synthesis and kinase phosphorylation of 4-deoxy- d - threo...

Synthesis and Kinase Phosphorylation of 4-Deoxy-D-threohexolose

CLIFFORD RAYMOND HAYLOCK AND KEITH NORMAN SLESSOR Department of Chemistry, Simon Fraser University, Burnaby 2, British CoIumbia

Received January 15, 1973

HAYLOCK, C. R., and SLESSOR, K. N. Synthesis and kinase phosphorylation of 4-deoxy-D- threohexulose. Can. J. Biochem. 51,969-972 (1973).

Synthesis of the only unknown deoxyfructose, 4-deoxy-D-threohexulose, is reported. Its preparation involved reductive lithium aluminum hydride ring opening of 3,4-anhydro-1,2:5,6- di-0-isopropylidene-wtalitol, followed by hydrolysis of the resulting epimeric deoxy diiso- propylidene hexitols and selective Acetobacter suboxydans oxidation of 3-deoxy-D-arabino- hexitol. Kinetic studies using 4-deoxy-D-threohexulose as substrate for yeast hexokinase support the premise that the C-4 hydroxyl is a binding group in formation of the enzyme-substrate complex. Enzymatic synthesis of 4-deoxy-D-threohexulose &phosphate and 4-deoxy-D-threo; hexulose 1 ,ddiphosphate has been achieved in low yield from 4-deoxy-D-threohexulose.

HAYLOCK, C . R., et SLESSOR, K. N. Synthesis and kinase phosphorylation of 4-deoxy-D- threohexulose. Can. J. Biochem. 51,969-972 ( 1973 ) .

Nous rapportons la synthbse du seul d6soxyfructose inconnu, le 4-dksoxy-D-thre'ohexulose. Sa preparation nkcessite les Ctapcs suivantes : ouverture de l'anneau du 3,4-anhydro- 1,2 : 5,6-di-0- isopropylidkne-D-talitol par rCduction avec l'hydrure d'aluminium et de lithium, hydrolyse des d6soxy-diisopropylid6ne hexitols kpimbres obtenus et oxydation sklective du 3-dksoxy-D-arabino- hexitol par Acetobacter subsxydans. Le comportement cinktique de l'hexokinase de levure en prCsence du 4-d6soxy-D-thre'ohexulose comme substrat confirme 19hypsth&se voulant que l'hy- droxyle en C-4 soit un groupement de liaison dans la formation du complexe enzyme-substrat, A partir du 4-dtsoxy-D-thrbohexulose, nous avons realisti, avec un faible rendement, la synthtse enzymatique du 4-dCsoxy-D-thre'ohexulose 6-phosphate et du 4-desoxy-D-thrbohexulsse 1,6- diphosphate. [Traduit par le journal]

Introduction Yeast hexskinase has been shown to have a

reasonably broad range of specificity including its main substrates 6-D-fructofuranose, D-glu- csse, and D-mannose ( 1 ) . Although this enzyme has been shown to be rather specific for the C-3 to C-6 region of the substrate, the only deoxy sugars employed were 3-deoxy-D-glucose and 3-desxy-D-mannme (2). The only substrates

used to test 42-4 were D-galactose and I,$- anhydro-D-glucitol ( 1 $4-ssrbitan) ( 3 ) . This work describes the synthesis of the only unknown deoxyfructose, 4-deoxy-D-threohexulose (Fig. 1, 4); the reaction of this sugar with yeast hexokinase, and subsequently phosphofructokinase, and the isolation of the sugar phosphates in low yield.

CH20H CHzOH CH20Rl

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HC--O\ /CHj HAOH HLoH HCOH 1 C

HZC--O/ \CHI I CH20H

I CH20H

I CH2OR1

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970 CAN. J. BIOCWBM. VOL. 51, 1973

Materials and Methods Adenosine triphosphate, yeast hexokinase (type V).

potato acid phosphatase (type %I), and rabbit muscle phosphofructokinase (type I) were purchased from Sigma Chemical Company. Bovine serum albumin was obtained from Nutritional Biochemicals.

Evaporations were carried out at reduced pressure, at bath temperatures not exceeding 45". Freeze-drying was done at -40°/0.3 Tsrr for 24 h. Optical rotations were measured on a Perkin-Elmer spectropolarimeter (P22) at 24". Mass spectra of freshly prepared pertrimethylsilyl derivatives (4) were recorded on a Hitachi Perkin-Elmer RMU-7 at an ionizing potential of 80 eV and an inlet temperature of 50".

Paper chromatography was performed on Whatman 3MM paper with the following solvent systems: A (descending) ethyl acetate - pyridine - water (5 :4: 3 ) , B (ascending) 1-butanol - 1-propanol - acetone - 80% formic acid - 30% trichloroacetic acid (40:20:25 :25 : 15) with tetrasodium EDTA (4.0 mg/ 125 ml) added and the solvent run twice in the same direction (51, C (descending) ethyl acetate - pyridine - water ( 10:4:3). Reducing sugars and simple polyhydric alcohols were detected on paper with silver nitrate- sodium hy- droxide. Phosphorylated compounds were chromato- graphed after conversion to their sodium salts by passage through Bowex 50 (Na+) and detected with acid-molybdate. Migration of the sugar phosphate was measured relative to inorganic phosphate (R,) . 4-Deoxy -D- t!sreohexudose (4)

3,4-Anhydro- 1,2 : 5,6-di-8-isopropy1idene-~-ta1ito1 (1) ( 6 ) ( 15 g ) was dissolved in 200 ml of ethyl ether, and 3 g of lithium aluminum hydride were added with care since a vigorous reaction results. 'The reaction was complete in 1 h at room temperature. A solution of 50 ml of 10% aqueous NHaCl was added dropwise to destroy excess hydride, followed by labdB rnl of acetone and 300 ml of ethyl acetate. The suspension was filtered through a Celite pad and the residue washed thoroughly with ethyl acetate. The combined ethyl acetate fractions were evaporated to dryness. The residue was dissolved in chloroform, washed with water, dried with CaCL, filtered, and evaporated to give 15 g of syrup. The syrup was dissolved in 100 mI of 0.05 Kg HCl and kept at 100" for 30 min. The solution was neutralized by passage through Dowex 3 (OH-) and evaporated to a symp presumably containing a mixture of 2 and 3 ( 10.3 g), [aID - 18.2" (c, 0.8 water).

Acetobacrs~. suboxyd~ns (ATCC 62 1 ) was used under aseptic conditions to oxidize 2 in the presence of 3. A broth containing 7.5 g of the deoxy hexitols (2 + 31, 0-32 g of yeast extract powder, and 63 ml of water was prepared in a 500 ml Erlenmeyer and autoclaved for 20 min at lb steam pressure. After cooling to room temperature, the broth was inoculated with a 48 h culture of A. s~~hroxydans grown on agar slants containing D-glucitol (7). The still culture was maintained at 30" for 7 days, shaken with 0.75 g of acti- vated charcoal, and filtered through Celite. The filtrate was evaporated to a syrup, and a portion (3 g) of this material was dissolved in 10 rnl of water and applied

to a column (5 x 30 cm) of Dowep: I (HS6,-; 200- 460 mesh) maintained at 25". Water, as the eluant, was passed through the column at a flow rate of 0.5 ml/min and 10-ml fractions were collected. Fractions 20-27 contained deoxyhexitol and fractions 29-42, 4-deoxy-D-i&areoke%anlose (4). The ketose fractions were pooled, passed through Dowex 3 (OH-), and evaporated to a chromatographicallyr pure (solvent A, 20R) syrup (I.$$ g of 4) [a]D -3.9" (c, 1.12 water). Several attempts to prepare a phenylosazone by standard procedures were unsuccessful.

Nuclear magnetic resonance (n.m.r.) spectroscopy of 4 in DnO showed no low-field aanorneric proton. Mass spectral analysis of the pertrimethylsilyl derivative showed the molecular ion presemt at m / e 452. A signal of high intensity was visible at m/e 349 amounting to 25% sf the base peak at m/e 73.

Measurement of Hexskinase Velocity The reaction system (6.0 ml), adjusted to pH 8.5

with aqueous NaOH, contained final concentrations of 4.3 x M ATP, 1.6 x M MgClz, and various concentrations of hexoses. The reaction was initiated by adding 0. l ml (8.3 1 mg) of yeast hexokinase dissolved in 0.2% bovine serum aIbumin, pH 8.5. A constant pH of 8.5 was maintained by a pH-stat containing 0.002 M NaOH. Initial velocities were deter- mined by subtracting the blank titration value from the observed value. Prom Lineweaver-Burk plots of the initial reaction velocities versus substrate concentrations, the relative Vm,, and Km were obtained.

4-Beoxy-~-threokext~10se 6-Bhospht~be (5) Compound 4 (0.$5 g, 5.2 mmol), 0.46 g (830 prnol)

of ATPQNan), and 0.57 g of MgC12 were dissolved in 180 ml of water and the solution was adjusted to pH 8.0 with 0.1 M NaOH. 'Phe reaction was initiated by the addition of 58 mg of hexokinase dissolved in 8 ml of 0.2% bovine serum albumin, pH 8.0. Constant pH (8.0) was maintained by a pH-stat containing 0.02 M NaOH. The reaction was stopped when base consurnp- tion equaled the blank titration value obtained in the absence of enzyme. The aqueous solution was placed in presoaked dialysis tubing and the tubing immersed in a chromatographic column containing water. Water was passed through the column at st flow rate of 0.5 ml/min and the eluant (1 liter) was collected and concentrated to a smaller volume. Acid-washed charcoal (30 g) was added, the resulting suspension filtered throaagh Celite, and the residue washed thoroughly with water. The filtrate and washings were adjusted to pH 6.5 and 10 ml of 1 M barium acetate were added. The barium salt of 4-desxy-D-threokexulose 6-phss- phate (5) was precipitated at 0" overnight with five volumes of ethanol, collected by centrifugation, and washed with 80% ethanol. The compound was dissolved in water, the pH adjusted to 7.0, md the solution freeze-dried. Paper chromatography of the freeze- dried residue (0.18 g) (solvent B, Rp 0.56) showed small amounts of another phosphorylated compound (solvent B, Rp 0.42). Nuclear magnetic resonance spectroscopy of the sodium salt in DaO showed a low- field anorneric proton present in small amounts,

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HAYLOCK AND SLESSOR: ~-DEOXY-D-FHWIEOHEXUL~SE

TABLE 1. PkosphoryIation parameters of yeast hexokinase

Substrate K I ~ Relative V,,,

*Literature values (3) in brackets.

indicative of an aldose impurity. Hydrolysis with acid phosphatase was carried out on the sodium salt essential by the method sf Wood (8) except depro- teinization was accomplished with 10% trichlsroacetic acid. Following filtration and neutralization to pH 7.0, the solution was deionized with Dowex 50 (H'), followed lay Dowex 3(OH-). Paper chromatography (solvent C, 1 % h) showed that the free sugar had a mobility identical with 4-deoxy-~-threohex~~10se (4).

4-Beoxy -D-shreollsxubse 1,6-Diphosphatf (6) The potassium salt of compound 5 (0.12 g, 428

pmol), 0.47 g (856 pmol) of ATP(Wa2), 0.15 g of MgC12, and 0.91 g of KC1 were dissolved in 150 ml of water, and the pH was adjusted to 8.5 with 0.1 M NaOH. Bhosphofhuctokinase (258 pg) was diluted with I rnl of 0.05 A4 cysteime hydrochloride, pH 8.5, and left for 5 min at room temperature to ensure maximal activity. Addition of the phosphofructokinase solution initiated the reaction and a constant pH of 8.5 was maintained by a pH-stat containing 8.802 M NaQM. Completion of the reaction and workup were as described for compound 5. The barium salt of 4- deoxy-D-threohexulose 1,6-diphosphate (6) was precipitated by cooling the reaction mixture to 0° , COB- lected by centrifugation, and washed with 30% ethanol. The residue was dissolved in aqueous acetic acid (pH 5) and the precipitation procedure repeated. The product (6) (37 mg) obtained was chromatographically pure (solvent B, Wp 0.44). Hydrolysis with acid phosphatase was carried out on the sodium salt of 6 by the method outlined for compound 5. Paper chromatography (solvent C, 12 h) showed the free sugar to be identical with 4-deoxy-~-fkreohexu10se (4).

Results and Discussions Hydride reduction of 3,4-anhydro- 1,2: 5,6-di-

0-isopropylidene-~-ta1itoI (I) results in the formation of two epirneric deoxyhexitol derivatives, 3-deoxy-D-arabirm (2) and 3-deoxy-D lyxohexitol (3). According to the pattern estab- lished by a Barge number of workers and stated as the Befiand-Hudson rule (9, 101, A. sub- o x y h n s will selectively oxidize a ~merythro system to provide a hexulose. Thus in our mixture, only 3-deoxy-D-aaabinohexitol is suscept- ible to oxidation and was converted to 4-deoxy- D-threohexulose (4) . Separation was readily

achieved on bisulfite resin ( 1 B ) utilizing the presence of a potential keto function in the hexulose.

The chemical and physical properties of 4 were in accord with its 4-deoxy-D-threshexdose structure. The absence of a low-field anomeric signal indicated a reducing hexulose to be present. The detection of the parent ion in the mass spectrum of the pertrimethylsilyl derivative sub- stantiated the molecular weight of 4, and the fragmentation pattern, in particular the m/e 349 signal (loss of CH20SiMe:3), was in accord with those observed for fructose 4 12).

The phosphorylation of 4 by yeast hexokinase was followed with a titrimetric pH-stat by deter- mining the acid equivalent of the transferred phosphoryl group. Table 1 shows the results obtained by this method and these agree favor- ably with those reported for glucose and fructose at pH 7.4 and 7.6 (3, 13).

The relative affinity of 4-deoxy-D-threohexulose with respect to fructose tends to support the contention that the 4-hydroxyl of the sugar is a binding site in yeast hexokinase action as has been suggested by the results with D-galactose (1). Although the 6-phosphate of 4 could be prepared enzymaticaUy, the yield was not high and the product was contaminated slightly by what probably was 4-deoxy-D-glucose B-phosphate. Phosphatase action on the deoxyhexulose 6-phosphate (5) gave materid identical with 4. The action of rabbit muscle phosphofructokinase on (5) yielded the 1,6-diphosphate in low yield ( M 28 5% ) . The material (6 ) was obtained chromatographically pure and on phosphatase treatment regenerated 4. The potential interest of 4-deoxy-D-threshexulose B,6-diphosphate (6) lies in its inability to be degraded by aldolase and thus possibly act as a competitive inhibitor of the glycolytic pathway.

We thank the National Research Council sf Canada for financial support.

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972 CAN. 4. BIOCHEM. VOL. 51, 1973

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synthesis and kinase phosphorylation of 4-deoxy- d - threo...

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