Preparation of glycosyl phosphates: β-D -glucopyranosyl phosphate

117

NOTES

Preparation

of glycosyl phosphates:

/?-D-glucopyranosyl

phosphate

The formation of glycosyl phosphates by reaction of a fully acetylated sugar with anhydrous phosphoric acid at moderate temperatures is a convenient procedure for the preparation of these esters, and a number of such phosphates of biochemical interest have been prepared in this way. For example, the potassium salts of a-Dglucopyranosyl phosphate and cr-D-galactopyranosyl phosphate were prepared from the corresponding pentaacetates of /?-rr-glucopyranose and /l-D-galactopyranose, approaching 60%. Subsequently, it was shown” that salts respectively, in yield@ of 2-acetamido-2-deoxy-a-D-glucopyranosyl phosphate and 2-acetamido-2-deoxy-a-Dgalactopyranosyl phosphate could be prepared from the /?-D-tetraacetates of the appropriate 2-acetamido sugars. In a later paper, O’Brien4 reported on a fusion reaction using n-acetamido-2deoxy-n-D-ghrcopyranose tetraacetate. This compound, having substituents on C-I and C-2 in the 1,2-h orientation, reacted much more slowly than its anomer having the 1,2-trm2.s (diequatorial) conformation, and reasonable yields of product (33%) were obtained only by raising the reaction temperature to 83” (as compared to 50°, originally used) and increasing the proportion of phosphoric acid. Recently, the pentaacetates of a-D-glucopyranose and a-D-, oalactopyranose have been shown to react readily only under the more strenuous conditions reported by O’Brien4, and the product isolated was, in each instance; the CC-D anome925. Several workers have reported the preparation cf salts of a-D-mannopyranosyl phosphate by using this general phosphorylating procedures. In the above experiments, the a-D (axial) anomer was generally the one isolated, although it is probable that, in most instances, both anomers are formed in the reaction. The isolation of only one anomer may be the result of loss of the other anomer (a) during the crystallization process, or (b) through the preferential hydrolysis of the (more labile) ,8-D (equatorial) anomer during the fractionation procedure4. The actual isolation of bcth anomers using an ion-exchange fractionation procedure was reported by O’Brien* for the anomeric 2-acetamido-2-deoxy-D-glucopyranosyl phosphates. The replacement of an acetoxy group on C-r of an acetylated sugar bearing acetoxy groups on C-I and C-2 in a trans cotiguration is greatly facilitated by neighboring-group participation of the acetoxy group on C-2. As a consequence, p-D-glucopyranose pentaacetate, for example, reacts much more rapidly than the corresponding cr-D anomer in various replacement reactions7, as has been amply shown in the preparation of glycosyl halides, as well as of glycosyl phosphates, as outlined above. In the preparation of a tetra-O-acetyl-a-D-glucopyranosyl halide from B-D-glucopyranose pentaacetate, it is to be expected that the tetra-U-acetylj3-6-D-glucopyranosylhalide will be the first product of the reaction. Under the conditions of the reaction, this compound would then be anomerized to the thermodynamically more stable CY-D anomer. The actual preparation of the /I-D anomer Carbohydrate Res.,3 (1966)117-120

NOTES

XI8

reqnires, in general, shorter reaction times, and conditions for such preparations

have

been publisheds. In an analogous way, it would be expected that, in the preparation of a Dglucosyl phosphate from fl-D-glucopyranose pentaacetate using the fusion procedure, the &St product would be a derivative of ,8-D-glucopyranosyl phosphate, and that this material would then be anomerized to the more stable CC-D anomer. In most of the previously published preparations of glycosyl phosphates by this method, the reaction has been allowed to proceed for up to two hours, Generally, only that anomer having a stable conformation (with an axial phosphate group) has been isolated, except in the case of the 2-acetamido-a-deoxy-D-glucose derivativea. We have now found that, as anticipated, a derivative of P-D-glucopyranosyl phosphate is an early and major product in the reaction of penta-O-acetyl-,8-Dglucopyranose with anhydrous phosphoric acid at 50”. After removal of the protecting acetyl groups, and purikation of the barium salt, an examination of the optical rotation showed that the yield of the more levorotatory anomer decreases as the duration of the fusion reaction is increased. Some results indicating this transformation are indicated in the Table. TABLE

I

EFFE(JTOF

RJXCTION

TIME

ON

FORMATION

OF THE

D-Ghcopyranosylphosphate

ANOMERS

D-GLUCOPYRANOSYL

PHOSPHATE

Time (mifz) 20

IO

5 Barium

OF

salt, yield % +Z

blD=

of fl anomerb, DipotassiumTV-, yield % %

talc.

88 I

k=dD

Di(cyclohexylammonium) /3-, yield WD

%

f73 &

+Z

+E

71 8 +63

48 31 +67

36

t-6.2

19

+5-S

~Calc. on basis of anhydrous salt. Wn the basis of reported molecular rotations 9, the rotations of the anhydrous barium salts would be +74” and +g.6” for the a: and fi anomer, respectively.

The barium salts were converted into the potassium salts, and the readily crystallizing dipotassium salt of a-D-glucopyranosyl phosphate dihydrate was obtained. Although no effort was made to purify this compound, the yield varied directly with the elapsed time for the fusion reaction. The salt of p-D-glucopyranosyl phosphate which remained in solution was then isolated by conversion into the di(cycIohexylammonium) salt, followed by crystallization. The air-dried salt was found to be anhydrous and to have a specific rotation of f5.g”. Putman and Hassids reported that their air-dried product, one molecule of which crystallized with one molecule of ethanol and one of water, had a spe~ifk rotation of +7.3” (corresponding to +8.3” for solvent-free material). Cdwhydrure

Res., 3 (xg66) rr7-120

rr9

DiCcydohexyZammonium) &mglucopyranosyl phosphate Ten g of anhydrous phosphoric acid (which had been dried overnight in vacua over magnesium perchlorate) was melted at 50”. and to the melt was added 5.00 g (12.8 mmoles) of powdered penta-U-acetyl+D-glucopyranose. The mixture was stirred magnetically in vacua at 50~; after 5 min, the mixture was rapidly cooled, and 205 ml of ice-cold 2 N lithium hydroxide was added. The contents of the flask were thoroughly mixed to disperse the sirup, and then allowed to stand overnight. The precipitated lithium phosphate was removed by filtration through Celite, and washed with cold, lithium hydroxide solution (ca. 0.01 N). The resulting solution contained 6.7 mmoles (52%) of acid-labile phosphatelO. The pH of the solution was lowered to ca. 8.5 with Dowex SOW-H+ and, after the resin had been removed by filtration, barium acetate (3.0 g) was added and the solution concentrated to about 50 ml. The barium salt was precipitated by the addition of four volumes of ethanol; after several hours at 5”, the solids were collected by centrifugation, washed successively with acetone and ether, and dried in vacua over calcium chloride_ The solid was dissolved in water (40 ml), faint traces of insoluble matter were removed by centrifugation, and the barium salt was reprecipitated by the addition of ethanol (120 ml). After a second such reprecipitation, there was obtained 3.92 g of barium salt containing 6.7 mmoles of phosphate and showing [LX]~ + 17” (c 2.6, water; calculated on the basis of the anhydrous barium salt). The salts were dissolved in water at ca. 5”. and the solution was passed through a precooled column of Dowex 50W-Hf (I x 25 cm) into water containing 1.2 g of potassium hydroxide. The column was washed with 50 ml of cold water, and the pH of the resulting combined effluent was adjusted to 9 with Dowex SOW-H+. The resin was removed by filtration, and the solution was concentrated to about 40 ml. The potassium salt crystallized at 5” on addition of 1.5 volumes of ethanol over a period of two days; it weighed 61.8 mg (1.3%) and showed [cz]~ + 73.3” (c 0.5, water). Pure a-o-glucopyranosyl dipotassium phosphate dihydrate shows” [o;Jg + 78”. The mother liquors remaining from crystallization of the potassium salt were concentrated under diminished pressure, and the residue was dissolved in 50 ml of cold water. This solution was passed through a precooled column of Dowex SOW-H+ (I x 25 cm) and the column was washed with IOO ml of water, the effluent being collected in water containing 2 ml of cyclohexylamine. The combined percolate was concentrated in vacua to ca. 2 ml, and IOOml of absolute ethyl alcohol was added. After two days at 5”, the crystalline salt was collected by filtration, washed with absolute ethanol, and air dried; yield 2.65 g (5.8 mmoles, 45%) [cc]~ + 8.8” (c 2, water). A further 0.23 g (0.5 mmole, 4%), showing [cc]~ + 28.6’, was obtained from the mother liquors. Recrystallization of the first crop gave 2.48 g (42%) of di(cyclohexylammonium) P-D-glucopyranosyl phosphate, showing [a]n + 5.9”. Carbohy&ate Res., 3 (x966) 117-120

NOTES

120

Pntman and HassidB reported that their air-dried product showed one molecule contained I molecule each of ethanol and water.

Anal. Calc. for ClaHs&09 Found*:

C, 47.10;

H, 8.81;

P (458.5): C, 47.15; N, 5.99; P, 6.87.

H, 8.57;

3-7.3”

and that

N, 6.11;

P, 6.76.

Health

Service

ACKNOWLEDGMENTS This work (GM 09756)

was supported

by grants

and from the Corn Industries

from Research

the U. S. Public Foundation.

Department of Chemistry and Science Research Institute, Oregon State University Corvallis, Oregon 97331 (U.S.A.)

D. L. MACDONALD**

REFERENCES I D. L. MACDONALD, J. Org. Chem., 27 (1962) 1107. 2 D. L. MACDONALD, in Complex Carbohydrates, a voIume of Methods Press, New York, 1966. 3 T. Y. KIM ANI) E. A. DAVIDSON, J. Org. Cllem., 28 (1963) 2475. 4 P. J. O’BRIEN, Biochim. Biophys. Acta, 86 (1964) 628. 5 D. L. MACDONALD, J. Org. Chem., 31 (1966) 513. 6 P. PERCHEMLIDES,E. A. DAVIDSON, AND N. ARONSON, Abstracts Papers 147 (1964) xc; A. D. ELBEIN AND E. C. HEATH, J. Biof. Chem., 240 AND C. E. BALLOU, J. Biol. Chem., 241 (x966) 895. 7 R. U. LEMIEUX, Ad~~rz. Curbohyd-ate Cbem., g (1954) I. 8 R. U. LEMIEUX, Methods Carbohydrate Chem., 2 (1963) 224. AND W. Z. HASSID, J. Am. Chem. Sot., 7g (1957) 5057. g E-W-PIO G. R. BARTLE~; J. Biol. Chem., 234 (1959) 466. II M. L. WOLFROM AND D. E. PLETCHER, J. Am. Chem. Sot-, 63 (Ig4r) (Received

May znd, 1966)

*Analyses by Elek Microanalytical Laboratories, Torrance, Celifomia. **Research Career Development Awardee, U. S. Public Health Service. Carboh~&Ife

Res.,

3 (1966) 117-120

in Enzymology,

Academic

Am. Chem. Sot. Meeting, (1965) 1926; D. L. HILL

1050.