Inhibition of fructose bisphosphatase and stimulation of phosphofructokinase by a stable isosteric phosphonate analog of fructose 2,6-bisphosphate

ARCHIVES

OF BIOCHEMISTRY

Vol. 245, No. 1, February

AND BIOPHYSICS

15, pp. 282-286,1986

COMMUNICATION Inhibition of Fructose Bisphosphatase Stable lsosteric Phosphonate

and Stimulation of Phosphofructokinase Analog of Fructose 2,6-Bisphosphate

by a

RONALD W. McCLARD,**Tl SOTIRIOS TSIMIKAS,* AND KENNETH E. SCHRIVERt” *Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 0.2167, and the TArthur F. Scott Laboratory of Chemistry, Reed College, Portland, Oregmz 97202 Received August 12, 1985, and in revised form November

11,1985

We describe the synthesis of a mixture of D-manno- and D-gluco-2,5-anhydro-l-deoxy1-phosphonohexitol 6-phosphate via a Horner-Emmons reaction of 2,3,5-tri-o-benzylfi-D-arabinofuranose followed by phosphorylation of the equivalent 6-position and subsequent deprotection. This mixture inhibits fructose-1,6-bisphosphatase; the concentration required for half-maximal effect in the presence of 25 PM AMP is approximately 6 j&M. The mixture of analogs also stimulates 6-phosphofructo-1-kinase from rabbit liver; the concentration required to reach one-half V,, was found to be ca. 25 I.LM at 0.25 mM fructose 6-phosphate and 50 PM AMP. These analogs have replaced the labile anomeric phosphate of fructose 2,6-bisphosphate with a stable methylenephosphonate, and could be of great interest due to their appropriate physiological effects and their chemical stability. 0 1386 Academic PM+ I~X.

During the past 5 years, significant interest has been focused on the regulation of glycolysis and gluconeogenesis by @n-fructose 2,6-bisphosphate (Fru 2,6-Pz, 1, Fig. 1): Fru 2,6-P, promotes glycolysis by stimulating 6-phosphofructo-1-kinase (PFK, EC 2.7.1.11). the enzyme that catalyzes the conversion of fructose 6-phosphate (Fru 6-P) into fructose l,&bisphosphate (Fru 1,6-P,); 1 attenuates gluconeogenesis by inhibiting fructose-1,6-bisphosphatase (FBPase, EC 3.1.3.11), the enzyme that catalyzes the hydrolysis of Fru 1,6-Pz to Fru 6-P and phosphate [for reviews see

i Correspondence should be addressed to this author at Reed College. Recipient of New Investigator Research Award (CA-33659) from the National Institutes of Health. 2Present address: Department of Chemistry and Biochemistry, University of California, Los Angeles, Calif. 90024. 3 Abbreviations used: Fru 2,6-Pz, fi-n-fructose 2,6bisphosphate; PFK, 6-phosphofructo-1-kinase; Fru 6P, fructose 6-phosphate; Fru 1,6-P*, fructose 1,6-bisphosphate; FBPase, fructose-1,6-bisphosphatase; Dl”l’, dithiothreitol. 0003-9861/86 $3.00 Copyright Q 1966 by Academic Press, Inc. All rights of reproduction in any form reaewad.

refs. (1, 2)]. The phosphate moiety at the anomeric carbon renders 1 susceptible to both enzymatic and hydrolytic degradation. For this reason, stable analogs of 1 would be of great potential utility in biochemical studies. Since we began our work, Maryanoff et al. (3) have recently reported the syntheses of o- and B-Darabinose 1,5-bisphosphate, analogs of Fru 2,6-Pz which lack the l-CHzOH substituent; these compounds were recognized by the target enzymes nonetheless. Replacement of the hemiacetal phosphate in Fru 2,6Pz with a methylenephosphonate moiety should result in compounds with considerably longer lifetimes. We report here the synthesis of a 21 mixture of D-mannoand n-gluco-2,5-anhydro-1-deoxy-1-phosphonohexitoi 6-phosphate (2 and 3, Fig. l), along with the results of preliminary in vitro biological studies using this mixture. These compounds may be considered as isosteric analogs to (Y- and P-D-Fru 2,6-P*, respectively, although they are lacking the hydroxymethyl substituent at the equivalent anomeric position.

EXPERIMENTAL General. Tris base [tris(hydroxymethyl)aminomethane], MgClz, NADP+, ATP, AMP, EDTA, NADH, 282

ISOSTERIC

PHOSPHONATE

ANALOG

dithiothreitol (DTT), Fru 6-P, Fru 1,6-Pz, Fru 2,6-Pz, phosphoglucose isomerase, glucose 6-phosphate dehyrogenase, rabbit liver FBPase (ca. 6 units/mg protein), rabbit liver PFK (ca. 30 units/mg protein), aldolase, triose phosphate isomerase, glycerol phosphate dehydrogenase, 2,3,5-tri-0-benzyl-O-D-arabinofuranose, and NaH were all purchased from Sigma. Diethylchlorophosphate and 10% Pd on charcoal were purchased from Aldrich and tetraethylmethylenebisphosphonate was from Strem Chemicals or synthesized by a published procedure (4). Bromotrimethylsilane was prepared as described by Gilliam et al (5). NMR spectra were obtained using a Varian EM360L (60.0 MHz, ‘H), a Varian FT-80 (80.0 MHz, ‘H; 20.0 MHz, 13C;32.2 MHz, 31P), or a Jeol FX-9OQ (22.5 MHz, i3C; 36.3 MHz, “P) instrument. Thin-layer chromatography (TLC) was performed using 0.2-mm sheets of silica gel 60 (Merck) that contained a fluor for detection of uv-absorbing species. One unit of enzyme activity is defined as that amount which will catalyze the formation of 1 pmol of product per minute. Synthesis of 2 and 3. All solvents were dried and redistilled prior to use. To 0.34 g (14.2 mmol, 1.2 eq) NaH suspended in 10 ml ethylene glycol dimethyl ether (glyme) at room temperature was added 3.77 g (13.1 mmol, 1.1 eq) of tetraethylmethylenebisphosphonate dissolved in 15 ml glyme. The addition took place over 30 min. The suspension was stirred under a nitrogen atmosphere at room temperature. After evolution of hydrogen gas ceased, the slightly cloudy solution was stirred for another 30 min. To this mixture was added all at once 5.0 g (11.9 mmol, 1.0 eq) of 2,3,5-tri-@benzyl-P-D-arabinofuranose dissolved in 50 ml glyme. The resulting light yellow solution was warmed to 35-40°C and stirred for 4 h after which TLC (ethyl acetate) revealed the disappearance of the starting sugar. Sodium diethylphosphate, the other product of the reaction, formed a sticky film on the inside of the reaction vessel. The desired reaction products, D-manno- and D-gluco-2,5-anhydro-3,4,6-tri0-benzyl-1-deoxy-1-(diethoxyphosphinyl)-hexitol (4 and 5, respectively), were extracted with methylene chloride, washed with aqueous phosphate buffer, and then chromatographed on a column of 175 g of silica gel 60 (Merck, 230-400 mesh). The compounds were eluted with 312 ethyl acetate/chloroform; the compounds eluted together with an Rfof ca. 0.57 (TLC, 3/ 2 ethyl acetate/chloroform). There is approximately 10% yield of an uncyclized trots olefin intermediate isolated with an &of ca. 0.23. The desired compounds were isolated in 85% yield based on the amount of sugar used in the reaction. The ratio of 4 to 5 was determined to be about 2:l by 13CNMR of this product. The -CHzP carbon of the D-manno compound (4) is downfield (6 = 29.9, Jcr = 138 Hz) of the -CHzP carbon of the D-gluco compound (5) (6 = 25.7, Jcr = 141 Hz) as would be predicted for a tram arrangement of substituents at the “anomeric” and adiacent ring carbons~~

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of such compounds (6). The alp shifts (relative to HaPO,) were (4) +27.4 ppm and (5) +28.5 ppm. The ‘H spectrum showed the expected double doublets centered at 2.21 ppm (Jrn = 19 Hz, &a = 7 Hz) for 4 and at 2.32 ppm for the minor product 5 (same splitting pattern); these resonances are assigned to the -CHIP and are also consistent with this assignment of configuration (6). The mixture of protected hexitol isomers from the above synthesis was then dissolved in 100 ml of ethanol. To this solution was added 0.5 g of 10% palladium catalyst. The material was hydrogenated at room temperature at slightly above 1 atm pressure Hz until complete removal of the benzyl groups was shown by TLC and ‘H NMR. Upon completion, the solution was filtered through Celite and the solvent removed by rotary evaporation under reduced presand sure. This yielded 2.7 g (100% yield) of D-mannOD-gluco-2,5-anhydro-l-deoxy-l-(diethoxyphosphinyl)hexitol. The mixture of isomers from the above synthesis was dissolved in 50 ml of dry pyridine. To this solution was added 1.8 g (1.1 eq) of diethylchlorophosphate at 0°C. The solution was allowed to warm to room temperature while stirring overnight. At this time, the reaction was complete and the pyridine was removed by rotary evaporation under reduced pressure. The final traces of pyridine were removed by azeotroping with toluene. The remaining oil was dissolved in 50 ml water and extracted twice with diethyl ether. The ether layer was discarded, and the pH of the aqueous layer was adjusted to 7 before the water was removed by rotary evaporation under reduced pressure at 50°C. The residue was then dissolved in methylene chloride and cooled to 0°C to induce precipitation of pyridinium chloride. The solution was filtered and the solvent removed by rotary evaporation under reduced pressure. The remaining oil was dissolved in 50 ml acetonitrile for use in the next reaction. To the solution of diethyl D-manno- and D-ghCO2,5-anhydro-l-deoxy-l-(Diethoxyphosphinyl)-hexitol 6-phosphate was added 5 eq of bromotrimethylsilane. The addition was made while stirring the solution at 0°C. The solution was stirred at this temperature for 1 h, then allowed to warm to room temperature, followed by stirring for 5 h. Acetonitrile and ethyl bromide byproduct were removed by rotary evaporation under reduced pressure, leaving a mixture of 2 and 3 as silyl derivaties. The silyl groups were cleaved by dissolving the light yellow oil in 20 ml water. The pH of the solution was adjusted to 7.5 with NaOH, and then the solution was applied to a column containing ca. 100 ml of Dowex 1 X -4 HCOB;. The column was rinsed with water, followed by elution with aqueous triethylammonium bicarbonate (TEAH+HCO;) (700 ml, 0.1 to 0.8 M gradient). The fractions were assayed for total phosphorus by the method of Ames (7). The three main fractions that eluted were identified to be, in order of elution, (1) unphosphorylated material, (2)

284

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partially esterified product, as determined (‘H NMR) by the presence of ethyl groups that could not be removed by cation exchange chromatography, and (3) the products 2 and 3. The last material, which eluted at about 0.5 M TEAH+HCOi, was pooled and the water removed by rotary evaporation under reduced pressure. Methanol was added to the residual oil and evaporated in order to remove TEAH+HCOi. This left 2 and 3 as TEAH+ salts; phosphorus analysis of the material showed there to be 0.21 pmol of analog/mg solid, based on the knowledge that 1 mol of analog contains 2 mol of P. The material was thus composed of about 15% of the TEAH+ salt of 2 and 3; the remainder was likely TEAH+HCOi and water. The ‘H NMR gave the expected peaks for the phosphonyl methylene protons and ring protons; conversion of the material to the ND: salt (Dowex 50) gave a substance that showed no ethyl resonance pattern in the ‘H NMR spectrum. ‘iP NMR of a sample filtered through Chelex 100 resin (Bio-Rad) showed the expected two sets of phosphonate and phosphate peaks of near-equal intensities. The assignments were (2) +3.4 ppm (gphosphate) and +23.2 ppm (phosphonate), and (3) +4.0 (6-phosphate) and +22.6 (phosphonate). As for 4 and 5, the ratio of 2 and 3 was determined to be roughly 2:l based on the relative intensities of the *iP resonances. Enzyme assays. Reaction mixtures for the assay of FBPase activity contained in a final volume of 1 ml (pH 7.5), 150 mM KCl, the following: 50 mM Tris-HCl 5 mM MgClz, 0.1 mM EDTA, 0.15 mM NADP+, phosphoglucose isomerase (1.2 units), glucose-gphosphate isomerase (1.2 units), FBPase (5 munit), and 5 PM Fru 1,6-Pz. The assays were initiated by the addition of Fru 1,6-Pz after equilibrating for several minutes at 30°C. The rate of FBPase is measured as the rate of increase in absorbance at 340 nm due to the reduction of NADP+. The assay for rabbit liver phosphofructokinase was the coupled aldolase assay adapted from

AND

SCHRIVER

FIG. 2. Effect of mixture of 2 and 3 on the activity of rabbit liver FBPase. Assays were performed using 5 pM Fru 1,6-Pz in the absence (0) or presence (M) of 25 PM AMP.

several different sources [see, for example, (8,9)]. All enzymes were extensively dialyzed against the enzyme buffer described below in order to remove ammonium ion. Enzyme buffer consisted of 50 mM Tris-HCl, pH 7.5, at 25”C, 0.1 mM ATP, 0.1 mM DTT, 1.0 rnrd EDTA, and 0.2 mg/ml bovine serum albumin. The assay mixture contained, in a final volume of 1 ml, 50 mM TrisHCl, pH 7.0, at 25’C, 1 mM ATP, 1 mrd EDTA, 2.5 mM DTT, 6.0 mM MgClz, 0.16 mM NADH, aldolase (0.4 unit), triose phosphate isomerase (4.0 units), glycerol phosphate dehydrogenase (0.4 unit), PFK from rabbit liver (5 munit), and various amounts of AMP, Fru 6P, and the mixture of 2 and 3 as indicated. The assays were initiated by addition of PFK. The decrease in absorbance at 340 nm was measured at 25’C. The rate of endogenous NADH oxidation was determined for each run individually during the equilibration period, each reported rate is measured 4 min after initiation and is corrected for the endogenous oxidation rate. The V,, for PFK was determined from reactions which contained 5 mM Fru 6-P and 50 PM AMP in the standard assay mixture. RESULTS

4 R,. 4. R=

-cbyh

R*. -C”*POww2

5 11,s -C”2PO(OEL,2. R*i -”

2 R*i -H. R2* -CH2P03 3 11,. -CHpa,.

R2- -n

FIG. 1. Structure of @-Fru 2,6,-Pz (1) and scheme for the synthesis of the analogs 2 and 3. Conditions were: a = CHz(P(O)(OEt)z)z, NaH, 35’C, b = Pd/C, Hz; c = 1 eq (EtO)zP(O)Cl, pyridine; d = MeaSiBr/H& e = chromatography on Dowex 1 (see Experimental for details). 8 = phosphate.

AND

DISCUSSION

The mixture of compounds 2 and 3 (approximately in a 21 ratio, respectively) was synthesized as described above from 2,3,5-tri-6benzyl-B-D-arabinofuranose via a Horner-Emmons reaction with tetraethylmethylenebisphosphonate to yield 4 and 6. The reaction proceeds through an acyclic olefin intermediate (6) which spontaneously cyclizes. The mixture of 4 and 5 was debenzylated, phosphorylated at the B-position, and deprotected by silylation and hydrolysis (see Fig. 1).

ISOSTERIC

PHOSPHONATE

ANALOG

The mixture of the phosphonate analogs 2 and 3 inhibits rabbit liver FBPase in the micromolar range as is shown in Fig. 2. The MO.6(concentration of modifier required for half-maximal effect) is significantly reduced from 12 to 6 axd when AMP is present at 25 PM. This effect is in keeping with that observed for Fru 2,6-P* (10, 11). For example, Van Schaftigen and Hers (11) found that the A& for Fru 2,6-Pa under similar conditions was about 3.3 PM and was lowered to about 0.3 PM in the presence of 25 PM AMP. The mixture of compounds 2 and 3 also stimulates the activity of liver PFK as shown in Fig. 3. At [Fru 6-P] = 0.25 mM and [ATP] = 1 mM, the analog mixture has an Mea of about 45 PM; that value is lowered to about 25 PM in the presence of 50 PM AMP. This synergism with AMP is as observed for the natural modifier, Fru 2,6-Pa (8,12). Under the latter conditions we found a half-maximal stimulation by about 0.05 pM Fru 2,6-P*. This observation is consistent with the range of concentrations required for the same effect under a variety of conditions (8, 12). Also analogous to observations of the action of Fru 2,6-P2 is the fact that the analogs exert their effect on PFK by shifting the S,,6 for Fru 6-P (Fig. 4). The .S,,.svalue is reduced from about 1.2 mM down to about 0.2 mM when 50 MM analogs is added to assays that contain 50 pM AMP. The mixture of 2 and 3 had a small stimulatory effect on PFK from rabbit muscle (data not shown). Triethylammonium chloride up to 25 mM had no significant effect on either PFK or FBPase. Thus we concluded that excess TEAH+HCO, in our preparations was inert. The responses of the two enzymes to these phosphonate analogs are very similar to those reported by Maryanoff et al. (3) for a- and 6-D-arabinose 1,5bisphosphate, which are different from 2 and 3 in

I

-

I

I

I

I

FIG. 3. Effect of mixture of 2 and 3 on the activity of rabbit liver PFK. Assay mixtures contained 0.25 mM Fru 6-P, 1.0 mM ATP, and either no AMP (0) or 50 PM AMP (0).

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\>

0

1

2 [Fru

4

3 6-P]

5

, mM

FIG. 4. Effect of mixture of 2 and 3 on affinity of PFK for Fru 6-P. Assays were done according to the following scheme: 1.0 mM ATP alone (A); 1.0 mM ATP and 50 PM AMP (A); 1.0 mM ATP, 50 PM AMP, and 50 gM analog mixture, 2 and 3 (m).

having an -0- in place of a -CHa-. The compounds described herein have the advantage of being relatively stable phosphonates instead of labile hemiacetal phosphates and the expected higher pK,, of the phosphonates apparently does not compromise their ability to stimulate PFK or inhibit FBPase. Comparison of 2 and 3 to &Fru 2,6-Pa reveals that the analogs are roughly 4-20 times less potent in inhibiting FBPase and approximately 500 times less potent in stimulating PFK. Clearly the l-CHaOH of Fru 2,6-P*, which is missing in our analogs and in arabinose l,&bisphosphate (3). contributes significantly to the metabolite’s ability to be recognized by these regulatory enzymes. It is unlikely that the minor component 3 competes with a-Fru 1,6-P*, the true substrate of FBPase (13), since the analog has a oneatom spacing between “anomeric” carbon and phosphorus and thus more closely mimics Fru 2,6-Pr for that reason. Our NMR data clearly indicate that the major phosphonate isomer is 2 which is the analog of the LY anomer of Fru 2,6-P*. Thus it is possible that pure 3 would give much lower M0.6 values for both FBPase and PFK. We have developed a stereospecific synthesis for 5, the precursor to 3, and shall report later on the effect of the single isomer, as well as additional analogs, on the kinase and phosphatase.’

* During review of this paper, Reitz et al. (14) reported the stereospecific synthesis of 3 by an approach similar to ours. They did not report any biochemical data for the compound.

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ACKNOWLEDGMENTS This work was funded by grants from the Juvenile Diabetes Foundation (184165), National Institutes of Health (CA-33659), and Boston College. The authors are grateful to Mr. Steven A. Jackson and Dr. Raymond R. Bard for their assistance in obtaining NMR spectra, and to the Oregon Graduate Center, where some of the NMR spectra were run.

REFERENCES 1. PILKIS, S. J., CHRISMAN, T., BURGESS,B., MCGRANE, M., COLOSIA, A., PILKIS, J., CLAUS, T., AND ELMAGHRABI, M. R. (1983) A&J. Enzyme Regd 21,147-172. 2. HERS, H. G., AND HUE, L. (1983) Annu. Rev. Biochem 52.617-653. 3. MARYANOFF, B. E., REITZ, A. B., TUTWEILER, G. F., BENKOVIC, S. J., BENKOVIC, P. A., AND PJLKIS, S. J. (1984) J. Amer. Chem Sot 106,7851-7853. 4. SCHWARZENBACH, G., AND ZURC, J. (1950) Manat.?chr. Chem 81,202-212.

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5. GILLIAM, W. F., MEALS, R. N., AND SAIJER, R. 0. (1946) J. Amer. Chem Sot. 68.1161-1163. 6. OHRUI, H., JONES, G. H., MOFFATT, J. G., MADDOX, M. L., CHRISTENSEN, A. T., AND BYRAM, S. K. (1975) J. Amer. Chem. Sot 97,4602-4613. 7. AMES, B. N. (1966) in Methods in Enzymology (Neufeld, E. F., and Ginsburg, V., eds.), Vol. 8, pp. 115-118, Academic Press, New York. 8. UYEDA, K., FURUYA, E., AND LUBY, L. (1981) .I Biol Chem. 256,8394-8399. 9. KEMP, R. G. (1971) J. Biol. Chem 246,245-252. 10. PILKIS, S. J., EL-MAGHRABI, M. R., PILKIS, S. J., AND CLAUS, T. H. (1981) J. Bid Ch.em 256,36193622. 11. VAN SCHAFTIGEN, E., AND HERS, H. G. (1981) Proc Nat1 Acad Sci. USA 78,2861-2863. 12. VAN SCHAFTIGEN, E., JE’IT, M. F., HUE, L., AND HERS, H. G. (1981) Proc. Nat1 Acud Sci USA 78,3483-3486. 13. FREY, W. A., FISHBEIN, R., DE MAINE, M. M., AND BENKOVIC, S. J. (1977) Biochemistry 16, 24792484. 14. REITZ, A. B., NORTEY, S. O., AND MARYANOFF, Z&t. 26,3915-3918. B. E. (1985) Tetrahedron