ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 288, No. 1, July, pp. 239-242, 1991

The Synthesis and Characterization of Uridine 5’-(P-L-Rhamnopyranosyl diphosphate) and Its Role in the Enzymic Synthesis of Rutin G. A. Barber Department

of

and E. J. Behrmanl Biochemistry,

The Ohio State University,

Received December 14, 1990, and in revised form February

484 West 12th Auenue,

8, 1991

MATERIALS Uridine 5’-(B-L-rhamnopyranosyl diphosphate) was synthesized by the condensation of uridine 5’-diphenylpyrophosphate and ,9-L-rhamnopyranosyl phosphate. That sugar l-phosphate was made via the phosphitylation of the hemiacetal hydroxyl group of 2,3,4-tetra-o-acetylP-L-rhamnopyranose. An enzyme preparation from the aureus) was primary leaves of mung bean (Phaseolus shown to catalyze the transfer of L-rhamnose from UDPP-L-rhamnose to the flavonol D-glucoside isoquercitrin 0 1991 Academic Press, Inc. to form rutin.

It has been demonstrated that enzyme extracts from various higher plants will catalyze the transfer of Lrhamnose from UDP-P[‘4C]-~-rhamnose to flavonol glycosides to form, for example, [14C]quercitrin [3-a-~rhamnosyl quercetin] (1) and rutin [ cu-L-rhamnosyl (1 + 6) ,8-D-glucosyl-3-quercetin] (2). In those examples the Lrhamnosyl nucleotide was itself synthesized enzymically with high specific activity but in exceedingly small amounts; hence, its characterization was limited. There is also the question in experiments of that kind whether any net synthesis occurred. To characterize the components of these glycosylation reactions more extensively and to show rates in vitro closer to those in uiuo, a larger supply of the nucleotide was required. To that end micromolar quantities of the compounds were synthesized by the chemical condensation of fl-L-rhamnopyranosyl phosphate and UMP, and Lrhamnose was transferred from it enzymically to quercetin-3-D-glucoside to form the flavonol glycoside rutin. Thus, for the first time, net rhamnosylation leading to a natural product was demonstrated in a cell-free plant system (Fig. 1). to either author. FAX: (614) 292-6773.

0003.9861/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

AND METHODS

Partition chromatography. Chromatographic isolation of nucleotides was carried out on Schleicher and Schuell No. 589 White Ribbon paper, and flavonols were chromatographed on Whatman No. 1 paper that had been washed by development overnight with 15% acetic acid. The following solvents were used: Solvent I, ethanol 95%: ammonium acetate 1 M, 7:3; Solvent II, aqueous acetic acid 15%; Solvent III, rz-butanol: acetic acidwater 52:13:35; Solvent IV n-propanol:ethyl acetate:water 7:1:2. Paper electrophoresis. Electrophoresis was performed with an apparatus constructed after that described by Crestfield and Allen (3) on Schleicher and Schuell No. 589 Orange Ribbon paper at 25-35 V/cm in: Buffer I, 0.05 M ammonium acetate, pH 5, or Buffer II, 0.05 M sodium tetraborate, pH9. Instrumentation. FAB mass spectra were obtained using a VG 70. 250 instrument. Proton NMR spectra were taken using a Bruker AM500 (500 MHz) instrument and 31P spectra with a Bruker MSL-300. Reagents. Quercetin&D-glucoside (isoquercitrin) was a gift from T. A. Geissman in 1960. Since it is only sparingly soluble in water, it was used in reaction mixtures as a suspension in 1% digitonin. All other reagents and solvents were obtained from the usual commercial sources. Plant material. Seeds of mung bean (Phuseolus aurem) were from a local natural foods store. They were germinated and the seedlings were grown in plastic trays of vermiculite (expanded mica) at room temperature under four 40-W “Gro-1ux” bulbs with a 12-h light/dark cycle. Primary leaves of mung bean lo-14 days Preparation of enzyme. old were washed and chilled, and 20 g were ground in a mortar with sea sand and 30 ml of buffer (0.1 M Tris-HCl, pH 7.5, 0.01 M P-mercaptoethanol). The homogenate was squeezed through four layers of cheesecloth and centrifuged 20 min at 10,OOOg.The supernatant solution was fractionated by the addition of (NH,),SO, as the solid salt. A green precipitate was obtained at 25-50% saturation and was the source of the enzyme. It was dissolved in 1 ml of dilute buffer (0.025 M Tris-HCI, pH 7.5, 0.01 M P-mercaptoethanol) and passed through a 1 X 15 cm column of Sephadex G-25 to remove endogenous phenolic compounds. Enzyme activity was recovered in the green fraction that was eluted from the column in the void volume. The eluate was made 50% saturated with (NH&SO,, and the precipitate was suspended in a small volume of the dilute buffer and dialyzed overnight against 2 X 500 ml volumes of that buffer. The dialyzed extract was stored in aliquots at -15°C.

RESULTS AND DISCUSSION Synthesis

1 Address correspondence

Columbus, Ohio 43210

of fl-L-rhamnopyranosyl

chemical synthesis of the naturally

The UDP-P-L-

phosphate.

occurring

239 Inc. reserved

240

BARBER

AND

BEHRMAN

HO 0 OH HO 0

HO OH

OH

UDP-p-L-Rhamnose

+UDP

lspquercitnn (Quercetvv3QGlucoside)

FIG.

1.

The conversion

rhamnose by conventional routes had been impossible for some years because of the unavailability of the precursor, /3-L-rhamnopyranosyl phosphate. A method was developed for the synthesis of that compound by Prihar and Behrman (4) in which the hemiacetal hydroxyl group of the sugar in the ,&configuration was rapidly phosphorylated by o-phenylenephosphorochloridate. In the work described here that process was modified such that the hemiacetal group was phosphitylated by the phosphorous compound 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite after the method of Westerduin et al, (5). The P(III) compound was subsequently oxidized to the phosphate by tert-butylhydroperoxide (5). The procedure for deacylation and isolation of the phosphorylated sugar after the oxidation was modified as follows: solvent acetonitrile was removed on a rotary evaporator at 25°C. In a typical reaction mixture in which 2 mmol of the starting material 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl bromide (4) was used, 6 ml of 1.5 M LiOH was added to the residue, and the mixture was stirred overnight at room temperature. Additional LiOH was added the next day to raise the pH from 7 to about pH 10. It was stirred for another hour and then passed through a lo-ml column of Dowex50 H+ to remove Li+ ions. That operation was conducted in the cold, and the effluent was collected in a beaker on ice containing an excess (6 mmol) of cyclohexylamine. The column was washed with 25 ml of water until the pH of the eluate had risen to 6, and the eluate and washings were concentrated to a few milliliters on the rotary evaporator at 30°C. Four volumes of acetone were added to the concentrate, and the mixture was allowed to stand at 5°C. Crystallization of the di(cyclohexylammonium) salt of /3-L-rhamnopyranosyl phosphate began within an hour. Yield 142 mg, about 15%. [oI]~ = +11.37 (c. 0.5 water), lit. [a!]~25 = f11.9 (c. 1, water) (4). Synthesis of a-L-rhamnopyranosyl phosphate. The di(cyclohexylammonium) salt of cr-L-rhamnopyranosyl

of isoquercitrin

to rutin.

phosphate was synthesized by the method of Chatterjee and MacDonald (6) [(~]n25 = -21.5 (c. 0.5 water), lit., [cr]? = -21.5 (c. 1, water) (6). Synthesis of UDP-/3-L-rhamnose and UDP-a-L-rhamnose. Those compounds were made by the condensation of UDP-diphenylphosphate (prepared from UMP and diphenylphosphorochloridate) with the appropriate anomer of L-rhamnopyranosyl phosphate by the method of Michelson (7) as modified by Shibaev et al. (8). The extent of the reaction was estimated by electrophoresis of an aliquot on paper in Buffer I and observation of the distribution of uv-absorbing compounds. They consisted of UDP-L-rhamnose, UMP, UDP, and UDPU (the condensation product of UDP-diphenylphosphate with UMP), the last of which, unfortunately, migrated very close to the sugar nucleotide. The sugar nucleotide could be separated from that contaminant, however, by chromatography on paper with Solvent I. Initial observations suggested a yield of 20-30%, but the lability of this sugar nucleotide made its isolation on a larger scale quite difficult. For example, when milligram quantities of the material were chromatographed with Solvent I on 1000 micron cellulose TLC plates (Avicel), it appeared to be separated from other uv-absorbing compounds. However, after elution of that area of the TLC plate with water and its concentration in uacuo, only a small amount of UDPL-rhamnose and a great deal of UMP could be detected. GDP-L-fucose has been reported to exhibit similar lability, that is, it broke down in high concentrations of salt even at neutral pH (9, 10). Since only small amounts of the sugar nucleotide were required for the experiments in mind, it was decided therefore to employ paper chromatography and electrophoresis for its isolation. Accordingly, from a reaction mixture starting with 25 pmol UDPdiphenylphosphate and 45 pmol @-L-rhamnopyranosyl phosphate, twelve 8 X 45 cm paper chromatograms were prepared and developed with Solvent I overnight. To remove residual ammonium acetate the papers were dried

UDP-P-L

RHAMNOSE

AND

RUTIN

241

SYNTHESIS

washed with methanol. The combined methanol extracts were let to dry in air and applied to a paper chromatogram which was developed with Solvent I for about 6 h. Under those conditions rutin moved ahead of its precursor quercetin-3-D-glucoside (R,, 0.43 and 0.34, respectively). The compounds were located by their characteristic fluorescence under uv light. With the best preparations, about 25% of the theoretical yield of rutin was obtained. There was no synthesis of rutin when UDP-a-L-rhamnose was supplied as the L-rhamnosyl donor. To obtain enough of the enzymically synthesized rutin for characterization, the reaction mixture was scaled up about 30 times. Methanol extracts were chromatographed with Solvent I on two sheets of washed paper 7.5 X 45 cm. About 0.15-0.2 pmol of rutin was made in this fashion. It migrated like rutin in Solvent I and Solvent II and on electrophoresis in Buffer II. These are systems that distinguish among a number of closely related flavonol glycosides (17). It gave absorption peaks in methanol at 259 and 360 nm with a spectrum characteristic of the quercetin aglycone. The positive-ion FAB spectrum (glycerol matrix) of the biosynthetic rutin showed ions at 611 (M + H), 633 (M + Na), and fragments corresponding to the data published by Crow et al. (18) and by de Koster et al. (19). This quantity of rutin was also enough for a proton NMR spectrum of sufficient quality to confirm the structure. Rutin from two chromatograms was dissolved in 1 ml of 99.8% D20. The solvent was evaporated. The residue was redissolved in 0.6 ml of 99.96% D20 to give a concentration of about 6 X 10m5M. Multiple scans with HDO suppression produced a spectrum in which it was possible to assign all of the aromatic protons, the anomeric protons, and the rhamnose methyl protons by comparison with an authentic sample taken at normal concentration in deuterated methanol. Integration showed the correct ratios within 20%. The data follow. Biosynthetic rutin: HDO at 6 4.65, H-2’ 6 7.54, H-6’ 6 7.46, H-5’ 6 6.90, H-8 6 6.3 (br), H-6 6 6.14, glucose-l 6 4.80 (d, J = 7 Hz), rhamnose-1 6 4.47, rhamnose-methyl 6 0.96 (d, J = 6 Hz). Authentic rutin recrystallized from 50% methanol-water,

in air for at least 5 h and then washed overnight in the chromatography vessels by development with 95% ethanol avoiding thereby high salt concentrations. The chromatographic procedure separated UDP-L-rhamnose from UMP and UDPU but not completely from L-rhamnopyranosyl phosphate. To effect that separation, the bands containing UDP-L-rhamnose were cut from the papers and eluted with water, and the eluates were combined and lyophilized to a small volume. The solution was then subjected to electrophoresis in Buffer I on three 17 X 59 cm paper strips. About 4 pmol of the product was recovered thereby as estimated by its absorption at 260 nm (yield 15% of theoretical). UDP-a-L-rhamnose was synthesized and isolated similarly beginning the process with the (Yinstead of the P-L-rhamnopyranosyl phosphate. Characterization of UDP-L-rhamnose by NMR. The anomeric purities of both (Y- and p- UDP-L-rhamnose were confirmed by partial analysis of their NMR spectra. These data are reported in Table I. The chemical shifts and coupling constants for the anomeric protons of the L-rhamnose residues were characteristic of a- and prhamnose derivatives (4, 11). The methyl group of ,(3linked rhamnose residues is characteristically downfield of methyl groups of a-linked residues (12,13). In addition, the phosphorus NMR shows that the pyrophosphate linkage was intact. The reported difference in chemical shift between the cy- and P-isomers for phosphorus is probably not significant due to the sensitivity of this shift to pH and ionic strength (14). The phosphorus-phosphorus couplings are in line with previously determined values (15, 16). The enzyme-catalyzed transfer of L-rhamnose from A typical UDP-(I-L-rhamnose to quercetin-3-D-glucoside. reaction mixture contained in a total volume of 34 ~1: 20 ~1 enzyme preparation (1.5-2 mg protein) pH 7.5, 0.02 pmol UDP-/3-L-rhamnose, 0.1 Fmol ATP, and 0.1 pmol quercetin-3-D-glucoside. The mixture was incubated in a sealed capillary tube for 90 min at 37°C and then added to a tube containing 0.8 ml methanol to extract the flavonols. A precipitate was removed by centrifugation and

TABLE

I

NMR Data for UDP-Rhamnose ‘H U-6 Alpha J Beta J

6

u-5

8.109

6.124 J5,6 = 8.1

6

Note.

8.100

6.130 Js,s = 8.1

‘H data at 500 MHz,

31P data at 121.5 MHz.

Ribose-1

Rhamnose-1

6.136 J,,, = 3.9

Rhamnose-6

5.576 J,,z = 1.7 J 1,p = 7.5 5.373 J1.* = 0.96 J 1.p = 8.8

6.140 J,,, = 4.4

Solvent,

31P

D,O with

acetonitrile

internal

P,

1.433 J5.6 = 6.3

-11.46

1.463 Js,6 = 6.1

-13.77

standard

at 2.058.

P,f -9.08 J P.P = 20.9 -11.55 Jp,p = 20.0

242

BARBER

AND

BEHRMAN

7. solvent CD,OD, TMS at 6 0, HDO at 6 4.81: H-2’ 6 7.668 8. = 2.1 Hz), H-6’ 6 7.629 (dd, Jortho = 8.4 Hz, Jmeta (4 Jmeta = 2.1 Hz), H-5’ 6 6.876 (d, Jortho = 8.3 Hz), H-8 6 6.396 (4 Jmta = 1.9 Hz), H-6 6 6.210 (d, Jmeta= 2.1 Hz), glucose9. 1 6 5.107 (d, J = 7.8 Hz), rhamnose-1 6 4.522 (d, J = 1.2 Hz), rhamnose-methyl 6 1.123 (d, J = 6.05 Hz). 10. ACKNOWLEDGMENTS NMR and mass spectra were obtained by C. Cottrell and D. Chang, respectively, at the Campus Chemical Instrument Center. The NMR spectrometer was funded in part by NIH Grant 1 SlO RR01458-OlAl.

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7, 35539.

103, 276-282. Chem.

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5. Westerduin, P., Veeneman, G. H., Marugg, J. E., van der Marel, G. A., and van Boom, J. H. (1986) Tetrahedron Lett. 27,1211-1214. 6. Chatterjee,

253-255.

A. K., and MacDonald,

D. L. (1968) Carbohydr.

Res. 6,

Michelson, A. M. (1964) Biochim. Biophys. Acta 91, l-13. Shibaev, V. N., Eliseeva, G. P., Kusov, Y. Y., Petrenko, V. A., Mishchenko, S. S., and Kochetkov, N. K. (1976) Bull. Amd. Sci. USSR, Diu. Chem.

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Gokhale,

U. B., Hindsgaul, O., and Palcic, M. M. (1990) Con. J. Chem. 68, 1063-1071. Nunez, H. A., and Barker, R. (1976) Biochemistry 15, 3843-3847. 11. DeBruyn, A., Anteunis, M., De Gussem, R., and Dutton, G. G. S. (1976) Carbohydr. Res. 47, 158-163. 12. Prihar, H. S., Tsai, J. H., Wanamaker, S. R., Duber, S. J., and Behrman, E. J. (1977) Carbohydr. Res. 56,315-324. Lett. 83313. Sinclair, H. B., and Sleeter, R. T. (1970) Tetrahedron

836. 14. Van Wazer, J. R., and Ditchfield, R. (1987) in Phosphorus NMR in Biology (Burt, C. T., Ed.) pp. 12-16, CRC Press, Boca Raton, FL. 15. Appleton, M. L., Cottrell, C. E., and Behrman, E. J. (1990) Carbohydr.

Res. 206,373-377.

16. Perlman, M. E., Davis, D. G., Gabel, S. A., and London, R. E. (1990) Biochemistry 29, 4318-4325. 17. Harborne, J. B., and Williams, C. A. (1975) in The Flavonoids (Harborne, J. B., Mabry, T. J., and Mabry, H., Eds.) pp. 376-441, Chapman and Hall, London. 18. Crow, F. W., Tamer, K. B., Looker, J. H., and Gross, L. (1986) Anal.

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Biochem.

19. de Koster, (1985)

C. G., Heerma, W., Dijkstra, G., and Niemann, Mass. Spect. 12, 596-601.

Biomed.

G. J.