Phosphorylated sugars. Part XIX. Synthesis of 3-deoxy-D-glycero-pent-2-ulosonic acid 5-(dihydrogen phosphate)

摘要

604 J.C.S. Perkin IPhosphorylated Sugars. Part X1X.l Synthesis of 3-Deoxy-~-g/ycero-pent-2-ulosonic Acid 5-( Dihydrogen Phosphate)By FranGois Trigalo and Ladislas Stab6, Equipe No. 55 du Centre National de la Recherche Scientifique,The title compound was obtained both by oxidation of ’ xylometasaccharinic acid ‘ 5-phosphate with sodiumchlorate-vanadium(v) oxide and by treatment of ’ isosaccharinic acid ’ 6-phosphate with periodate. Its stabilitytowards acid and base is similar to that of higher homologues.lnstitut de Biochimie, Universite de Paris-Sud, 91 405 Orsay, France3-DEOXY-D- AND L-g~ymO-PENT-2-ULOSONIC ACIDS are or pyruvate PIUS glycolaldehyde or g1yCOliC acid,lknown to be intermediates in D- and L-arabinose metabol- formed through the action of an ald~lase.~ The synthe-ism in Pseudomonads,2 the end products being either a- sis of 3-deoxy-~-glycero-pent-2-ulosonic acid &phosphateoxoglutaric acid, formed from the ulosonic acid via 2,4- (I) (‘ 2-keto-3-deoxy-~-arabonic acid &phosphate ’), .. - -&oxopentanoic acid (‘ a-ketoglutarate semialdehyde ’)1 Part XVIII, A. Chiron and P. Szabo. weceding Paper.A. C. Stoolmiller and R. H. Abeles, J. Biol. Chem., 1966,241,5764.a N. J. Palleroni and H. Doudoroff, J. s o l . Che&.,*1666, 223, 4 A. S. Dahms and R. L. Anderson, Biochem. Biophys. Res.499. Comm., 1969, 36, 8091975 605higher homologues of which have been prepared earlier:is described here.The phosphorylated 3-deoxypentulosonic acid wasprepared by two independent syntheses.In the first,xylometasaccharinic acid ' 5-phosphate (a mixtureof 3-deoxy-~-evytho- and -D-threo-pentonic acid 5-phos-phates) (11), obtained by alkaline treatment of D-xylose 3,5-phosphate,7 was oxidised with chlorate andvanadium(v) oxide 8 and the ct-oxo-acid (I) formed wasisolated as the lithium salt after ion-exchange chromato-graphy.6" In the second, ' isosaccharinic acid ' 6-phos-phate [a mixture of 2-C-(hydroxymethyl)-~-erythro- and-D-threo-pentonic acid 6-phosphates] (111), obtained byCOzH CO, H CO2HI I Ico C H,OH CO H,CH, OHI I ICH2 I CH2 I CH2 IHCOH HCOH HCOHI I IH, COPO,H, H2 COPO, H, H, COP0,H2( 1 ) (nl cm,alkaline degradation of D-glucose 4,6-~hosphate,~ wastreated with 1 mol. equiv. of periodate and the 3-deoxy-pentulosonic acid phosphate (I) formed was isolated asabove.In the semicarbazide test both products gavea molar absorption coefficient value of 10,OOO and in thethiobarbiturate reaction,1° carried out after treatmentwith an acid phosphatase, a value of 90,000 was obtained,as expected; 1 mol. equiv. of formaldehyde was formed(Figure 1) during the periodate-thiobarbiturate reaction.When treated with O-lN-sodiurn hydroxide at 50°,compound (I) undergoes cleavage of the C(3)-C(4) bond,pyruvic acid aud (presumably) glycolaldehyde phosphateFIGURE 1 Formaldehyde formation and reaction kinetics of 3-deoxy-D-glycero-pentulosonic acid &phosphate in the periodate-thiobarbiturate test after enzymic dephosphorylation : pen-tulosonate 6 x ~O-*M, NaIO, 36 x 1 0 - 4 ~ in O.~N-H~SO, a t 0"being formed.Although the latter eventually yieldsinorganic phosphate, pyruvate and mineral phosphate are5 (a) F. Trigalo, W. Jachymczyk, J. C . Young, and L. Szabb,J.C.S. Perkin I , 1975, 593; (b) D. B. Sprinson, J. Rothschild,and J. M. Sprecher, J . Biol. Chem., 1963, 238, 3170; F. Trigalo,M. Level, and L. Szab6, J.C.S. Perkin I , 1975, 600.W. Jachymczyk, L. MCnager, and L. Szab6, Tetrahedron,1966, 21, 2049.7 J. G. Moffat and H. G. Khorana, J . Amer. Chew. Soc., 1957,79, 1194.not produced in parallel (Figure 2), as is the case when 3-deoxy-~-erythro-hex-2-ulosonic acid is treated similarly.0.751/.0 .' r I I I I L I I I I I Ilo t l h 20FIGURE 2 Formation of pyruvic acid (PA) and inorganicphosphate (Pi) during the treatment of 3-deoxy-~-glyfxro-pentulosonic acid &phosphate with OlN-NaOH at 60"The discrepancy is explained by the rapid formation ofinorganic phosphate from triose phosphate by p-elimin-ation in the latter case, a mechanism is not operativewith glycolaldehyde phosphate .230 240 250 260 270 280 290 300h /nmFIGURE 3 Evolution of the absorption spectrum of 3-deoxy-D-glycero-pentulosonic acid &phosphate (4-16 mg) treated withO-~N-HC~ (25 ml) a t 96"; spectra were taken at the timesindicatedWhen exposed to OSlN-acid at 9 5 O , compounds absorb-ing at 230 and 255 nm (Figure 3) and inorganic phosphate(Table) are formed, as is the case with S-deoxy-~-erythro-hex-2-ulosonic acid 6-phosphate ; 5a however both reac-tions proceed much more slowly with the pentulosonateFormation of inorganic phosphate (Pi) from 3-deoxy-~-glycero-pent-2-ulosonic acid &phosphate (4.16 mg)treated with O-~N-HCI (25 ml) at 95'Time (t/h) 1 2.6 5.5 7 2s% Pi 7 16.6 38 46 90than with the hexulosonate: for instance, half-lives forester-bound phosphate are about 4 and 9 h for the phos-phorylated 3-deoxy-hexulosonic and -pentulosonic acids,* P.P. Regna and B. P. Caldwell, J. Amer. Chem. SOC., 1944,66, *243.J. McGee and M. Doudoroff, J . Biol. Chem., 1964, 210, 617.l o D. Charon, R. S. Sarfati, D. R. Strobach, and L. Szabb,European J . Biochem., 1969, 11, 364606 J.C.S. Perkin Irespectively. Since, according to the mechanism pro-posed l1 for the acid-catalysed degradation of 3-deoxy-aldulosonic acids, the formation of 2-furoic acid must bepreceded by the departure of the phosphate group in thecase of 3-deoxypentulosonic acids, the relatively lowrate of acid-catalysed hydrolysis of the phosphate groupmay be the f_actor responsible for the overall slackeningof the degradation.EXPERIMENTALAll evaporations were carried out under reduced pressurebelow 40".3-Deoxy-~-glycero-~ent-2-uloson~c A cid 5- (DihydrogenPhosphate) (I) .-(a) A suspension of ' xylometasaccharinicacid ' &phosphate, barium salt 6 (450 mg) in water (10 ml)was treated with Amberlite IR120 (H+) resin (15 ml) ; themixture was stirred (5 min) and the solids were filtered offand washed.&Lithium hydroxide was added to thefiltrate, which was then heated on a water-bath until the pHremained constant a t 9.The solvents were then removedand commercial vanadium(v) oxide (3 mg) and water (1 ml),containing phosphoric acid (85%; 0.035 ml; d 1.71), wereadded to the dry residue. The pH of the solution wasadjusted to 4.6-4-8 with pyridine or 8.5% phosphoric acidand the mixture was stirred in a closed vessel for 5 days.It was then passed through a column (30 ml) of AmberliteIR120 (H') resin and the pH of the acid effluent and wash-ings (50 ml) was adjusted to 7.5 with aqueous N-ammonia.As estimated with sernicarba~ide,~ the solution contained0.35 mmol of a-oxo-acid. It was passed through a column(5 ml) of Dowex 1 x 8 resin (Cl-; 100-200 mesh); thecolumn was washed with water and eluted with 0.01~-hydrochloric acid (60 ml h-l).Collected fractions (each12.5 ml) were tested for phosphorus content.12 When thiswas nil, the elution was continued with 0-0%-hydrochloricacid and the a-oxo-acid and phosphorus contents of thefractions were estimated. Those containing the title com-pound were pooled, brought to pH 6.9 with lithium hydrox-ide, and concentrated to 3 ml. The pH was adjusted to 7.2with N-lithium hydroxide and the lithium salt of the phos-phorylated pentulosonic acid was precipitated with ethanol(60 ml). The precipitate (80 mg, 30%) was collected bycentrifugation, washed with ethanol until free of chloride(3-4 times) and once with acetone, dried in vacuo, andequilibrated in air. It had [a)D22 +1.6" (c 0-5 in water)(Found: C, 22-65; H, 2.8; P, 11.5. C,H,Li,O,P,H,O re-quires C, 22.7; H, 3.0; P, 11.7%).(b) To a stirred suspension of ' isosaccharinic acid ' 6-phosphate,s barium salt, (481 mg, 1 mmol) in water (10 ml),Amberlite IR120 (H+) resin (15 ml) was added; 15 min laterthe resin was filtered off and washed.N-Lithium hydroxidewas added to the pooled effluent and washings, which werethen heated on a water-bath until the pH remained constanta t 9. The solvent was then removed and the residue dis-solved in water (5 ml); to the stirred solution an aqueoussolution (10 ml) of sodium periodate (214 mg) was addeddropwise during 30 min. Stirring was continued for another60 min, and the solution was passed through a column ofDowex 1 x 8 resin (Cl-; 100-200 mesh; 10 ml). Thecolumn was washed with water (50 ml) and then eluted(60 ml h-1) with OaOlN-hydrochloric acid. After the re-moval of iodate, 20 more fractions (each 12.5 ml) were col-lected and discarded; the product (200 mg, 76%) was theneluted with O.O2~-hydrochloric acid and isolated as in (a)(Found: C, 22.7; H, 2.8; P, 11.6%).[4/129S Received, 28th June, 19743l1 D. Charon and L. Szab6, J.C.S. Perkin I, 1973, 1178.l2 M. Macheboeuf and J. Delsal, BUZZ. SOC. chim. biol., 1943, 25,116