ANALYTICAL BIOCHEMISTRY 20, 58-64 (1x7)

A Simple Chemical Synthesis

of a-D-Glucose 1 ,&Diphosphatel

W. A. KHAN, T. BALDWIN, U. BRODBECK, AND K. E. EBNER

Department of Biochemistry, Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma 74074

Received January 6, 1967

A requirement for n-glucose 1,6diphosphate in the phosphoglucomutase reaction (EC 2.7.5.1) was first demonstrated by Leloir el al. (l), and recent studies by Ray and Roscelli (2) have suggested that a continuous replenish- ment of the cofactor is required during the enzymic reaction. Phospho- glucomutase isolated from Bacillus cereus and Micrococcus lysodeikticus has an absolute requirement for glucose 1,6-diphosphate (3).

Several enzymes that synthesize glucose l$diphosphate have been isolated. Phosphoglucokinase (EC 2.7.1.10) from muscle and yeast catalyzes the ATP dependent phosphorylation of n-glucose (4). Glucose-l-phosphate dismutase (EC 2.7.1.41) from Escherichia coli (5) and rabbit muscle (6) catalyzes the dismutation of glucose l-phosphate or glucose 6-phosphate to form glucose 1,6diphosphate. n-Glucose 1,6diphosphate hydrolyzing enzymes are present in guinea pig liver (8).

It is apparent that the biological role of n-glucose 1,6-diphosphate is under active investigation, but the preparation of this compound at present is difficult and time consuming. n-Glucose 1,6diphosphate may be syn- thesized chemically (9) by phosphorylating 2,3,4-triacetyl-a-n-glucosyl- bromide 6-diphenylphosphate with silver diphenyl phosphate or with trisilver phosphate. Both a-n-glucose l,&diphosphate and 8-n-glucose 1,6-diphosphate are formed and may be separated by fractional crystalliza- tion of the brucine salts or they may more readily be separated by ion- exchange chromatography (2). a-D-Glucose 1,6-diphosphate may be isolated from human erythrocytes by ion-exchange column chromatography with yields between 0.19 and 0.24 Mmole/ml of blood (10). Glucose 1,6diphos- phate may be isolated from glucose l-phosphate by ion-exchange chroma- tography and yields of 20-25 pmoles from 5 gm of glucose l-phosphate have been reported (3).

The present procedure is an adaptation of the MacDonald synthesis

1 Supported in part by a grant from the National Science Foundation, GB-3465.

58

SYNTHESIS OF a-D-GLUCOSE 1,6-DIPHOSPHATB 59

which has been successfully used to prepare various glycosyl l-phosphates (11, 12). Glucose 6-phosphate was acetylated to yield 1,2,3,4-tetraacetyl- &n-glucose 6-phosphate, which was phosphorylated by anhydrous phos- phoric acid to yield 2,3,4-triacetyl-cr-n-glucose 1,6-diphosphate. The acetyl groups were easily removed by hydrolysis with lithium hydroxide. The over-all yield of the cyclohexylammonium salt was between 20 and 25y0 when based on glucose 6-phosphate.

MATERIALS ANIl METHODS

Glucose &phosphate, glucose l-phosphate (Type V, free of glucose l,Bdiphosphate), glucose-6-phosphate dehydrogenase (Type 1’) and crystalline phosphoglucomutase (rabbit muscle) were obtained from Sigma Chemical Co. Anhydrous phosphoric acid was from Fluka. All other chemicals were of reagent grade. Phosphate assays were determined by the Fiske-SubbaRow method (13), and reducing sugars by the method of Somogyi (14). Inorganic phosphate and acid labile phosphate were detected on chromatograms by use of a HC1G4-HC”l ammonium molybdate spray (15), and reducing sugars were detected by an aniline phthalate spray (16). Elemental analyses were performed by Galbraith Laboratories, Knoxville, Tennessee. a-n-Glucose l,&diphosphate, sodium salt, was a gift from Dr. P. Handler, Duke University, and was isolated from glucose l-phosphate (3). Enzymic rates were measured on a (‘ary recording spectro- photometer, model 14, at, 25”.

RESVLTS

Synthesis of a-D-Ghcosc I$-l)iphosphulr

(1) 1,2,3,4-Tetraacety&3-D-glucose 6-Phosphate. Freshly fused sodium acetate (0.246 gm, 3 mmoles) was mixed with 3.0 ml of acetic anhydride and t,he mixture was heated to boiling. Portions of glucose 6-phosphate! disodium salt (1.07 gm, 3 mmoles), were added to the acetylation mixt’ure, which was kept boiling by intermittent, heating. When the addition of glucose 6-phosphat,e was eompIete, the mixttrrr was reflused for 10 mm, after which 15 ml of absolute ethanol was added to destroy the excess acetic anhydride. The solution was evaporated to dryness under vacuum and the slightly colored product, was recrystallized from absolute et#hanol after treatment with decolorizing carbon. The product was dried i?l vacua over magnesium perchlorate. The yield of the hygroscopic product, was 1.4 gm (quantitative yield as the free acid). The infrared spectrum com- pared favorably with that of P-n-glucose pent’aacetate (17), indicating that the acetate in the one position was of the 0 configuration (absence of absorption at 8.7 II).

60 KHAN, BALDWIN, BRODBECK, AND EBNER

(2) Phosphorylation oj 1 ,d,S,&Tetraacetyl-@-glucose &Phosphate. 1,2,3,4- Tetraacetyl-W-glucose 6-phosphate (1.4 gm, 3 mmoles) and anhydrous phosphoric acid (4.0 gm, 41 mmoles) were placed separately in two opposite limbs of Thunberg tubes over magnesium perchlorate and dried in vacua. The two limbs were connected and evacuated and the limb containing the anhydrous phosphoric acid was immersed in a water bath at 50”. When the phosphoric acid had melted, 1,2,3,4-tetraacetyl+n-glucose 6-phosphate was added in small portions. There was an immediate vigorous bubbling as acetic acid was liberated. The reaction was continued for 2 hr at 50” and by this time the bubbling had virtually ceased.

(3) cu-w%xose 1,6-Diphosphate. The reaction mixture was extracted with 10 ml of dry tetrahydrofuran and then poured into 200 ml of ice-cold 1.0 N lithium hydroxide. The mixture was allowed to stand overnight at room temperature. The precipitated lithium phosphate was removed from the alkaline solution by filtration and the clear filtrate was passed through a Dowex 50 (H+) column (2 X 25 cm) at 4”. The column was washed until the effluent was neutral. The solution was made basic (pH 8.9) with cyclo- hexylamine and then evaporated to dryness on a rotary evaporator (4’). The residue was washed three times with absolute ethanol, which was removed in VUCUO. The slightly colored crystalline solid was mixed with 200 ml of dry 2-propanol at room temperature to dissolve the cyclohexyl- ammonium acetate. The mixture was centrifuged and the product was washed four times with 30 ml portions of 2-propanol. Next, the product was washed with dry ether and dried in vacua over magnesium perchlorate. A small amount of inorganic phosphate was present in the product and this was removed as magnesium ammonium phosphate by adding mag- nesium acetate (0.5 gm) to a solution of the sugar phosphate in 40 ml of 1.5 N ammonium hydroxide. Cations were removed on a small column (1 X 10 cm) of Dowex 50 (H+). The percolate was made basic (pH 8.5) with cyclohexylamine and the solution was evaporated to dryness under vacuum. The residue was washed with absolute ethanol and dried in vacua. No inorganic phosphate was present in the product (0.47 gm, 21%). The ratio of 7 min phosphate to total phosphate was 1:2.3.

The cyclohexylammonium salt (0.284 gm in 150 ml of HzO) was further purified on a Dowex 1 (Cl) column (2 X 26 cm). The column was washed with 100 ml of water and then eluted with a linear gradient of LiCl (O-500 millimolar with 500 ml in each vessel). The tubes containing 7 min labile phosphate (eluting at 150 millimolar) were pooled and lyophilized.

The resulting syrup was washed three times with 10 ml of absolute ethanol which was removed by evaporation in vacua. The white crystalline solid was washed repeatedly with 1 :l mixture of absolute ethanol and acetone until chloride ion was absent (AgN03 test). The product was dried

SYNTHESIS OF a-D-GLUCOSE 1,6-DIPHOSPHATE 61

over magnesium perchlorate in V~CUO and a yield of 100 mg (70%) of the lithium salt was obtained. The chemical composition calculated for C~H12012Li2P2.2H~0 in percent was: C 18.6, 114.12, and P 15.9; observed- C 19.52, H 3.85, and P 15.67. The ratio of 7 nun phosphate to total phosphate to reducing sugar was 1: 2 : 1. The sodium salt of a-n-glucose 1,8diphosphate was prepared readily from the cyclohexylammonium salt. The cyclohexylammonium salt (125 mgj was dissolved in 20 ml of wat,er and passed through a Dowex 50 (H+) column (1 X 20 cm at 4”). The column was washed with water until the effluent was neutral. The pH of the effluent was adjusted to 8.5 with 0.5 N T;iaOH and the solution was concentrated to a small volume (2-5 ml). Three to five volumes of absolute ethanol was added and upon cooling and rubbing with a glass rod the sticky solid turned into a crystalline sohd. The product was washed with dry ether and dried in vacua over magnesium perchlorate (70 mg).

Characterization of cu-~-Glucose 1,6-Diphosphate

The product was characterized to determine if any P-n-glucose 1,6- diphosphate was present in the final preparation. The (Y and /3 isomers of glucose 1,8diphosphate have been separated on a Dowex 1 formate column (2) and, accordingly, the product was chromatographed on Dowex 1 formate and eluted with a linear gradient of 0.75 to 1.5 M formic acid-pyridine, pH 3.0. Only one peak having 7 min phosphate was observed and the ratio of total phosphate to 7 min phosphate was 2 : 1 in the peak t’ubes.

The first-order rate constant for the hydrolysis of glucose l,A-diphosphate was determined in 2 N H&O4 at 30” and was found to he ‘7.6 X 10M4 min-I. The reported first-order rate constant for a-D-glucose 1,6diphosphate under these conditions is 7.8 X 1O-4 and for @-D-&K’Ose 1,6diphosphate is 3.15 X 1O-3 min-’ (18).

12 sample of the product was hydrolyzed for 7 min in 1 N H2S04 at 100’ and assayed for 7 min phosphate, total phosphate, reducing sugar, and, after neutralization to pH 7.0, for glucose 6-phosphate by glucose-6-phosphate dehydrogenaxe (19). The observed ratios were 1 .O : 2.0 : 1.2 : 1.1. hTo reducing sugar or glucose 6-phosphate was present in the unhydrolyzed product. The migration of glucose 1,6-diphosphate on electrophoresis (Table I) and on paper and thin-layer chromatography (Table 2) was compared to authentic ol-n-glucose 1,6diphosphate (sodium salt) isolated from commercial glucose 1 -phosphate.

Dephospho-phosphoglucomutase was prepared from rabbit muscle phos- phoglucomutase (20) and was used to determine enzymically the concen- tration of glucose 1,6diphosp

7 ate. The enzymic assay measured glucose

6-phosphate formation with g ucose-gphosphate dehydrogenase (2). The cofactor assay for glucose 1,Bdiphosphate depended on the ability of

KHAN, BALDWIN, BRODBECK, AND EBNER

TABLE 1

Electrophoresis of Sugar Phosphates

Distance moved, cm

Compound pH 3.60 0.05 M borateb

Glucose 1,6-diphosphate (Na) 23.0 19.6

Glucose 1,6-diphosphate (standard, Na) 23.0 20.0 Glucose 1,bdiphosphate (Li) 22.9 20.2

Glucose-l-P 15.8 13.6 Glucose&P 15.8 14.2

Pi 23.2 21.0

a Pyridine-acetic acid-Hz0 (1: 10:89, pH 3.6), 1 hr at 2500 V, Whatman 3 MM. b 0.05 M Na borate, 45 min at 2500 V, Whatman 3 MM.

glucose 1,6-diphosphate to stimulate t.he dephospho-form of phospho- glucomutase and was previously described (7). Calculation of the amount of glucose 1,6diphosphate (Li salt) was based on the K, (6 X lo-* M) and measurement of the initial velocity at a concentration equal to Km. V,,,

TABLE 2 Paper and Thin-Layer Chromatography of Sugar Phosphates

Compound

R(Pi)

Papera Thin-Layer’

Pi 1.00 1.00

Glucose-l-P 0.57 0.44

Glucose 1,6-diphosphate (Li) 0.72 0.55 Glucose 1,6-diphosphate (Na) 0.68 0.56

Glucose 1,6-diphosphate (standard, Na) 0.67 0.55

Glucose-6-P 0.74 0.55

a Solvent: methanol-formic acid-water (80 : 15 : 5 v/v) containing 0.2 gm EDTA/lOO ml; acid-washed Whatman 3 MM, ascending.

b MN 300 (Brinkman) cellulose plates in the same solvent as for paper chromatography.

was determined under standard conditions. Under identical assay condi- tions, the determined V,,, was equal to an absorbance change of O.Oll/min at 340 rnp, whereas the velocity measured with 6 X 1W8 M glucose 1,6- diphosphate was O.O56/min, thus agreeing well with the expected velocity of +v,*, (O.O55/min). The sodium salt was also active at comparable concentrations.

DISCUSSION

~-n-Glucose 1,6diphosphate was synthesized chemically in a simple two-step reaction from readily available starting materials by adapting the MacDonald synthesis which has been used widely for the synthesis of

SYNTHESIS OF a-D-GLUCOSE 1,6-DIPHOSPHATE 63

sugar l-phosphates (11). The present method should also be useful for the synthesis of other hexose 1,6-diphosphates.

Various sugar l-phosphates have been prepared by the MacDonald procedure : cY-N-acetylglucosamine l-phosphate and a-IV-acet’ylgalacto- samine l-phosphate (21), N-acetyl-cY- and A;-acetyl+glucosamine l-phos- phates (22) and N-acetylchondrosine a-1-phosphat,e (23). MacDonald (12) has reported that acetylated sugars with the 1,2-trans diequatorial con- figuration (e.g., the fl-pentaacetate of galactose) readily yield the a-l-phos- phates whereas in acetylated reducing sugars with the 1,2-cis configuration the acetate in the one position is difficult to displace. The phosphorylation of a-n-pentaacetylgalactopyranose gave low yields of cy-n-galactopyranose l-phosphate and no /3 isomer was reported (12). There appears to be no re- port of the occurrence of a P-l-phosphate when the corresponding P-penta- acetate was phosphorylated, though lack of this observation may be due in part to the fact that the /3 isomer is usually more labile than the correspond- ing (Y isomer. Even though the mechanism of the MacDonald reaction is not fully delineated, the present evidence indicates t,hat phosphorylation of a p-I-acetylated sugar gives rise to the corresponding a-l-phosphate.

The data presented in this study on the characterization of a-n-glucose 1,6-diphosphate show that the chemically synthesized compound is of the 1~ configuration and no evidence for the presence of the fi isomer was found. Chromatography on paper and thin-layer chromatography and electro- phoresis showed that the migrations of the synthesized compound were identical to the reference compound. The first-order rate constant for hydrolysis was similar to the reported value. Perhaps the strongest evidence that the product is a a-D-ghlCOSe 1,Bdiphosphate comes from the enzymic experiments, where the product was required for the activation of dephos- pho-phosphoglucomutase. Under standard conditions, the product assayed enzymically as the (Y isomer. The /3 isomer is unreactive and is inhibitory in the phosphoglucomutase reaction (2).

The over-all yield of a-n-glucose 1,6-diphosphate based on glucose 6-phosphate was between 20 and 25%. Further experiments have shown that purification of the product on Dowex 1 Cl was not necessary since the sodium salt prepared directly from the cyclohexylammonium salt was active in the phosphoglucomutase reaction at low concentrations (6 X I O-8 M) .

SUMMARY

cu-n-Glucose 1,6-diphosphate was synthesized from glucose B-phosphate by a simple two-step chemical reaction. Glucose 6-phosphate was acetylated with fused sodium acetate and hot acetic anhydride to yield 1,2,3,4tetra- acetyl-P-n- glucose 6-phosphate, which was phosphorylat,ed with anhydrous

64 KHAN, BALDWIN, BRODBECK, AND EBNER

phosphoric acid to yield 2,3,4-triacetyl-a-D-glucose 1,6diphosphate. The acetyl groups were removed by hydrolysis with lithium hydroxide. The yield of the cyclohexylammonium salt of cr-D-glucose 1,6diphosphate was between 20 and 25%.

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