Synthesis of sorbistin analogues

D-Galactose was converted into the glycosylating agents 4-azido-2,3-di-O-benzyl-4-deoxy-6-O-propionyl-alpha-D-glucopyranosyl chloride (11) and the methyl beta-D-thiopyranoside 19. Condensation of 11 with 2,5-diazido-1,6-di-O-benzoyl-2,5-di-deoxy-L-iditol in the presence of mercury salts gave 24% of 2,5-diazido-3-O-(4-azido-2,3-di-O-benzyl-4-deoxy-6-O-propionyl-alp ha-D- glucopyranosyl)-1,6-di-O-benzoyl-2,5-dideoxy-L-iditol. Methyl trifluoromethanesulfonate-promoted glycosylation of 1,3-diazido-2-O-benzyl-1,3-dideoxy-5,6-O-isopropylidene-D-gulit ol with 19 in the presence of 2,6-di-tert-butyl-4-methylpyridine gave 1,3-diazido-4-O-(4-azido-2,3-di-O-benzyl-4-deoxy-6-O-propionyl-alp ha-D- glucopyranosyl)-2-O-benzyl-1,3-dideoxy-5,6-O-isopropylidene-D-gulitol (42), whereas, in the absence of base, migration of the O-isopropylidene group occurred, affording 1,3-diazido-6-O-(4-azido-2,3-di-O-benzyl-4-deoxy-6-O-propionyl-alp ha-D- glucopyranosyl)-2-O-benzyl-1,3-dideoxy-4,5-O-isopropylidene-D-gulitol in addition to 42.     

Synthesis of potent Ins(1,4,5)P3 5-phosphatase inhibitors by modification of myo-inositol 1,3,4,6-tetrakisphosphate

Three myo-inositol tetrakisphosphate analogues were synthesised based upon myo-inositol 1,3,4,6-tetrakisphosphate: 2,5-di-O-methyl myo-inositol-1,3,4,6-tetrakisphosphate 19 and its phosphorothioate derivative 22, together with myo-inositol 1,3,4,6 tetrakisphosphorothioate 25. These compounds were prepared by phosphitylating 2,5-di-O-methyl-myo-inositol and 2,5-di-O-benzyl-myo-inositol followed by oxidation with t-butylhydroperoxide or sulfoxidation at room temperature using sulfur in a mixed solvent of DMF and pyridine. Sulfoxidation was complete within 15 min; however, without DMF, the reaction was much slower, and required overnight. When evaluated against Ins(1,4,5)P(3) 5-phosphatase, 3-kinase and for Ca(2+) release at the Ins(1,4,5)P(3) receptor, only weak activity was observed for Ca(2+) release. 22 and 25 are potent 5-phosphatase inhibitors and 25 is a moderate inhibitor of 3-kinase. Thus, we have synthesised potent enzyme inhibitors, which do not mobilise Ca(2+) and devised conditions for quick, clean and inexpensive sulfoxidation of inositol polyphosphite intermediates.     

Metabolism of inositol-1,3,4,6-tetrakisphosphate to inositol pentakisphosphate in adrenal glomerulosa cells

Angiotensin II stimulates rapid formation of inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) in bovine adrenal glomerulosa cells. In addition to being rapidly metabolized to lower inositol phosphates, Ins-1,4,5-P3 is converted to Ins-1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) and Ins-1,3,4-P3 which is in turn phosphorylated to a further Ins-P4 isomer, namely Ins-1,3,4,6-P4. In bovine adrenocortical cytosol [3H]Ins-1,3,4,5-P4 and [3H]Ins-1,3,4-P3 were converted to Ins-1,3,4,6-P4 and inositol pentakisphosphate (Ins-P5) in a metabolic sequence suggesting that unlike Ins-1,3,4,5-P4, Ins-1,3,4,6-P4 is a direct precursor of Ins-P5. Consistent with this assumption, [3H]Ins-1,3,4,6-P4 was converted to Ins-P5 in electropermeabilized adrenal glomerulosa cells. These findings demonstrate that Ins-1,3,4,6-P4 is an intermediate link between InsP3 metabolism and the higher inositol phosphates detected in several tissues.     

A convenient synthetic route to the disaccharide repeating-unit of peptidoglycan

Glycosylation of the readily accessible benzyl 2-acetamido-6-O-benzyl-2-deoxy-3-O-[(R)-1-(methoxycarbonyl)ethyl]-alpha- D- glucopyranoside with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl chloride (2), using the silver triflate method in the absence of a base, afforded 65-70% of the fully protected [beta-D-GlcNPhth-(1----4)-MurNAc] methyl ester derivative 4, the structure of which was ascertained on the basis of 500-MHz 1H-n.m.r. data. 2,2'-Dideoxy-2,2'-diphthalimido-beta,beta-trehalose hexa-acetate was a by-product. Removal of the Phth group from 4, followed by acetylation, yielded 90% of the acetylated 1,6-di-O-benzyl derivative 5, which, on saponification and catalytic hydrogenation, afforded 2-acetamido-4-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-3-O-[(R)-1- carboxyethyl]-2-deoxy-D-glucopyranose. Similarly, 5 was converted into the acetylated methyl ester derivative, which, on selective removal of the methyl ester group, gave benzyl 2-acetamido-4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D- glucopyranosyl)-6-O-benzyl-3-O-[(R)-1-carboxyethyl]-2-deoxy-alpha-D- glucopyranoside. An alternative route for the preparation of 2 is described.     

The regulation of the phosphorylation of inositol 1,3,4-trisphosphate in cell-free preparations and its relevance to the formation of inositol 1,3,4,6-tetrakisphosphate in agonist-stimulated rat parotid acinar cells

High performance liquid chromatography analysis of supernatants from acid-quenched [3H]inositol-labeled parotid acinar cells revealed an inositol pentakisphosphate and three inositol tetrakisphosphates. Two of the latter were identified as the 1,3,4,5 and 1,3,4,6 isomers, whereas the third was probably a mixture of unknown proportions of the 3,4,5,6/1,4,5,6 enantiomeric pair. Methacholine (100 microM) produced a 40-50-fold increase in the levels of inositol trisphosphate (mainly the 1,3,4 isomer) and inositol 1,3,4,5-tetrakisphosphate, but inositol 1,3,4,6-tetrakisphosphate only increased 5-fold. Levels of inositol 3,4,5,6/1,4,5,6-tetrakisphosphate and inositol pentakisphosphate were unaffected by agonist stimulation. Thus, in parotid cells, an agonist-induced increase in both inositol trisphosphate and inositol 1,3,4,6-tetrakisphosphate formation does not result in an increase in the rate of formation of inositol pentakisphosphate. Following the addition of 100 microM atropine to methacholine-stimulated parotid cells, the levels of [3H]inositol 1,3,4,5-tetrakisphosphate fell rapidly, returning to basal levels within 5 min. Inositol trisphosphate was metabolized more slowly and was still elevated 20-fold above basal 5 min after the addition of atropine. Inositol 1,3,4,6-tetrakisphosphate was metabolized much more slowly (t1/2 approximately 15 min). Inositol 1,3,4-trisphosphate metabolism was examined in parotid homogenates as well as in 100,000 x g cytosolic and particulate fractions. Inositol 1,3,4-trisphosphate was both dephosphorylated and phosphorylated. Two inositol tetrakisphosphate products were formed, namely the 1,3,4,6 and 1,3,4,5 isomers. Over 90% of both kinase and phosphatase activities were found in the cytosolic fractions. The ratio of activities of kinase to phosphatase decreased as the levels of inositol 1,3,4-trisphosphate substrate were increased from 1 nM to 10 microM. These data led to the conclusion that the kinetic parameters of the inositol 1,3,4-trisphosphate kinases and phosphatases are such that in stimulated cells, dephosphorylation of inositol 1,3,4-trisphosphate is greatly favored. Inositol 1,3,4-trisphosphate kinase activity was potently inhibited by inositol 3,4,5,6-tetrakisphosphate (IC50 = 0.1-0.2 microM), which leads us to propose that inositol 3,4,5,6-tetrakisphosphate is an endogenous inhibitor of the kinase.     

Origins of myo-inositol tetrakisphosphates in agonist-stimulated rat pancreatoma cells. Stimulation by bombesin of myo-inositol 1,3,4,5,6-pentakisphosphate breakdown to myo-inositol 3,4,5,6-tetrakisphosphate

In the rat pancreatoma cell line, AR4-2J, three inositol tetrakisphosphate isomers were identified, (1,3,4,6), (1,3,4,5), (3,4,5,6), which were increased during activation of phospholipase C by bombesin. Two other isomers were identified, (1,4,5,6) and a fifth isomer which was either (1,2,3,4) or (1,2,3,6), which have not previously been detected in any cell type. To study the metabolic interrelationships between these compounds and inositol 1,3,4,5,6-pentakisphosphate in the intact cell, their turnover was assessed under different protocols of [3H]myo-inositol labeling; the inositol phosphates were labeled to near steady state or under conditions where either rapidly or slowly turning over inositol polyphosphates were preferentially labeled. The relative specific radioactivities of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,3,4-trisphosphate, and inositol 1,3,4,6-tetrakisphosphate were very similar in bombesin-stimulated cells, consistent with the pathway for the conversion of inositol 1,4,5-trisphosphate to the other three inositol polyphosphates. Compared with these inositol phosphates, the turnover of inositol 1,3,4,5,6-pentakisphosphate was slow. An accumulation of radioactivity into inositol 1,3,4,5,6-pentakisphosphate was observed only under labeling conditions where its relative specific radioactivity was substantially below that of inositol 1,3,4,6-tetrakisphosphate. This indicated that the precursor for de novo synthesis of inositol 1,3,4,5,6-pentakisphosphate was inositol 1,3,4,6-tetrakisphosphate. Bombesin stimulated the net breakdown of inositol 1,3,4,5,6-pentakisphosphate and increased the level of inositol 3,4,5,6-tetrakisphosphate; the relative specific radioactivities of these two compounds were similar under all conditions. These data led to the novel proposal that inositol 3,4,5,6-tetrakisphosphate is the product of inositol 1,3,4,5,6-pentakisphosphate breakdown. This reaction was apparently stimulated by a regulated change in the enzyme(s) which interconvert inositol 1,3,4,5,6-pentakisphosphate and inositol 3,4,5,6-tetrakisphosphate.     

The pathway of myo-inositol 1,3,4-trisphosphate phosphorylation in liver. Identification of myo-inositol 1,3,4-trisphosphate 6-kinase, myo-inositol 1,3,4-trisphosphate 5-kinase, and myo-inositol 1,3,4,6-tetrakisphosphate 5-kinase

Inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) metabolism has been studied in liver homogenates and in 100,000 x g supernatant and particulate fractions. When liver homogenates were incubated in an "intracellular" medium containing 5 mM MgATP, equal proportions of Ins(1,3,4)P3 were dephosphorylated and phosphorylated. Two inositol tetrakisphosphate (InsP4) products and an inositol pentakisphosphate (InsP5) were detected. The InsP4 isomers were unequivocally identified as inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) and inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4) by high performance liquid chromatography separation of inositol phosphates, periodate oxidation, alkaline hydrolysis, and stereo-specific polyol dehydrogenase. Ins(1,3,4)P3 5-kinase is a novel enzyme activity and accounted for 16% of the total Ins(1,3,4)P3 phosphorylation. Ins(1,3,4,6)P4 was also shown to be further phosphorylated to inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) by a kinase not previously known to occur in liver. About 75% of Ins(1,3,4)P3 kinase activities were soluble and were partly purified by anion-exchange fast protein liquid chromatography. The two Ins(1,3,4)P3 kinase activities eluted as a single peak that was well resolved from Ins(1,3,4)P3 phosphatase, Ins(1,3,4,6)P4 5-kinase, and Ins(1,3,4,5)P4 5-phosphatase activities. A further novel observation was that 10 microM Ins(1,3,4,5)P4 inhibited Ins(1,3,4)P3 kinase activities by 60%.     

Synthesis of 6-deoxy-5-thio- -glucose

Three routes were investigated for the conversion of -glucose into the title compound. In the first approach, reduction of the 5,6-thürane ring of 5,6-dideoxy-5,6-epithio-1,2-O-isopropylidene α- -glucofuranose (17) as well as that of its 3-O-allyl derivative (13) with lithium aluminium hydride was investigated; 17 afforded the corresponding 6-deoxy derivative besides di-, tri-, and poly-mers, whereas only polymers were formed from 13. In the second approach, the oxirane ring of

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was reduced by sodium borohydride and the resulting 6-deoxy derivative was converted into the 5-thiobenzoate; the corresponding hex-4-enofuranose was formed as a byproduct. In the third approach partial mesylation of methyl 5-thio-α- -glucopyranoside was attempted, but the 6-mesylate 27 could be isolated only in modest yield (28%) together with rearranged 2,5-thioanhydromannofuranoside derivatives. The mechanism of this rearrangement is discussed in detail. The 6-mesylate 27 was converted via the 6-iodo derivative into the title compound.     

Synthesis of 4-substituted phenyl 2,5-anhydro-1,6-dithio-alpha-D-gluco- and -alpha-L-guloseptanosides possessing antithrombotic activity

Two independent approaches were investigated for the synthesis of 3,4-di-O-acetyl-1,6:2,5-dianhydro-1-thio-D-glucitol (18), a key intermediate in the synthesis of 1,3,4-tri-O-acetyl-2,5-anhydro-6-thio-alpha-D-glucoseptanose (13), needed as glycosyl donor. In the first approach 1,6-dibromo-1,6-dideoxy-D-mannitol was used as starting material and was converted via 2,5-anhydro-1,6-dibromo-1,6-dideoxy-4-O-methanesulfonyl-3-O-tetrahydropy ranyl-D-glucitol into 18. The second approach started from 1,2:5,6-di-O-isopropylidene-D-mannitol and the allyl, 4-methoxybenzyl as well as the methoxyethoxymethyl groups were used, respectively, for the protection of the 3,4-OH groups. The resulting intermediates were converted via their 1,2:5,6-dianhydro derivatives into the corresponding 3,4-O-protected 2,5-anhydro-6-bromo-6-deoxy-D-glucitol derivatives. The 1,6-thioanhydro bridge was introduced into these compounds by exchanging the bromine with thioacetate, activating OH-1 by mesylation and treating these esters with sodium methoxide. Among these approaches, the 4-methoxybenzyl protection proved to be the most suitable for a large scale preparation of 18. Pummerer rearrangement of the sulfoxide, obtained via oxidation of 18 gave a 1:9 mixture of 1,3,4-tri-O-acetyl-2,5-anhydro-6-thio-alpha-L-gulo- (12) and -D-glucoseptanose 13. When 12 or 13 were used as donors and trimethylsilyl triflate as promoter for the glycosylation of 4-cyanobenzenethiol, a mixture of 4-cyanophenyl 3,4-di-O-acetyl-2,5-anhydro-1,6-dithio-alpha-L-gulo- (58) and -alpha-D-glucoseptanoside (61) was formed suggesting an isomerisation of the heteroallylic system of the intermediate. A similar mixture of 58 and 61 resulted when 18 was treated with N-chloro succinimide and the mixture of chlorides was used in the presence of zinc oxide for the condensation with 4-cyanobenzenethiol. When 4-nitrobenzenethiol was applied as aglycon and boron trifluoride etherate as promoter, a mixture of 4-nitrophenyl 3,4-di-O-acetyl-2,5-anhydro-1,6-dithio-alpha-L-gulo- (60) and -alpha-D-glucoseptanoside (62) was obtained. Deacetylation of 58, 61 and 62 according to Zemplen afforded 4-cyanophenyl 2,5-anhydro-1,6-dithio-alpha-L-glucoseptanoside (59), 4-cyanophenyl 2,5-anhydro-1,6-dithio-alpha-D-glucoseptanoside (63) and 4-nitrophenyl 2,5-anhydro-1,6-dithio-alpha-D-glucoseptanoside (66), respectively. The 4-cyano group of 63 was transformed into the 4-aminothiocarbonyl, and the 4-(methylthio)(imino)methyl derivative and the 4-nitro group of 66 into the acetamido derivative. All of these thioglycosides displayed a stronger oral antithrombotic effect in rats compared with beciparcil, used as reference.     

Stimulus-Induced Accumulation of Inositol Tetrakis-, Pentakis-, and Hexakisphosphates in N1E-115 Neuroblastoma Cells

When [3H]inositol-prelabelled N1E-115 cells were stimulated with carbamylcholine (CCh) (100 microM), high K+ (60 mM), and prostaglandin E1 (PGE1) (10 microM), a transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached its maximum level at 15 s and had declined to the basal level at 2 min. CCh, high K+, and PGE1 also caused accumulations of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], [3H]inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4], and [3H]inositol hexakisphosphate (InsP6). Muscarine and CCh induced accumulations of [3H]Ins(1,4,5)P3, [3H]-Ins(1,3,4,6)P4, [3H]InsP5, and [3H]InsP6 with a similar potency and exerted these maximal effects at 100 microM, whereas nicotine failed to do so at 1 mM. With a slower time course, CCh, high K+, and PGE1 caused accumulations of [3H]-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In an N1E-115 cell homogenate, [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3 were converted to [3H]InsP5 through [3H]-Ins(1,3,4,6)P4. The above results indicate that Ins(1,3,4,6)P4, InsP5, and InsP6 are rapidly formed by several kinds of stimulants in N1E-115 cells.     

Fluorinated carbohydrates as potential plasma membrane modifiers and inhibitors. Synthesis of 2-acetamido-2,6-dideoxy-6-fluoro-D-galactose

Reaction of benzyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-mesyl-alpha-D-galactopyran oside with cesium floride gave benzyl 2-acetamido-3,6-anhydro-4-O-benzyl-2-deoxy-alpha-D-galactopyranoside instead of the desired 6-fluoro derivative. Acetonation of benzyl 2-acetamido-2-deoxy-6-O-mesyl-alpha-D-galactopyranoside gave the corresponding 3,4-O-isopropylidene derivative. The 6-O-mesyl group was displaced by fluorine with cesium fluoride in boiling 1,2-ethanediol, and hydrolysis and subsequent N-acetylation gave the target compound. In another procedure, treatment of 2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-alpha-D-galactose with N-(diethylamino)sulfur trifluoride gave 2-acetamido-1,3,4-tri-O-acetyl-2,6-dideoxy-6-fluoro-D-galactose which, on acid hydrolysis followed by N-acetylation, gave 2-acetamido-2,6-dideoxy-6-fluoro-D-galactose.     

Sulfonate protecting groups. Regioselective sulfonylation of myo-inositol orthoesters-improved synthesis of precursors of D- and L-myo-inositol 1,3,4,5-tetrakisphosphate,myo-inositol 1,3,4,5,6-pentakisphosphate and related derivatives

The regioselectivity of sulfonylation of myo-inositol orthoesters was controlled by the use of different bases to obtain the desired sulfonate. Monosulfonylation of myo-inositol orthoesters in the presence of one equivalent of sodium hydride or triethylamine resulted in the sulfonylation of the 4-hydroxyl group. The use of pyridine as a base for the same reaction resulted in sulfonylation of the 2-hydroxyl group. Disulfonylation of these orthoesters in the presence of excess sodium hydride yielded the 4,6-di-O-sulfonylated orthoesters. However, the use of triethylamine or pyridine instead of sodium hydride yielded the 2,4-di-O-sulfonylated orthoester. Sulfonylated derivatives of myo-inositol orthoesters were stable to conditions of O-alkylation but were cleaved using magnesium/methanol or sodium methoxide in methanol to regenerate the corresponding myo-inositol orthoester derivative. These new methods of protection-deprotection have been used: (i) for the efficient synthesis of enantiomers of 2,4-di-O-benzyl-myo-inositol, which are precursors for the synthesis of D- and L-myo-inositol 1,3,4,5-tetrakisphosphate; (ii) for the preparation of 2-O-benzyl-myo-inositol which is a precursor for the preparation of myo-inositol 1,3,4,5,6-pentakisphosphate.     

Synthesis of O-alpha-L-fucopyranosyl-(1----3)-O-beta-D-galactopyranosyl-(1----4)-2- acetamido-2-deoxy-D-glucopyranose (N-acetyl-3'-O-alpha-L-fucopyranosyllactosamine)

Methyl 2-O-benzyl-beta-D-galactopyranoside (6) was obtained in five, good yielding steps from methyl beta-D-galactopyranoside (1). Treatment of 1 with tert-butylchlorodiphenylsilane in N,N-dimethylformamide in the presence of imidazole afforded a 6-(tert-butyldiphenylsilyl) ether, which was converted into its 3,4-O-isopropylidene derivative (3). Benzylation of 3 with benzyl bromide-silver oxide in N,N-dimethylformamide, and subsequent cleavage of its acetal and ether groups then afforded 6. On similar benzylation, followed by the same sequence of deprotection, benzyl 2-acetamido-3,6-di-O-benzyl-4-O-[6-O-(tert-butyldiphenylsilyl)-3,4 -O- isopropylidene-beta-D-galactopyranosyl]-2-deoxy-alpha-D-glucopyranoside gave the 2-O-benzyl derivative (10). Compound 10 was converted into its 4,6-O-benzylidene acetal (11). Glycosylation (catalyzed by halide-ion) of 11 with 2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl bromide afforded the fully protected trisaccharide derivative (13). Cleavage of the benzylidene and then the benzyl groups of 13 furnished the title trisaccharide (16). The structure of 16 was established by 13C-n.m.r. spectroscopy.     

Synthesis of 6-deoxy-5-thio-d-glucose

Three routes were investigated for the conversion of d-glucose into the title compound. In the first approach, reduction of the 5,6-thürane ring of 5,6-dideoxy-5,6-epithio-1,2-O-isopropylidene α-d-glucofuranose (17) as well as that of its 3-O-allyl derivative (13) with lithium aluminium hydride was investigated; 17 afforded the corresponding 6-deoxy derivative besides di-, tri-, and poly-mers, whereas only polymers were formed from 13. In the second approach, the oxirane ring of was reduced by sodium borohydride and the resulting 6-deoxy derivative was converted into the 5-thiobenzoate; the corresponding hex-4-enofuranose was formed as a byproduct. In the third approach partial mesylation of methyl 5-thio-α-d-glucopyranoside was attempted, but the 6-mesylate 27 could be isolated only in modest yield (28%) together with rearranged 2,5-thioanhydromannofuranoside derivatives. The mechanism of this rearrangement is discussed in detail. The 6-mesylate 27 was converted via the 6-iodo derivative into the title compound.     

Inositol tetrakisphosphates as second messengers induce Ca(++)-dependent chloride currents in Xenopus laevis oocytes.

Microinjection of inositol 1,3,4,5-tetrakisphosphate or inositol 1,4,5-trisphosphate induced distinct chloride membrane currents in defolliculated Xenopus laevis oocytes. To decide whether these Cl(-)-currents were due to the injected compounds or their metabolic products, [3H]Ins(1,3,4,5)P4 or [3H]Ins(1,4,5)P3 were injected into oocytes and their metabolites were analyzed by HPLC. Our results indicate that Ins(1,3,4,5)P4 itself or its metabolite Ins(1,3,4,6)P4 is able to induce Cl(-)-membrane currents, most likely by increasing the cytosolic Ca(++)-concentration.     

Glycosidation of 2,5-anhydro-3,4-di-O-benzyl-D-mannitol with different glucopyranosyl donors: a comparative study

2,5-Anhydro-3,4-di-O-benzyl-D-mannitol was glycosylated using different donors such as tetra-O-acetyl-alpha-D-glucopyranosyl bromide in the presence of Hg(CN)(2), the corresponding beta-thiophenylglycoside in the presence of NIS and TfOH as well as the alpha- and beta-trichloroimidate with TMSOTf as promoter. The resulting mixtures were analyzed by HPLC and the following main components were isolated and characterized: 2,5-anhydro-3,4-di-O-benzyl-1-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-d-mannitol; 6-O-acetyl-2,5-anhydro-3,4-di-O-benzyl-1-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-D-mannitol; 2,5-anhydro-3,4-di-O-benzyl-1,6-bis-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-D-mannitol; 2,5-anhydro-3,4-di-O-benzyl-1-O-[-2-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-3,4,6-tri-O-acetyl-beta-D-glucopyranosyl]-6-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-D-mannitol and 2,5-anhydro-3,4-di-O-benzyl-1,6-bis-O-(3,4,6-tri-O-acetyl-1,2-O-ethylidene-2'-yl-alpha-D-glucopyranosyl)-D-mannitol. The latter compound representing a bis-orthoester might be a common intermediate in all the investigated reactions, as its rearrangement and/or decomposition can yield all of the isolated compounds.     

Inositol polyphosphate multikinase regulates inositol 1,4,5,6-tetrakisphosphate

The human inositol phosphate multikinase (IPMK, 5-kinase) has a preferred 5-kinase activity over 3-kinase and 6-kinase activities and a substrate preference for inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4) over inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). We now report that the recombinant human protein can catalyze the conversion of inositol 1,4,5,6-tetrakisphosphate (Ins(1,4,5,6)P4) to Ins(1,3,4,5,6)P5 in vitro; the reaction product was identified by HPLC to be Ins(1,3,4,5,6)P5. The apparent Vmax was 42 nmol of Ins(1,3,4,5,6)P5 formed/min/mg protein, and the apparent Km was 222 nM using Ins(1,3,4,6)P4 as a substrate; the catalytic efficiency was similar to that for Ins(1,4,5)P3. Stable over-expression of the human protein in HEK-293 cells abrogates the in vivo elevation of Ins(1,4,5,6)P4 from the Salmonella dublin SopB protein. Hence, the human 5-kinase may also regulate the level of Ins(1,4,5,6)P4 and have an effect on chloride channel regulation.     

Ca influx evoked by inositol-3,4,5,6-tetrakisphosphate in ras-transformed NIH/3T3 fibroblasts

Infusion of inositol-3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P₄) from the patch pipette into the cytoplasm, produced a biphasic intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase in ras-transformed NIH/3T3 (DT) cells. The Ins(3,4,5,6)P₄-induced increase in DT cells depended upon extracellular Ca²⁺ and was enhanced by membrane hyperpolarization. Identical [Ca²⁺]_i increases were observed with intracellular application of inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄) and inositol-1,3,4,6-tetrakisphosphate but not with inositol-1,2,4,5-tetrakisphosphate, inositol-1,4,5-trisphosphate or inositol-1,3,4,5,6-pentakisphosphate. Stimulation of DT cells with bradykinin increased the levels of Ins(3,4,5,6)P₄ and Ins(1,3,4,5)P₄. These results suggest that Ins(3,4,5,6)P₄ may serve as a second messenger for continuous Ca²⁺ influx along with other tetrakisphosphates downstream from bradykinin receptors in DT cells.     

Accumulation of inositol polyphosphate isomers in agonist-stimulated cerebral-cortex slices. Comparison with metabolic profiles in cell-free preparations.

1. Basal and carbachol-stimulated accumulations of isomeric [3H]inositol mono-, bis-, tris- and tetrakis-phosphates were examined in rat cerebral-cortex slices labelled with myo-[2-3H]inositol. 2. In control samples the major [3H]inositol phosphates detected were co-eluted on h.p.l.c. with Ins(1)P, Ins(4)P (inositol 1- and 4-monophosphate respectively), Ins(1,4)P2 (inositol 1,4-bisphosphate), Ins(1,4,5)P3 (inositol 1,4,5-tris-phosphate) and Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate). 3. After stimulation to steady state with carbachol, accumulation of each of these products was markedly increased. 4. Agonist stimulation, however, also evoked much more dramatic increased accumulations of a second [3H]inositol trisphosphate, which was co-eluted on h.p.l.c. with authentic Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) and of three further [3H]inositol bisphosphates ([3H]InsP2(s]. 5. Examination of the latter by chemical degradation by periodate oxidation and/or h.p.l.c. allowed identification of these as [3H]Ins(1,3)P2, [3H]Ins(3,4)P2 and [3H]Ins(4,5)P2 (inositol 1,3-, 3,4- and 4,5-bisphosphates respectively), which respectively accounted for about 22%, 8% and 3% of total [3H]InsP2 in extracts from stimulated tissue slices. 6. By using a h.p.l.c. method which clearly resolves Ins(1,3,4,5)P4 and Ins(1,3,4,6)P4 (inositol 1,3,4,6-tetrakisphosphate), only the former isomer could be detected in extracts from either control or stimulated tissue slices. Similarly, [3H]inositol pentakis- and hexakis-phosphates were not detectable either in the presence or absence of carbachol under the radiolabelling conditions described. 7. The catabolism of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 by cell-free preparations from cerebral cortex was also studied. 8. In the presence of Mg2+, [3H]Ins(1,4,5)P3 was specifically dephosphorylated via [3H]Ins(1,4)P2 and [3H]Ins(4)P to free [3H]inositol, whereas [3H]Ins(1,3,4)P3 was degraded via [3H]Ins(3,4)P2 and, to a lesser extent, via [3H]Ins(1,3)P2 to D- and/or L-[3H]Ins(1)P and [3H]inositol. 9. In the presence of EDTA, hydrolysis of [3H]Ins(1,4,5)P3 was greater than or equal to 95% inhibited, whereas [3H]Ins(1,3,4)P3 was still degraded, but yielded only a single [3H]InsP2 identified as [3H]Ins(1,3)P2. 10. The significance of these observations with cell-free preparations is discussed in relation to the proportions of the separate isomeric [3H]inositol phosphates measured in stimulated tissue slices.     

Synthesis of myo-inositol 1,3,4,5,6-pentakisphosphate from inositol phosphates generated by receptor activation.

myo-[3H]Inositol 1,3,4,5,6-pentakisphosphate can be made from myo-[3H]inositol 1,4,5-trisphosphate in a rat brain homogenate or soluble fraction. Although D-myo-inositol 3,4,5,6-tetrakisphosphate can be phosphorylated by a soluble rat brain enzyme to give myo-inositol 1,3,4,5,6-pentakisphosphate, it is not an intermediate in the pathway from myo-inositol 1,4,5-trisphosphate. The intermediates in the above pathway are myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4-trisphosphate and myo-inositol 1,3,4,6-tetrakisphosphate [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147; Balla, Guillemette, Baukal & Catt (1987) J. Biol. Chem. 262, 9952-9955], and it is catalysed by soluble kinase activities of similar anion-exchange mobility and Mr value. Compounds with chromatographic and chemical properties consistent with the structures myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4,6-tetrakisphosphate and myo-inositol 3,4,5,6-tetrakisphosphate are present in avian erythrocytes, human 1321 N1 astrocytoma cells and primary-cultured murine bone-marrow-derived macrophages. The amounts of these inositol tetrakisphosphates rise upon muscarinic cholinergic stimulation of the astrocytoma cells or stimulation of macrophages with platelet-activating factor.