic acid alsoappears during the metabolism of D-glucuronic andD-galacturonic acids 5a-e in bacteria; it is the keyintermediate for D-glucose metabolism in those organismsin which the lack of certain enzymes precludes the useof both the pentose phosphate cycle and the glycolyticPart XIV, P.Szab6, J.C.S. Perkin I , 1974, 920.J. MacGee and M. Doudoroff, J . Biol. Chem., 1954, 210, 617.J. DeLey and M. Doudoroff, J . Biol. Chem., 1957, 227, 745.4 C. W. Schuster and M. Doudoroff, Arch. Mikvobiol.. 1967,59,279.ti (a) W. W. Kilgore and M. P. Starr, J . Biol. Chem., 1959,234,2227; (b) G. Ashwell, A. J. Wahba, and J. Hickman, ibid., 1960,285, 1669; (c) J. Hickman and G. Ashwell. ibid., p. 1666; (d) J. D.Smiley and G. Ashwell, ibid., p. 1571.H. P. Meloche and W. A. Wood, Methods Enzymol., 1966, 9,61.A. Weissbach and J. Hurwitz, J .Biol. Chem., 1959,254, 705.' J. F. Wilkinson and M. Doudoroff, Scieme, 1964,144, 669.pathway. Although of importance for biochemicalinvestigations, both phosphorylated acids have, hitherto,only been prepared by enzyme-catalysed reactions a 7and are thus not easily accessible; nor have theirchemical reactions been studied. The chemical syn-theses and some reactions of 3-deoxy-~-erythro- and -D-threo-hexulosonic acid 6-phosphates are now described.Of the many synthetic methods elaborated forobtaining aldulosonic acids two have acquired practicalinterest: in the first, aldonic acids 298-13 are treated withchlorate in the presence of a vanadium oxide catalyst ; 144bin the second aldehydes are condensed with oxalacetic9 D. B. Sprinson, J.Rothschild, and M. Sprecher, J . Biol.Chem., 1963, 238, 3170.10 D. T. Williams and M. B. Perry, Canad. J . Biochem., 1969,47, 491.11 D. T. Williams and M. B. Perry, Canad. J . Biochem., 1969.47, 983.1s M. B. Perry and A. C. Webb, Canad. J . Chem., 1969, 47,2893.13 ( a ) D. Charon, R. S. Sarfati, D. R. Strobach, and L. Szab6.Euvopean J . Biochem., 1969,11, 364; ( b ) D. Charon and L. Szab6,J.C.S. Perkin I , 1973, 1175.14 (a) P. P. Regna and B. P. Caldwell, J . Amer. Chem. SOL,1944, 66, 243; (b) H. S . Isbell, J . Res. Nat. Bur. Stand., 1944, 88,45594 J.C.S. Perkh Iacid in a basecafalysed As it is wellknown that phosphate ester systems situated p to acarbonyl group, rapidly undergo elimination in alkalinemedium,18u-c the condensation of D-glyceraldehydephosphate with oxalacetic acid to give the requiredphosphorylated 3-deoxyaldulosonic acids was not con-sidered feasible.Therefore the oxidation route, whichhas already been successfully employed for the synthesisof phosphorylated aldulosonic acids l7 and 3-deoxy-ddulosonic acid^,^ was used, the starting materialsbeing the 6-phosphates of glucometasaccharinic acid lBb(I) and of 3-deoxy-~-xyh-hexoseIn the original method of Regna and Caldwell lb theoxidation by chlorate was carried out in the presence ofphosphoric acid and catalysed by vanadium pentoxide ;the reaction was allowed to proceed for 4 days. Sprinsonand his colleagues: when applying this procedure to theoxidation of 3-deoxy-~-gluco-heptonate 7-phosphateJobtained erratic results and therefore elaborated a newmethod for the preparation of the catalyst: theydissolved the vanadium oxide in concentrated hydro-chloric acid, added pyridine to the solution, and usedthe suspension thus obtained after adjustment of itspH to 3.2.The phosphorylated aldulosonic acid wasformed in yields of 40-60% after a reaction time ofonly 16.5 h. -The same catalyst has been used at aslightly higher pH value (4.6-4.8) for the oxidation ofD-gluconic acid 6-phosphate to D-arabino-hexulosonicacid 6-ph0~phate.l~ When glucometasaccharinic acid6-phosphate (I) was oxidised with this catalyst, an(XVI) .CO2H1COZH1CO2HICHOH co co (IIT)I I IIHCOHIHCOH2 x 10 ion-exchange resin with a formic acid-ammoniumformate system failed, the keto-acid being completelydestroyed during the removal of ammonium formate.Clean separation and excellent recovery were, however,obtained when a O~OS~-chloroacetic acid-O*125~-sodiurnchloroacetate buffer of pH 3.8 was used, the chloroaceticacid being removed batchwise, by ether extraction,after addition of small amounts of an acid ion-exchange25 50 75t l h0.750.500.2 5FIGURE 1 Formation of inorganic phosphate (Pi) and of CL-keto-acid (SC) during the treatment of glucometasaccharinicacid f3-phosphate wirh KC10,-V,O, (Sprinson type catalyst)resin to the pooled fractions containing the phosphoryl-ated keto-acid, which was then isolated as the alcohol-insoluble barium salt.It gave satisfactory analyticalfigures, and, in the semicarbazide test a molar absorbancyof 10,200 (pyruvic and a-ketoglutaric acids: 10,200).However, when submitted to enzymic analysis only50--6070 of the calculated amounts of triose phosphate(IV) and pyruvate (111) were formed.In view of theanalytical data it was clear that the preparation con-sisted, in fact, of a mixture of positioialisomers pre-sumably resulting from phosphate migration, and itI OH- I - 3- was possible to demonstrate this by ion-exchange HCOH - CHOH +PO,a-keto acid was readily formed (judged by the semi-carbazide test carried out as described previously 17).As with gluconic acid 6-phosphate,17 the formation ofinorganic phosphate (Figure 1) and of u.v.-absorbingmaterial was observed when the oxidation was carriedmt for prolonged periods; the highest yields of a-keto-acid were obtained when the oxidation was allowed toproceed for 20-25 h (Figure 1).Attempts to isolate the phosphorylated keto-acidafter separation by column chromatography on Dowex16 ( a ) J.W. Cornforth, M. E. Firth, and A. Gottschalk, Bio-chem. J., 1968, 88, 67; (b) M. A. Ghalambor, E. M. Levine, andE. C. Heath, J . Biol. Chem., 1968, 241, 3207; (c) C. Hershberger,M. Davis, and S. B. Binkley, ibid., 1968, 243, 1686; (d) D.Charon and L. Szab6, European J . Biochem., 1972, 29, 184.16 ( a ) D. M. Brown, F. Hayes, and A. R. Todd, Chem. Bey.,1967, 90, 936; (b) St. Lewak and L. Szab6, J . Chem. Sot., 1963,3976; (c) W. Jachymczyk, L. MBnager, and L. Szab6, Tetra-hedron, 1966, 21, 2049.chromatography ; however no information regardingthe number of isomers present and their structurescould be obtained by that method.In Part XIIm a method is described by whichisomeric phosphate esters of polyhydroxy-compoundscan be identified.As applied to sugar phosphates, themethod calls for reduction of the carbonyl function (toavoid interference of esters produced from cyclic forms),periodate oxidation of the resulting phosphorylatedpolyol, borohydride reduction of the aldehyde groupsthus formed and, finally, separation of the phosphoryl-ated fragments by paper electrophoresis. The mixtureof the isomeric phosphate esters of 3-deoxyhexulosonicacids was analysed by this method.3-Deoxyhexulosonic acids can form three isomeric17 F.Trigalo and L. Szab6, European J . Biochem., 1972, 25,18 K. Antonakis, A. Dowgiallo, and L. Szab6, Bull. SOC. chine.19 L. Szabo, Amer. Chem. SOC. Advances in Chemistry Series,20 F. Trigalo, P. Szab6, and L. Szab6, J . Chem. SOC. ( C ) , 1968,336.France, 1962, 1366.1968, no. 74, p. 86.9011975 595phosphate esters (4-, 5-, and 6-phosphates), and each ofthese can be expected to yield specific phosphate estersin the above reaction sequence. Indeed (see Scheme)the 6-phosphate (VI) should yield, as the sole phosphateester, ethylene glycol phosphate (VII), and no otherCO2HIcoICH2 III( YI 1 tyn 1HCOHHCOH CH2OHC H ,OPO,H 2.;,ii.iil. IH2COP03H2CHZOHIH2COHHCOH HCOHCH2OHICHOHI1 I1HCOPO jH2H2CO H( Y J I I ) I: IX 1H2COHI IIHZCOHI( X I ) ( X I I )HCOPO3Hz HCOPOjH2H,CO HCO2HICHOHICO,H1coI ICH2 I i.ii, i i i IHCOP03H2HCOHII ’H2COHt X l I I , H2COH( X Y )SCHEME Reagents: i, NaBH,; ii, NaIO,; iii NaBH,isomer can yield this fragment.The 5-phosphate(VIII), bearing no vicinal diol group, will appear largelyas glucometasaccharinic acid &phosphate (IX) ; how-ever, as a-hydroxy-acids are cleaved, albeit very slowly,by periodate,21 2-deoxyribitol 4-phosphate (X) shouldalso be detectable, as should be a 3-deoxyhexitolphosphate (XII), the latter arising from reductionof the lactone (XI) formed under the acidic conditionsof the periodate cleavage during the relatively long(48 h) exposure to this reagent.Neither of these com-pounds can be derived from the other two isomers.Finally, the 4-phosphate (XIII) should yield a phos-phorylated 3-deoxypentonic acid (XIV) together with asmall amount of 2-deoxytetritol phosphate (XV).Paper electrophoresis (pH 5 ; pyridinium acetate0 . 2 ~ ) of the products formed from the isomeric mixtureof phosphorylated 3-deoxyhexulosonic acids and revel-ation of phosphate esters gave the following results:besides the major spot of glycol phosphate (Mpi 0.86),which proved the presence of 3-deoxyhexulosonic acid6-phosphate in the isomeric mixture, phosphate estershaving the same mobilities ( M ) as 2-deoxyribitolphosphate (Mp, 0.64) and glucometasaccharinic acid5-phosphate (Mpi 1.03) were present, as well asanother phosphate ester (detectable with the periodate-benzidine spray22) which had a mobility lower thanthat of the periodate-benzidine-negative 2-deoxypentitolphosphate and identical with that of a 3-deoxyhexitolphosphate. The presence of these esters clearly indi-cated that 3-deoxy-~-erythro-hexulosonic acid 5-phos-phate (VIII) was contaminating the 6-phosphate.The absence of the 4-phosphate (XIII) in the originalmixture was ascertained in a separate experiment inwhich the postulated breakdown products, namely3-deoxypentonic acid phosphate 16c and 2-deoxytetritolphosphate, were used as markers: the mobilities ofthese two compounds (Mpi 1.12 and 0.72 for 3-deoxy-D-erythro-pentonic acid phosphate and 2-deoxy-D-glycero-tetritol 4-phosphate, respectively) are quite differentfrom those of the phosphate esters present in the mixture.As it was suspected that phosphate migration occurredduring the ion-exchange procedure, isolation of theketo-acid was next carried out on a Dowex 1 x 8 resin(Cl- form) and the phosphate was isolated as the lithiumsalt, but again material of only 70-80% purity, asassayed by enzymic analysis, was obtained.Nophosphate migration was detected in these preparationsbut in the tbiobarbiturafe reac€ion the enzymicallydephosphorylated compounds’ molar absorption co-efficient was only 80,000 instead of the theoretical95,000.It was then considered that the very active catalystmight have initiated some unidentified reaction (cj.following paper).The oxidation was therefore carriedout under Regna and Caldwell’s conditions l4= previouslyused for obtaining 3-deoxy-~-manno-octu~osonic ,I3a 3-deoxy-~-arab~no-heptu~osonic,~~~ and 3-deoxy-~-threo-hexulosonic acids.13b Although it was necessary toallow the oxidation to proceed for about 5 days toobtain reasonable yields (3040%), the a-keto-acid,which was separated from other material by ion-exchangechromatography in the chloride system and isolated as21 P. F. Fleury, G. Poirot, and Y. FiCvet, Compt. rend., 1946,220, 664; P. F. Fleury, J. E. Courtois, R. Perlks, and L. Le Dizet,Bull. SOC. chim. France, 1964, 347.zp J. A. Cifonelli and F. Smith, Arralyf. Chem., 1964, 28, 1132596 J.C.S. Perkin Ithe lithium salt, gave correct elemental and functionalanalyses and also assayed for more than 98% of 3-deoxy-D-erythro-hexulosonic acid 6-phosphate whentreated with the specific aldolase.For obtaining 3-deoxy-~-th~eo-hexulosonic acid 6-phosphate, 3-deoxy-~-xylo-hexose 6-phosphate l8 (XVI)was first oxidised by bromine to the correspondingCHO CO2H COzHI 1 II I Ico HCOH HCOHc H* CH* CH2[OI I & IHOCHIHOCH - HOCHphosphorylated deoxyaldonic acid (XVII), which wasthen further oxidised under Regna and Caldwell'sconditions to the phosphorylated aldulosonic acid(XVIII); it gave correct analytical figures both asregards composition and functional groups, but thepurity of the compound could not be checked byenzymic methods because the pure, specific aldolase wasnot available.Both compounds gave only very weak reactions withperiodate-thiobarbiturate indicating that no free vicinal4,5-&01 system was present ; however, after treatmentwith acid phosphatase both compounds gave theFIGURE 2 Kinetics of (A) 3-deoxy-~-erythvo- and (B) 3-deoxy-~-thveo-hexulosonic acid 6-phosphates in the periodate-thiobarbiturate test 130 after enzymic dephosphorylationtheoretical molar absorption coefficient of 93 x 103 inthe thiobarbituric acid test carried out as previouslydescribed ; ls0 but while the compound with D-erythYO-configuration reached this value within 5-6 h, thetho-analogue required 40 h exposure to periodate(Figure 2); their rates of reaction in this test maythus be used to distinguish between the two isomers.As measured by the semicarbazide test,2 S-deoxy-D-erythro-hexulosonic acid 6-phosphate is relativelystable in N-hydrochloric acid at 50" (Figure 3); nomeasurable amount of inorganic phosphate is formedduring 7 h.However, at higher temperature (e.g. atnon-phosphorylated deoxyaldulosonic acids (appear-ance of absorption bands at 230 and 260 nm, accom-panied in the present case by formation of inorganic- 251 2 3 4 5 6 1 0t t hFIGURE 3 Destruction of a-keto-acid during the treatment of3-deoxy-~-eryfhro-hexulosonic acid 6-phosphate with N-HClat 60"phosphate) are observed, indicating that similar re-actions occur in both cases. Formation of inorganicphosphate parallels that of the substance absorbing at260 nm (Figure 4).CI nh) cn 0 s15 20 1 ° t / h 5FIGURE 4 Formation of (A) inorganic phosphate and (B)material absorbing at 260 nm during acid treatment (O-IN-HC~ ;96") of 3-deoxy-~-u~ub~no-hexulosonic acid 6-phosphateThe same phosphorylated deoxyaldulosonic acid isvery unstable towards alkali: the kinetics of inorganicphosphate formation, catalysed by different concen-trations of sodium hydroxide at 50" are shown inFigure 5.The appearance of inorganic phosphate inthe early stages of the reaction at a rate incompatiblewith the generally low rate of base-catalysed hydrolysisof phosphomonoesters 23 (the latter is observed duringthe later stages of the reaction) suggested that an0 75 1 4 iir-*N , , ~ 0-01 N25.- -- 10 20 30 LO0t l hKinetics of inorganic phosphate formation duringthe treatment of 3-deoxy-~-erythro-hexulosonic acid 6-phos-phate with various concentrations of NaOH at 60"elimination reaction may be occurring. Such anelimination reaction could occur if, as is the case withFIGURE 62s C.A. Bunton, D. R. Llewellvn. K. G. Oldham. and C. A.96" in O-IN-HC~), phenomena similar to those seen-kith Vernon, J. Chem. SOC., 1968, 36741975D-arabino-hexulosonic acid 6-phosphate,17 pyruvate (111)and D-glyceraldehyde 3-phosphate (IV) were formed fromthe phosphorylated 3-deoxyhexulosonic acid by aldolcleavage, the phosphorylated fragment than undergoinga p-elimination of the phosphate to yield DL-lactic acid(V) and inorganic phosphate. That this is indeed thecase can be seen from the Table : formation of inorganicMol ofinorganic pyruvic lacticphosphate acid acidformed per mol of 3-deoxy-~-erythro-hexalulosonic acidt/h in 0-1N-NaOH at 50"1 0.395 0.34 0.3282 0-557 0.49 0.4983.76 0.714 0.62 0.604.5 0.749 0-66 0.646.5 0.776 0.676 0-656in O-h-NH,OH at 50'18 0.12 0.10 0-0324 0.14 0.13 0.0540 0.2 1 0.175 0.11phosphate is accompanied by the appearance of equiva-lent amounts of pyruvic and lactic acids as estimated bythe appropriate enzymes (as the enzyme used is specificfor L-lactic acid, the amount shown is twice that actuallymeasured).Towards 0-lwammonium hydroxide a t 50" , thecompound is far more stable (Table) : 12% of inorganicphosphate and 10% of pyruvic acid, but only 3% ofDL-lactic acid are formed in 18 h.The presence ofequimolar amounts of pyruvate and inorganic phosphateindicates that here, too, a retro-aldol reaction is operative.The formation of small amounts of lactic acid could beaccounted for on the basis of the observation thatalthough 3-O-methyl-~-glucose is degraded to 3-deoxy-D-erythro-hexosulose, the ultimate product is an imidazolederivative rather than metasaccharinic acid, the reactionof the intermediary a-dicarbonyl compound withammonia to yield the heterocycle being faster than thebenzylic acid type of rearrangement .24 Inorganicphosphate is also liberated when the phosphorylateddeoxyaldulosonic acid is treated, at 50°, with 0.1~-cyclohex ylamine or tet ramet hyl- or tet ra-n-butyl-ammonium hydroxide.As can be seen from Figure 5, only 50-60y0 of thetotal phosphate is released by this mechanism and itcan be shown by paper electrophoresis that, at the timewhen the rapid phase of phosphate release is terminated,two phosphate esters are present which do not give thecharacteristic reactions of the starting material andwhich both have an absorption maximum at 256 nm.Their structure is being investigated.EXPERIMENTALAll evaporations were carried out under reduced pressurebelow 40".3-Deoxy-~-xylo-hexonic Acid 6-Plzosphate (XVII) .-Toa stirred, aqueous solution (10 ml) of 3-deoxy-~-,~yZo-hexose6- (dilithium phosphate) dihydrate (obtained from thecalcium salt 18) (1.25 g) were added barium carbonateM.R. Grimmett, R. Hodges, and E.L. Richards, Austval. J .Chcm., 1968, 21, 606.(2 g ) and then, dropwise, saturated aqueous bromine, untila slight colour persisted. Stirring was continued for 1 h.The mixture was kept overnight a t 4O, then centrifuged;the sediment was washed with water (2 x 15 ml) and thecombined supernatant liquid was decationised on a columnof Amberlite IR120 (H+) resin (50 ml). The effluent wasneutralised with barium hydroxide solution, then heated(60-70"), and the pH of the solution was simultaneouslyadjusted to 7.5 until no further addition of base wasnecessary. The solution was concentrated (10 ml) andthe product precipitated with ethanol. The solid (1.5 g)collected by centrifugation and washed free of bromidewith ethanol had +8.8" (c 1 in 0*1~-HCl) (Found:C, 14.1; H, 3.0; P, 6.0.Calc. for C,H,oBa,.,0Q,3H,0:C, 13.9; H, 3.1; P, 6.0y0). The calcium salt, prepared byneutralising the decationised solution with aqueous calciumhydroxide as above, had +11.2' (c 1 in 0 . 1 ~ -HCl) (Found: C, 19-3; H, 4.1; P, 8.35. Calc. forC,H,oCal.,0QP,3H20: C, 19.4; H, 4.3; P, 8.35%).3-Deoxy-~-erythro-hexulosonic Acid 6-Phosphate [(II),(VI)] .-(a) Oxidation with vanadiurn(v) oxide. A mixtureof glucometasaccharinic acid 6-phosphate trilithium salt(940 mg, 3 mmol), commercial vanadium(v) oxide (9 mg),and potassium chlorate (129 mg, 0.99 mmol) wastreated with water (3 ml) containing phosphoric acid(0-105 ml; 85%; d 1-71). The pH of the mixture wasadjusted to 4.6-4.8 with either pyridine or 85% phosphoricacid and the reaction was allowed to proceed in a closedtube with constant stirring for 5 days.The mixture wasthen percolated through a column (2-8 x 15 cm) ofAmberlite IR120 (H+) resin and the pH of the effluentbrought to 7.5 by addition of concentrated ammoniumhydroxide solution (ca. 300 mi). The solution, which bythe semicarbazide test contained about 1 mmolof a-keto-acid, was slowly percolated through a column(1.2 x 8 cm) of Dowex 1 x 8 resin (100-200 mesh;C1-) and the column was washed with water (ca.100 ml). It was first eluted with 0-Oh-hydrochloric acid(80 ml h-l) and the fractions (12.5 ml) were analysed fortotal phosph~rus.~~ When the phosphorus content of thefractions became negligible, elution was continued withO~O2~-hydrochloric acid, the fractions being analysed forboth phosphorus and a-keto-acid. The pooled fractionscontaining the keto-acid were neutralised (pH 6.9) withN-lithium hydroxide and concentrated (to ca.5 ml). ThepH of the solution was adjusted to 7.6 and the lithium saltof the title compound precipitated with ethanol (100 ml) ,collected by centrifugation, washed free of lithium chloridewith ethanol, washed with acetone, and dried in vucuo a troom temperature (P,O,). After equilibration in air I s bthe compound (300 mg) had [,IDz2 + 6" (c 1 in H,O) (Found :C, 23.3; H, 3.7; P, 10.1. Calc. for C,H,,Li,O,P,ZH,O:C, 23.1; H, 3.85; P, 9.9%). When treated with 2-keto-3-deoxy-6-phosphogluconic aldolase i t yielded 0.98-0.99mol.equiv. of both pyruvate and D-glyceraldehyde phos-phate. In the thiobarbituric acid test i t had, after enzymicdephosphorylation, a molar absorption coefficient of(b) Oxidation with a Sprinson-type catalyst.s A sus-pension of commercial vanadium(v) oxide (160 mg) inconcentrated hydrochloric acid (d 1-19; 9 ml) was stirredfor 1 h. To the red solution pyridine (9 ml) was addedslowly, whereupon the colour changed through green to25 M. Macheboeuf and J. Delsal, Bull. SOC. Chim. biol., 1943, 25,116.93 x 103598 J.S.C. Perkin Iblue and a slight precipitate appeared. The catalyst wasused immediately. A solution of ghcometasaccharinicacid 6-phosphate lithium salt dihydrate (940 mg, 3 mmol)was decationised with Amberlite IR120 (H+) resin, thepH of the solution was brought to 9 with cyclohexyl-amine, and the mixture was heated on a water-bath,the pH being re-adjusted to 9 until it remained constant.The solution was then concentrated (15 mi) (5 ml per mmolof metasaccharinic acid phosphate), sodium chlorate ( 160mg, 0.47 mmol per mmol of substrate) and catalyst (4.5 ml)were added and, if necessary, the pH of the mixture wasadjusted to 4.6-4.8 with pyridine or concentrated hydro-chloric acid as required, The deep green mixture wasstirred in a closed vessel a t room temperature for 24 h.The pale green solution was passed through a column(3 x 30 cm) of Amberlite IR120 (H+) resin and thepercolate and washings were pooled, brought to pH 6.9with BN-lithium hydroxide and concentrated (10 ml).After adjustment of the pH to 7, ethanol (150 ml) wasadded and the precipitate was collected by centrifugation,washed free of lithium chloride with ethanol and dried.The mixed lithium salts (ca.860 mg), which contained400-450 mg of a-keto-acid (semicarbazide test) weredissolved in water (250 ml) and passed through a column(1.2 x 8 cm) of Dowex 1 x 8 resin (100-200 mesh; Cl-).The resin was washed with water (100 ml) and eluted(80 ml h-l) with 0 . 0 1 ~ - and 0.02n-hydrochloric acid and thephosphorylated a-keto-acid (300 mg) was isolated as above ;[a],,*, +6.5" (c 1 in H,O) (Found: C, 23-0; H, 4.0; P, 10.1.C,H8Li,0,P,2H,0 requires C, 23.1; H, 3.85; P, 9.9%).When treated with the specific aldolase it yielded 0-6-0.8mol. equiv. of pyruvate, and in the thiobarbituric acid testi t had, after dephosphorylation, a molar absorbance of75-85 x lo3.3-Deoxy-~-threo-hexucloson~c Acid 6-Phosphate (XVIII) .-The lithium salt of the title compound was obtained from3-deoxy-~-xyZo-hexonic acid 6-phosphate (lithium salt ;630 mg, ca. 2 mmol) by the procedure (a) described above.It has [a]D22 -8-2" (c 0.5 in H,O) (Found: C, 23.0; H, 3.6;P, 9.8. Calc. for C,H8Li30,P,2H,0: C, 23.0; H, 3.9; P,9.9%). The barium salt, prepared by passing a solution ofthe lithium salt through a column of Amberlite IR120 (H+)resin, raising the pH of the acid efAuent to 7 with bariumhydroxide solution, and precipitating the salt with ethanol,had [a]D22 +3.4" (c 0.5 in O-~N-HC~) (Found: C, 14.5; H,2.4; P, 6-0. Calc. for C6H,Ba,.,O,P,2H,O: C, 14.6; H,2.4; P, 8.2%).We thank Drs. M. Doudoroff and H. P. Meloche for theenzymic analyses and the French Government for fellow-ships (to W. J. and J. C. Y.).[4/1026 Received, 29th May, 1974
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