2-Deoxy-2-Fluoro-d-Glucose as a Functional Probe for NMR: The Unique Metabolism Beyond Its 6-Phosphate

Abstract: Epimeric conversion of 2-deoxy-2-fluoro- d -glucose (FDG) to its 2-epimer 2-deoxy-2-fluoro- d -mannose (FDM) proved by ¹⁹F NMR has been shown to reflect the brain activity. To examine the feasibility of FDG as a new NMR probe for in vivo functional monitoring, we studied here the fundamental NMR properties of metabolites, spectral assignments, and reliability of NMR quantification. Metabolites confirmed in brain besides FDM-6-phosphate were as follows: FDG-1-phosphate, FDG-1,6-bisphosphate, FDM-1-phosphate, FDM-1,6-bisphosphate, and FDG and FDM derivatives of nucleotide diphosphate. NMR quantification of these metabolites was evaluated in comparison with the method of ¹⁸F-labeled FDG. In the NMR functional study using FDG, where a high dose is inevitable, the dose dependence of uptake was investigated. FDG uptake in mouse brain was shown to be in the range of interpretation using the biochemical parameters of enzymes for glucose uptake as long as a dose of <200 mg/kg was used.     

Noninvasive In Vivo Demonstration of 2-Fluoro-2-Deoxy-d-Glucose Metabolism Beyond the Hexokinase Reaction in Rat Brain by 19F Nuclear Magnetic Resonance Spectroscopy

The metabolism of 2-fluoro-2-deoxy-D-glucose (FDG) in vivo was observed noninvasively in rat brain using 19F nuclear magnetic resonance (NMR) spectroscopy following an intravenous injection of FDG (400 mg/kg). At 3 h after infusion, four resonances with discrete chemical shifts were resolved. Chemical shift analysis of these resonances suggested the chemical identity of two of the resonances to be FDG and/or FDG-6-phosphate and 2-fluoro-2-deoxy-delta-phosphogluconolactone and/or 2-fluoro-2-deoxy-6-phosphogluconate. The chemical identities of the other two resonances remain to be elucidated. The present study indicates that the metabolism of FDG in vivo is more extensive than is previously recognized and demonstrates the feasibility of using 19F NMR spectroscopy to follow the 19F-containing metabolites of FDG in vivo.     

Uptake of 2-fluoro-2-deoxy-d-[U-14C]-glucose during chemotherapy in murine Lewis lung tumor

Mice bearing intramuscular Lewis lung tumor were treated with BCNU and doxorubicin (ADM) to study chemotherapy-induced changes in the uptake of 2-fluoro-2-deoxy-[U-¹⁴C]glucose (FDG). A decreased FDG uptake, tumor regression and a diminished proportion of aneuploid versus diploid cells as evaluated by DNA flow cytometry were seen after treatment with BCNU but not with ADM; HPLC indicated that most of the ¹⁴C activity in tumors was from FDG6-phosphate. The results suggest that changes in FDG uptake reflect the effectiveness of antitumor therapy. FDG may be valuable in follow-up studies of cancer treatment.     

In vivo metabolism of 3-deoxy-3-fluoro-D-glucose

Recent studies have demonstrated that 3-deoxy-3-fluoro-D-glucose (3-FG) is metabolized to 3-deoxy-3-fluoro-D-sorbitol (3-FS), via aldose reductase, and 3-deoxy-3-fluoro-D-fructose (3-FF), via the sorbitol dehydrogenase reaction with 3-FS, in rat cerebral tissue (Kwee, I. L., Nakada, T., and Card, P. J. (1987) J. Neurochem. 49, 428-433). However, the biochemistry of 3-FG in other mammalian organs has not been investigated making the application of 3-FG as a metabolic tracer uncertain. To address this issue we investigated 3-FG metabolism and distribution in isolated cell lines and in rabbit tissues in vivo with 19F NMR and gas chromatography-mass spectrometry. In general, the production of 3-FS is well correlated with the known distribution of aldose reductase in all the systems studied. Further metabolism of 3-FS to 3-FF was verified to occur in cerebral tissue. Surprisingly, two new fluorinated compounds were found in the liver and kidney cortex. These compounds are identified as 3-deoxy-3-fluoro-D-gluconic acid, which is produced via glucose dehydrogenase activity on 3-FG, and 3-deoxy-3-fluoro-D-gluconate-6-phosphate. Based on enzyme studies, it is argued that the 3-deoxy-3-fluoro-D-gluconate-6-phosphate is derived directly from 3-deoxy-3-fluoro-D-gluconic acid and not as a product of pentose phosphate activity. Direct oxidation and reduction are the major metabolic routes of 3-FG, not metabolism through glycolysis or the pentose phosphate shunt. Thus, 3-FG metabolism coupled with 19F NMR appears to be very useful for monitoring aldose reductase and glucose dehydrogenase activity in vivo.     

Metabolism and actions of 2-deoxy-2-fluoro-D-galactose in vivo

The synthetic D-galactose analog 2-deoxy-2-fluoro-D-galactose (dGalF) offers unique advantages for studies of the D-galactose pathway by non-invasive techniques using 19F-NMR spectroscopy or positron emission from the 18F-labeled compound. The metabolism of 2-deoxy-2-fluoro-D-galactose was studied in rodents using the unlabeled, the 18F-labeled, and the 14C-labeled D-galactose analog. Analyses for the metabolites of 2-deoxy-2-fluoro-D-galactose were performed by HPLC, enzymatic methods, and 19F-NMR spectroscopy in vivo and in vitro. The metabolism of 2-deoxy-2-fluoro-D-galactose was most active in the liver which took up the major part of the administered dose of the 14C-labeled D-galactose analog, but renal excretion was also pronounced. This was confirmed by in vivo scanning of the rat using the 18F-labeled sugar (1.5 microCi/g; 25 nmol/g) and examination by positron-emission tomography and gamma camera. The dose dependence of the levels of the hepatic metabolites of 2-deoxy-2-fluoro-D-galactose was investigated for doses between 25 nmol/g body mass and 1 mumols/g body mass. After 1 h, the major part of the acid-soluble uracil nucleotides consisted of UDP-2-deoxy-2-fluoro-D-hexoses when the dose was at least 0.1 mumols/g. With higher doses, 2-deoxy-2-fluoro-D-galactose 1-phosphate became the predominant initial metabolite. After a dose of 1 mumols/g 2-deoxy-2-fluoro-D-galactose 1-phosphate accumulated rapidly (5.3 +/- 0.4 mumols/g liver after 30 min) followed by the formation of UDP-2-deoxy-2-fluoro-D-galactose and UDP-2-deoxy-2-fluoro-D-glucose (0.7 +/- 0.1 mumols/g and 1.8 +/- 0.1 mumols/g, respectively, after 5 h). The diversion of uridylate, due to the accumulation of UDP-2-deoxy-2-fluoro-D-hexoses, was associated with a rapid depletion of hepatic UTP, UDP-glucose, and UDP-galactose. The UTP content was decreased to 11 +/- 6% of normal within 15 min after administration of 2-deoxy-2-fluoro-D-galactose at a dose of 1 mumols/g. The UTP-depleting action was minimal, however, at a dose of 25 nmols/g or less, indicating that interference in uridylate metabolism would be negligible at the doses required for positron-emission tomography of the liver using the 18F-labeled compound. At higher doses, the UTP deficiency induced by 2-deoxy-2-fluoro-D-galactose could be useful in the chemotherapy of D-galactose-metabolizing tumors such as hepatocellular carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of 1-phosphofructokinase from Pseudomonas putida.

The 1-phosphofructokinase (1-PFK, EC 2.7.1.56) from Pseudomonas putida was partially purified by a combination of (NH4)2SO4 fractionation and DEAE-Sephadex column chromatography. In its kinetic properties, this enzyme resembled the 1-PFK's from other bacteria. With the substrates fructose-1-phosphate (F-1-P) and adenosine triphosphate (ATP) Michaelis-Menten kinetics were observed, the Km for one substrate being unaffected by a variation in the concentration of the other substrate. At pH 8.0, the Km values for F-1-P and ATP were 1.64 X 10(-4) M and 4.08 X 10(-4) M, respectively. At fixed concentrations of F-1-P and ATP, an increase in the Mg2+ resulted in sigmoidal kinetics. Activity was inhibited by ATP when the ratio of ATP:Mg2+ was greater than 0.5 suggesting that ATP:2 Mg2+ was the substrate and free ATP was inhibitory. Activity of 1-PFK was stimulated by K+ and to a lesser extent by NH4+ and Na+. The reaction rate was unaffected by 2 mM K2HPO4, pyruvate, phosphoenolpyruvate, adenosine monophosphate, adenosine 3',5'-cyclic monophosphate, fructose-6-phosphate, glucose-6-phosphate, 6-phosphogluconate, 2-keto-3-deoxy-6-phosphogluconate, or citrate. The results indicated that the 1-PFK from P. putida was not allosterically regulated by a number of metabolites which may play an important role in the catabolism of D-fructose.     

19F NMR investigations of the catalytic mechanism of phosphoglucomutase using fluorinated substrates and inhibitors.

The complexes of phosphoglucomutase with a number of fluorinated substrate analogues have been investigated by 19F NMR and the effects of the binding of Li+ and Cd2+ to these complexes determined. Very large downfield chemical shift changes (-14 to -19 ppm) accompanied binding of the inhibitors 6-deoxy-6-fluoro-alpha-D-glucopyranosyl phosphate and alpha-glucosyl fluoride 6-phosphate to the phosphoenzyme. Smaller shift changes were observed for ligands substituted with fluorine at other positions. Addition of Li+ to enzyme/fluorinated ligand complexes caused a 10(2)- to 10(3)-fold decrease in ligand dissociation constants as witnessed by the change from intermediate to slow-exchange conditions in the NMR spectra. Measurement of the 19F NMR spectra of complexes of the Li(+)-enzyme with each of the fluoroglucose 1-phosphates and 6-phosphates has provided some insight into the environment of each of these fluorines (thus also parent hydroxyls) in each of the complexes. Results obtained argue strongly against a single sugar binding mode for the glucose 1- and 6-phosphates. Two enzyme-bound species were detected in the 19F NMR spectra of the complexes formed by reaction of the Cd(2+)-phosphoenzyme complex with the 2- and 3-fluoroglucose phosphates. These are tentatively assigned as the fluoroglucose 1,6-bisphosphate species bound in two different modes to the dephosphoenzyme. Only one bound species was observed in the case of the 4-fluoroglucose phosphates.(ABSTRACT TRUNCATED AT 250 WORDS)     

(19)F-heptuloses as tools for the non-invasive imaging of GLUT2-expressing cells

Suitable analogs of d-mannoheptulose are currently considered as possible tools for the non-invasive imaging of pancreatic islet insulin-producing cells. Here, we examined whether (19)F-heptuloses could be used for non-invasive imaging of GLUT2-expressing cells. After 20 min incubation, the uptake of (19)F-heptuloses (25 mM) by rat hepatocytes, as assessed by (19)F NMR spectroscopy, ranged from 0.50 (1-deoxy-1-fluoro-d-mannoheptulose and 3-deoxy-3-fluoro-d-mannoheptulose) to 0.25 (1,3-dideoxy-1,3-difluoro-d-mannoheptulose) and 0.13 (1-deoxy-1-fluoro-d-glucoheptulose, 3-deoxy-3-fluoro-d-glucoheptulose and 1,3-dideoxy-1,3-difluoro-d-glucoheptulose) μmol per 3×10(6)cells. (19)F MRI experiments also allowed the detection of 1-deoxy-1-fluoro-d-mannoheptulose in rat hepatocytes. All three (19)F-mannoheptuloses cited above, as well as 7-deoxy-7-fluoro-d-mannoheptulose and 1-deoxy-1-fluoro-d-glucoheptulose inhibited insulin release evoked in rat isolated pancreatic islets by 10mM d-glucose to the same extent as that observed with an equivalent concentration (10mM) of d-mannoheptulose, while 3-deoxy-3-fluoro-d-glucoheptulose and 1,3-dideoxy-1,3-difluoro-d-glucoheptulose (also 10mM) were less potent than d-mannoheptulose in inhibiting insulin release. The 1-deoxy-1-fluoro-d-mannoheptulose and 3-deoxy-3-fluoro-d-mannoheptulose only marginally affected INS-1 cell viability. These findings are compatible with the view that selected (19)F-heptuloses may represent suitable tools for the non-invasive imaging of hepatocytes and insulin-producing cells by (19)F MRI.     

Microdialysis and 2-[18F]fluoro-2-deoxy-D-glucose (FDG): a study on insulin action on FDG transport,uptake and metabolism in rat muscle,liver and adipose tissue

A combination of microdialysis (MD) and 2-[18F ]fluoro-2-deoxy-D-glucose (FDG) was used to assess FDG uptake, phosphorylation and the glucose metabolic index (Rg') in certain tissues of fed and fasting anesthetized Sprague-Dawley rats which received an i.v. bolus injection of insulin or saline during the course of the study. The relative recovery for FDG for the MD probes was also measured as a function of flow rate and temperature. The elimination half-life (T(1/2 FDG)) of FDG from the plasma and the extracellular fluid of muscle and liver was studied with MD. The phosphorylation of FDG in muscle, liver, subcutaneous fat and mesenteric fat from homogenates of these tissues was analyzed by a radioHPLC-method and the Rg' was calculated. The results show that the nutritional status does not affect the T(1/2 FDG), the total uptake of FDG 6-phosphate or the Rg' values in the studied tissues at ambient glucose. Insulin stimulation decreased T(1/2 FDG), and increased the total FDG 6-P accumulation and Rg' in the muscle of fed and fasted rats. In adipose tissues the insulin stimulation enhanced the phosphorylation but in muscle the proportion of FDG 6-P remained unchanged. Rg' in adipose tissue was higher after insulin administration in fed rats than without insulin but with fasted rats there were no differences in Rg' values with or without insulin, although the proportion of FDG 6-P did increase. The Rg' values for the livers were unaffected by any of the manipulations, but fasted rats accumulated proportionately more FDG 6-P after insulin administration than did fed rats. These results indicate that the combination of MD and FDG is a valuable and reliable tool when studying glucose metabolism in physiological and pathological models in vivo.     

Enzymatic synthesis of UDP-(3-deoxy-3-fluoro)-D-galactose and UDP-(2-deoxy-2-fluoro)-D-galactose and substrate activity with UDP-galactopyranose mutase

The novel UDP-sugar uridine 5'-(3-deoxy-3-fluoro-D-galactopyranosyl diphosphate) (1) and UDP-(2-deoxy-2-fluoro)-D-galactose (2) have been prepared enzymatically and tested as substrate analogues for the enzyme UDP-galactopyranose mutase (UDP-Galp mutase EC 5.4.99.9). Turnover of both 1 and 2 by UDP-Galp mutase was observed by HPLC and 19F NMR. The HPLC elution profile and 19F chemical shift of the products are consistent with the formation of the predicted furanose forms of 1 and 2. The Km values for compounds 1 and 2 were similar to those of the natural substrate UDP-Galp (0.26 mM for 1, 0.2 mM for 2, and 0.6 mM for UDP-Galp), but the values for kcat were substantially different (1.6/min for 1, 0.02/min for 2, and 1364/min for UDP-Galp). A correlation was also observed between the equilibrium yield of product formed during turnover of UDP-sugar by UDP-Galp mutase (UDP-Galp, compound 1 or compound 2), and the amount of furanose present for the free sugar at thermal equilibrium in aqueous solution, using 1H and 19F NMR spectroscopy. The implications of these results to the mechanism of the unusual enzymatic reaction are discussed.     

The effect of glucose, insulin and noradrenaline on lipolysis and on the concentrations of adenosine 3′:5′-cyclic monophosphate and adenosine 5′-triphosphate in adipose tissue

1. The deoxyfluoro-d-glucopyranose 6-phosphates were prepared from the corresponding deoxyfluoro-d-glucoses and ATP by using hexokinase. 2. 3-Deoxy-3-fluoro- and 4-deoxy-4-fluoro-d-glucose 6-phosphate were substrates for glucose phosphate isomerase, and in addition the products of this reaction, 3-deoxy-3-fluoro- and 4-deoxy-4-fluoro-d-fructose 6-phosphate respectively, were good substrates for phosphofructokinase. 3. Some C-2-substituted derivatives of d-glucose 6-phosphate were found to be competitive inhibitors of glucose phosphate isomerase. 4. The possible role of the hydroxyl groups in the binding of d-glucose 6-phopshate to glucose phosphate isomerase is discussed.     

Inhibition of in vitro biosynthesis of n-acetylneuraminic acid by n-acyl- and n-alkyl-2-amino-2-deoxyhexoses

The biosynthesis of N-acetylneuraminic acid is markedly inhibited by 2-deoxy-2-propionamido-d-glucose (GlcNProp) and to a much lesser extent by 2-deoxy-2-propionamido-d-mannose (ManNProp), but not by 2-deoxy-2-propionamido-d-galactose and N-methylated derivatives of 2-amino-2-deoxy-d-glucose. 2-Deoxy-2-trimethylamino-d-glucose is a weak inhibitor of 2-acetamido-2-deoxy-d-mannose metabolism. When incubated in a cell-free system from rat liver, GlcNProp gives the 6-phosphate, which is converted into N-propionylneuraminic acid. Evidence is presented which shows that it is the metabolites GlcNProp-6-P and ManNProp-6-P which are the competitive inhibitors, and not GlcNProp itself.     

Cellular release of [18F]2-fluoro-2-deoxyglucose as a function of the glucose-6-phosphatase enzyme system

[(18)F]-2-Fluoro-2-deoxyglucose (FDG) is a glucose analog currently utilized for positron emission tomography imaging studies in humans. FDG taken up by the liver is rapidly released. This property is attributed to elevated glucose-6-phosphatase (Glc-6-Pase) activity. To characterize this issue we studied the relationship between Glc-6-Pase activity and FDG release kinetics in a cell culture system. We overexpressed the Glc-6-Pase catalytic unit in a Glc-6-Pase-deficient mouse hepatocyte (Ho-15) and in A431 tumor cell lines. Glc-6-Pase enzyme activity and FDG release rates were determined in cells transfected with the Glc-6-Pase gene (Ho-15-D3 and A431-AC3), in mock-transfected cells of both cell lines, and in wild-type mouse hepatocytes (WT10) as control. Although the highest level of Glc-6-Pase activity was measured in A431-AC3, Ho-15-D3 cells showed much faster FDG release rates. The faster FDG release correlated with the level of glucose 6-phosphate transporter (Glc-6-PT) mRNA, which was found to be expressed at higher levels in Ho-15 compared with A431 cells. Overexpression of Glc-6-PT in A431-AC3 produced a dramatic increase in FDG release compared with control cells. This study gives the first direct evidence that activity of the Glc-6-Pase complex can be quantified in vivo by measuring FDG release. Adequate levels of Glc-6-Pase catalytic unit and Glc-6-PT are required for this function. FDG-positron emission tomography may be utilized to evaluate functional status of the Glc-6-Pase complex.     

The sphingolipid degradation product trans-2-hexadecenal forms adducts with DNA

Sphingosine 1-phosphate, a bioactive signaling molecule with diverse cellular functions, is irreversibly degraded by the endoplasmic reticulum enzyme sphingosine 1-phosphate lyase, generating trans-2-hexadecenal and phosphoethanolamine. We recently demonstrated that trans-2-hexadecenal causes cytoskeletal reorganization, detachment, and apoptosis in multiple cell types via a JNK-dependent pathway. These findings and the known chemistry of related α,β-unsaturated aldehydes raise the possibility that trans-2-hexadecenal may interact with additional cellular components. In this study, we show that it reacts readily with deoxyguanosine and DNA to produce the diastereomeric cyclic 1,N(2)-deoxyguanosine adducts 3-(2-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8R-hydroxy-6R-tridecylpyrimido[1,2-a]purine-10(3H)one and 3-(2-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8S-hydroxy-6S-tridecylpyrimido[1,2-a]purine-10(3H)one. Thus, our findings suggest that trans-2-hexadecenal produced endogenously by sphingosine 1-phosphate lyase can react directly with DNA forming aldehyde-derived DNA adducts with potentially mutagenic consequences.     

Effect of regional hyperemia on myocardial uptake of 2-deoxy-2-[(18)F]fluoro-D-glucose

2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) may be used to predict glucose kinetics when the factor relating differences in transport and phosphorylation between compounds remains constant ("lumped constant"). It is not clear whether hyperemia alters that factor. In anesthetized swine, myocardial FDG uptake was estimated by positron emission tomography, during an intracoronary infusion of either adenosine, ATP, or bradykinin (40 microg x kg(-1) x min(-1), 40 microg x kg(-1) x min(-1), and 2 nmol x kg(-1) x min(-1), respectively; n = 6 for all groups). In controls during normal perfusion (n = 6), FDG uptake was 0.78 +/- 0.32 micromol x g(-1) x min(-1), whereas glucose uptake by Fick was 0.71 +/- 0.25 micromol x g(-1) x min(-1) (r = 0.73; P < 0.05). Adenosine increased blood flow from 1.29 +/- 0.43 to 4.80 +/- 2.19 ml x g(-1) x min(-1) (P < 0.05) and glucose uptake from 1.16 +/- 1.10 to 3.35 +/- 2.12 micromol x g(-1) x min(-1) (P < 0.05), whereas FDG uptake in the hyperemic region was lower than remote regions (0.46 +/- 0.29 and 0.95 +/- 0.55 micromol x g(-1) x min(-1), respectively; P < 0.05). In the ATP and bradykinin groups, blood flow increased four- and twofold, respectively, with no net change in glucose uptake. FDG uptake in the hyperemic region was also significantly lower than remote regions. For all animals, the ratio of blood flow in the hyperemic region relative to remote region was inversely proportional to the ratio of FDG uptake in the same regions (r(2)=0.73; P < 0.001). Because nitric oxide elaboration during hyperemia could potentially alter substrate preference and FDG kinetics, six additional swine were studied during maximal adenosine before and after intracoronary N(G)-monomethyl-L-arginine (1.5 mg/kg). Inhibition of nitric oxide had no effect on either regional myocardial substrate uptake or FDG accumulation. In conclusion, hyperemia decreased regional myocardial FDG uptake relative to normally perfused regions and this effect on the lumped constant was independent of nitric oxide.     

Identification of the 22-phosphate esters of ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone from newly laid eggs of the desert locust, Schistocerca gregaria.

The four major ecdysteroid (insect moulting hormone) conjugates present in the newly laid eggs of the desert locust, Schistocera gregaria, have been purified by reversed-phase and anion-exchange high-performance liquid chromatography. The steroid moieties were identified as ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone. Phosphate analysis of acid-hydrolysed samples showed a steroid:phosphate ratio of approx. 1:1 for all four compounds. The intact conjugates were identified as ecdysone 22-phosphate, 2-deoxyecdysone 22-phosphate, 20-hydroxyecdysone 22-phosphate and 2-deoxy-20-hydroxyecdysone 22-phosphate by fast atom bombardment mass spectrometry and 1H, 13C and 31P n.m.r. The significance of ecdysteroid phosphates as a source of free hormone during embryogenesis is discussed.     

Two-step regio- and stereoselective syntheses of [19F]- and [18F]-2-deoxy-2-(R)-fluoro-beta-D-allose

Replacement of specific hydroxyl groups by fluorine in carbohydrates is an ongoing challenge from chemical, biological, and pharmaceutical points of view. A rapid and efficient two-step, regio- and stereoselective synthesis of 2-deoxy-2-(R)-fluoro-beta-d-allose (2-(R)-fluoro-2-deoxy-beta-d-allose; 2-FDbetaA), a fluorinated analogue of the rare sugar, d-allose, is described. TAG (3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol or 3,4,6-tri-O-acetyl-d-glucal), was fluorinated in anhydrous HF with dilute F(2) in a Ne/He mixture or with CH(3)COOF at -60 degrees C. The fluorinated intermediate was hydrolyzed in 1N HCl and the hydrolysis product was purified by liquid chromatography and characterized by 1D (1)H, (13)C, and (19)F NMR spectroscopy as well as 2D NMR spectroscopy and mass spectrometry. In addition, (18)F-labeled 2-deoxy-2-(R)-fluoro-beta-d-allose (2-[(18)F]FDbetaA) was synthesized for the first time, with an overall decay-corrected radiochemical yield of 33+/-3% with respect to [(18)F]F(2), the highest radiochemical yield achieved to date for electrophilic fluorination of TAG. The rapid and high radiochemical yield synthesis of 2-[(18)F]FDbetaA has potential as a probe for the bioactivity of d-allose.     

Di-tert-butyl diethylphosphoramidite as the phosphitylating reagent in the preparation of 3-deoxy-3-C-methylene-D-ribo-hexose-6-phosphate and 3-deoxy-3-C-methylene-D-erythro-pentose-5-phosphate.

3-Deoxy-3-C-methylene-D-ribo-hexose-6-phosphate and 3-deoxy-3-C-methylene-D-erythro-pentose-5-phosphate were prepared from a common intermediate 3-deoxy-3-C-methylene-1,2-O-isopropylidene-alpha-D-ribo-hexofuranose. The preparation of the phosphorylated unsaturated sugars employed di-tert-butyl diethylphosphoramidite as the phosphitylating reagent. The removal of all the protecting groups was done under acidic conditions in the ultimate step. The unsaturated sugar phosphates were competitive inhibitors but neither substrates nor inactivators of glucose-6-phosphate and ribose-5-phosphate isomerases.     

A comparative study on diurnal changes in metabolite levels in the leaves of three crassulacean acid metabolism (CAM) species, Ananas comosus, Kalancho? daigremontiana and K. pinnata.

A comparative study on diurnal changes in metabolite levels associated with crassulacean acid metabolism (CAM) in the leaves of three CAM species, Ananas comosus (pineapple), a hexose-utilizing species, and Kalancho? daigremontiana and K. pinnata, two starch-utilizing species, were made. All three CAM species showed a typical feature of CAM with nocturnal malate increase. In the two Kalancho? species, isocitrate levels were higher than citrate levels; the reverse was the case in pineapple. In the two Kalancho? species, a small nocturnal citrate increase was found and K. daigremontiana showed a small nocturnal isocitrate increase. Glucose 6-phosphate (G-6-P), fructose 6-phosphate (F-6-P) and glucose 1-phosphate (G-1-P) levels in the three CAM species rose rapidly during the first part of the dark period and decreased during the latter part of the dark period. The levels of the metabolites also decreased during the first 3 h of the light period, then, remained little changed through the rest of the light period. Absolute levels of G-6-P, F-6-P and G-1-P were higher in pineapple than in the two Kalancho? species. Fructose 1,6-bisphosphate (F-1,6-P(2)) levels in the three CAM species increased during the dark period, then dramatically decreased during the first 3 h of the light period and remained unchanged through the rest of the light period. The extent of nocturnal F-1,6-P(2) increase was far greater in the two Kalancho? species than in pineapple. Absolute levels of F-1,6-P(2) were higher in the two Kalancho? species than in pineapple, especially during dark period. Diurnal changes in oxaloacetate (OAA), pyruvate (Pyr) and phosphoenolpyruvate (PEP) levels in the three CAM species were similar.