Ca2(+)-stimulatable and protein kinase C-inhibitable accumulation of inositol 1,3,4,6-tetrakisphosphate in human platelets.

Thrombin-stimulated (10 s) human platelets produce Ins(1,4,5)P3 and an additional inositol trisphosphate (InsP3), in approximately a 1:20 ratio. The major InsP3 co-migrates with Ins(1,3,4)P3 on strong-anion-exchange h.p.l.c. To identify this species unequivocally, we treated putative Ins(1,3,4)P3 obtained from thrombin-stimulated myo-[3H]inositol-labelled platelets with NaIO4/NaBH4 or 4-phosphomonoesterase. The products indicate that the major InsP3 is at least 90% D-Ins(1,3,4)P3. D-[3H]Ins(1,3,4)P3 added to saponin-permeabilized platelets is hydrolysed to an InsP2 (7.8%) and phosphorylated by a kinase to yield an inositol polyphosphate (0.9%) in 5 min. The phosphorylation product co-migrates with Ins(1,3,4,6)P4 on Partisphere WAX h.p.l.c. Under similar conditions, L-[3H]Ins(1,3,4)P3 is dephosphorylated but not phosphorylated. Relative phosphatase:kinase ratios are 8.7:1 (Vmax. values) and 0.86:1 (Km values) with respect to D-Ins(1,3,4)P3. The kinase activity is predominantly cytosolic (96.8% of total activity) in freeze-thaw-disrupted platelets, and the accumulation of its product is Ca2(+)-dependent. The activity is identified as a 6-kinase on the basis of its product's insensitivity to 5-phosphomonoesterase, resistance to periodate oxidation and co-migration with standard Ins(1,3,4,6)P4 on h.p.l.c. Incubation of platelets with beta-phorbol dibutyrate (beta-PDBu, 76 nM), causing activation of protein kinase C, results in a 57.5% inhibition (reversible by the protein kinase C inhibitor staurosporine) of Ins(1,3,4,6)P4 accumulation. alpha-PDBu, which does not stimulate protein kinase C, has no effect. Stimulation of intact platelets with thrombin results in the production of Ins(1,3,4,6)P4 (1.4-fold rise in 30 s) and Ins(1,3,4,5)P4, with the latter being the major InsP4 species. Accumulation of Ins(1,3,4,6)P4 is slightly delayed in comparison with Ins(1,3,4)P3 and is relatively small. We propose that the major route of Ins(1,3,4)P3 metabolism in stimulated human platelets is via phosphatase action.     

The human homolog of the rat inositol phosphate multikinase is an inositol 1,3,4,6-tetrakisphosphate 5-kinase

We have demonstrated that the human homolog of the rat inositol phosphate multikinase is an inositol 1,3,4,6-tetrakisphosphate 5-kinase (InsP(4) 5-kinase). The cDNA of the human gene contained a putative open reading frame of 1251 bp encoding 416 amino acids with 83.6% identity compared with the rat protein. The substrate specificity of the recombinant human protein demonstrated preference for Ins(1,3,4,6)P(4) with a catalytic efficiency (V(max)/K(m)) 43-fold greater than that of Ins(1,3,4,5)P(4) and 2-fold greater than that of Ins(1,4,5)P(3). The apparent V(max) was 114 nmol of Ins(1,3,4,5,6)P(5) formed/min/mg of protein, and the apparent K(m) was 0.3 microm Ins(1,3,4,6)P(4). The functional homolog in yeast is Ipk2p, and ipk2-null yeast strains do not synthesize Ins(1,3,4,5,6)P(5) or InsP(6). Synthesis of these compounds was restored by transformation with wild-type yeast IPK2 but not with human InsP(4) 5-kinase. Thus the human gene does not complement for the loss of the yeast gene because yeast cells do not contain the substrate Ins(1,3,4,6)P(4), and the reaction of the human protein with Ins(1,3,4,5)P(4) is insufficient to effect rescue or synthesis of InsP(5) and InsP(6). Therefore the major activity of human InsP(4) 5-kinase is phosphorylation at the D-5 position, and the pathways for synthesis of Ins(1,3,4,5,6)P(5) in yeast versus humans are different.     

A concise synthesis of 4-nitrophenyl 2-azido-2-deoxy- and 2-acetamido-2-deoxy-D-mannopyranosides

4-nitrophenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha- and beta-D-mannopyranosides were prepared from methyl 4,6-O-benzylidene-alpha-D-glucopyranoside and 1,3,4,6-tetra-O-acetyl-alpha-D-glucopyranose, respectively. Chemoselective reduction of both azides with hydrogen sulfide readily afforded 4-nitrophenyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-alpha-D- and -beta-D-mannopyranosides in higher yields than reduction with triphenylphosphine or a polymer-supported triarylphosphine. Subsequent de-O-acetylation yielded 4-nitrophenyl 2-acetamido-2-deoxy-alpha-D-mannopyranoside and 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-mannopyranoside in 20% and 44% overall yields, respectively.     

Access to tetrachlorophthalimide-protected ethyl 2-amino-2-deoxy-1-thio-beta-D-glucopyranosides.

Ethyl 2-deoxy-2-tetrachlorophthalimido-1-thio-beta-D-glucopyranoside (7) was prepared from glucosamine hydrochloride in four steps with a 20-25% overall yield. Formation of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-tetrachlorophthalimido-beta-D- glucopyranoside (5) was found to be crucial for this reaction sequence since the corresponding alpha-1-acetate did not react in Lewis-acid-catalyzed ethylthio glycosidations. Formation of the beta-1-acetate (5) was achieved by treatment of 3,4,6-tri-O-acetyl-2-deoxy-2-tetrachlorophthalimido-alpha-D-glucop yranosyl bromide (4) with acetic acid under silver zeolite promotion. This was necessary because conditions normally used for beta-1-acetate formation were not tolerated by the tetrachlorophthalimido (TCP) group.     

Improved synthesis of 1,3,4,6-tetra-O-acetyl-2-azido-2-deoxy-alpha-D-mannopyranose

By improved (anhydrous) work-up conditions of a triflate displacement reaction, the yield in the preparation of the versatile synthetic intermediate 1,3,4,6-tetra-O-acetyl-2-azido-2-deoxy-alpha-d-mannopyranose has been significantly enhanced. This important precursor is now available in three efficient steps from d-glucose.     

Product-precursor relationships amongst inositol polyphosphates. Incorporation of [32P]Pi into myo-inositol 1,3,4,6-tetrakisphosphate, myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 3,4,5,6-tetrakisphosphate and myo-inositol 1,3,4,5,6-pentakisphosphate in intact avian erythrocytes.

Avian erythrocytes were incubated with myo-[3H]inositol for 6-7 h and with [32P]Pi for the final 50-90 min of this period. An acid extract was prepared from the prelabelled erythrocytes, and the specific radioactivities of the gamma-phosphate of ATP and of both the myo-inositol moieties (3H, d.p.m./nmol) and the individual phosphate groups (32P, d.p.m./nmol) of [3H]Ins[32P](1,3,4,6)P4,[3H]Ins[32P](1,3,4,5)P4, [3H]Ins[32P](3,4,5,6)P4 and [3H]Ins[32P](1,3,4,5,6)P5 were determined. The results provide direct confirmation that one of the cellular InsP4 isomers is Ins(1,3,4,5)P4 which is synthesized by sequential phosphorylation of the 1,4,5 and 3 substitution sites of the myo-Ins moiety, precisely as previously deduced [Batty, Nahorski & Irvine (1985) Biochem. J. 232, 211-215; Irvine, Letcher, Heslop & Berridge (1986) Nature (London) 320, 631-634]. This is compatible with the proposed synthetic route from PtdIns via PtdIns4P, PtdIns(4,5)P2 and Ins(1,4,5)P3. The data also suggest that, in avian erythrocytes, the principle precursor of Ins(1,3,4,5,6)P5 is Ins(3,4,5,6)P4. Furthermore, if the gamma- (and/or beta-) phosphate of ATP is the precursor of the phosphate moieties of Ins(3,4,5,6)P4, then this isomer must be derived from the phosphorylation of Ins(3,4,6)P3. If the gamma- (and/or beta-) phosphate of ATP similarly acts as the ultimate precursor to all of the phosphates of Ins(1,3,4,6)P4, then, in intact avian erythrocytes, the main precursor of Ins(1,3,4,6)P4 is Ins(1,4,6)P3. This contrasts with the expectation, based on results with cell-free systems, that Ins(1,3,4,6)P4 is synthesized by the direct phosphorylation of Ins(1,3,4)P3.     

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.     

An analysis of myo-[3H]inositol trisphosphates found in myo-[3H]inositol prelabelled avian erythrocytes.

Evidence is presented to show that acid extracts of avian erythrocytes prelabelled for 24-48 h with myo-[3H]inositol contain the following myo-[3H]inositol trisphosphates (expressed as a percentage of total myo-[3H]inositol trisphosphates extracted): 36% myo-[3H]inositol 1,4,5-trisphosphate; 33.7% myo-[3H]inositol 1,3,4-trisphosphate; 13% myo-[3H]inositol 3,4,5-trisphosphate; 9.7% myo-[3H]inositol 3,4,6-trisphosphate; 4.4% myo-[3H]inositol 1,4,6-trisphosphate and 3.3% myo-[3H]inositol 1,3,6-trisphosphate. The only phosphatidyl-myo-[3H]inositol bisphosphate that could be detected in [3H]Ins-prelabelled avian erythrocytes was phosphatidyl-myo-[3H]inositol 4,5-bisphosphate. Cellular myo-[3H]inositol 3,4,5-trisphosphate may be synthesized by dephosphorylation of myo-[3H]inositol 3,4,5,6-tetrakisphosphate. D- and L-myo-[3H]inositol 1,4,6-trisphosphate and D- and L-myo-[3H]inositol 1,3,6-trisphosphate may be dephosphorylation products of myo-[3H]inositol 1,3,4,6-tetrakisphosphate.     

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.     

Changes in the hemicellulosic polysaccharides of rye-grass with increasing maturity

Reaction of the C-2 mercurated methyl hexopyranoside acetates 1–3 with an excess of iodine resulted in nearly quantitative replacement of mercury by iodine with retention and inversion of configuration at C-2. Similar replacement was observed with 2-acetoxymercuri-3,4,6-tri-O-acetyl-2-deoxy-α-d-glucopyranose (4). In the iodinolysis of 2-acetoxymercuri-1,3,4,6-tetra-O-acetyl-2-deoxy-α-d-glucopyranose (5) in methanol, however, replacement at C-2 was accompanied to a considerable extent by solvolysis of the 1-acetoxyl group, and a mixture of 1,2-trans isomers of methyl 3,4,6-tri-O-acetyl-2-deoxy-2-iodo-hexopyranosides having the d-gluco and d-manno configurations was obtained, together with 1,3,4,6-tetra-O-acetyl-2-deoxy-2-iodo-α-d-mannopyranose.     

First synthesis of tepidopterin [2'-O-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-L-threo-biopterin

N(2)-(N,N-Dimethylaminomethylene)-1'-O-(4-methoxybenzyl)-3-[2-(4-nitrophenyl)ethyl]-L-threo-biopterin (14) was prepared from L-xylose in an 11-step-sequence. The first synthesis of tepidopterin (3) was achieved by treatment of 14 with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl bromide in the presence of silver triflate and tetramethylurea, followed by removal of the protecting groups.     

Synthesis of 1,3,4,6-tetra-O-acetyl-2-[3-alkyl-(aryl)-thioreido]-2-deoxy-α-d-glucopyranoses and their transformation into 2-alkyl(aryl)amino-(1,2-dideoxy-α-d-glucopyrano)[2,1-d]-2-thiazolines

1,3,4,6-Tetra-O-acetyl-2-deoxy-2-isothiocyanato-α-d-glucopyranose, produced from 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-α-d-glucopyranose hydrochloride, thiophosgene, and calcium carbonate, was condensed with alkyl- and aryl-amines in ether to afford the crystalline 1,3,4,6-tetra-O-acetyl-2-[3-alkyl(aryl)-thioureido]-2-deoxy-α-d-glucopyranoses (2). Compounds 2 and the β anomers 3 were converted in high yield into 2-alkyl(aryl)amino-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-d-glucopyrano)[2,1-d]-2-thiazoline hydrobromides (4) by hydrogen bromide-promoted cyclisation. The O-deacetylated thiazoline hydrobromide 5 was also isolated and converted into 2-[N-(4-methoxyphenyl)acetamido]-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-d-glucopyrano)[2,1-d]-2-thiazoline (8). Conformational studies of 4 and 8 were made by ¹H-n.m.r. spectroscopy.     

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.     

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.     

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%.     

Metabolic and signaling properties of an Itpk gene family in Glycine max

We have cloned and characterized four Itpk genes from soybean. All four recombinant Itpk proteins showed canonical Ins(1,3,4)P3 5/6-kinase activity, but a kinetic analysis raised questions about its biological significance. Instead, we provide evidence that one alternative biological role for soybean Itpks is to interconvert the Cl(-) channel inhibitor, Ins(3,4,5,6)P4, and its metabolic precursor, Ins(1,3,4,5,6)P5, within a substrate cycle. The soybean Itpks also phosphorylated Ins(3,4,6)P3 to Ins(1,3,4,6)P4 which was further phosphorylated to Ins(1,3,4,5,6)P5 by soybean Ipk2. Thus, soybean Itpks may participate in an inositol lipid-independent pathway of InsP6 synthesis.     

The synthesis of p-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl) P2-ficaprenyl pyrophosphate

2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α,β-D-glucopyranosylammonium phosphate was prepared by the action of crystalline phosphoric acid on 2-acetamido-1,3,4,6-tetra-O-acetyl-β-D-glucopyranose. The α-D anomer (3) was the main product, and was isolated pure by preparative thin-layer chromatography or by removal of the β-D anomer (6) by selective acid hydrolysis. Ficaprenyl phosphate was prepared from ficaprenol, obtained as an isomeric mixture (mainly C₅₅) from an extract of Ficus elastica. Compound 3 was converted into the free acid and then into the tributyl-ammonium salt, which was treated with P¹-diphenyl P²-ficaprenyl pyrophosphate to give the acetylated pyrophosphate diester 8, characterized by analytical, spectral, and hydrogenolytic studies. Deacetylation of 8 gave the synthetic “lipid intermediate”, P¹-(2-acetamido-2-deoxy-D-glucopyranosyl) P²-ficaprenyl pyrophosphate (9), the properties of which were compared with those of natural substances considered to be active in the biosynthesis of teichoic acids.     

Addition of halogenoazides to glycals

Addition of chloroazide to 3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-d-lyxo- (1) and -d-arabino-hex-1-enitol (2) under u.v. irradiation proceeds regio- and stereo-selectively yielding mainly O-acetyl derivatives of 2-azido-2-deoxy-d-galactopyranose and -d-glucopyranose, respectively. 3,4,6-Tri-O-acetyl-2-chloro-2-deoxy-α-d-galactopyranosyl azide and 3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-d-talopyranose (from 1), and 1,3,4,6-tetra-O-acetyl-2-chloro-2-deoxy-α-d-glucopyranosyl azide and 1,3,4,6-tetra-O-acetyl-2-azido-2-deoxy-α-d-mannopyranose (from 2) are byproducts. 1,5-Anhydro-3,4,6-tri-O-benzyl-2-deoxy-d-lyxo- and -d-arabino-hex-1-enitol reacted more rapidly with chloroazide, to give, under irradiation, derivatives of 2-azido-2-deoxy-d-galactose and -d-glucose, respectively. However, reaction in the dark gave mainly O-benzyl derivatives of 2-chloro-2-deoxy-α-d-galacto- and -α-d-glucopyranosyl azide. The difference between the products obtained may depend on the existence of two parallel processes, one radical (under irradiation), and the other ionic (reaction in the dark).     

Synthesis of mycothiol, 1D-1-O-(2-[N-acetyl-L-cysteinyl]amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol, principal low molecular mass thiol in the actinomycetes

Members of the actinomycetes produce 1D-1-O-(2-[N-acetyl-L-cysteinyl]amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol or mycothiol 1 as principal low molecular mass thiol. Chemical synthesis of a biosynthetic precursor of mycothiol, the pseudodisaccharide 1D-1-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol 13 was achieved by the following steps: (1) Enantioselective synthesis gave the glycosyl acceptors (-)-2,3,4,5,6-penta-O-acetyl-D-myo-inositol D-7 and the corresponding L-isomer L-7. (2) Condensation of D-7 and L-7 with the glycosyl donor 3,4,6-tri-O-acetyl-2-deoxy-2-(2,4-dinitrophenylamino)-alpha-D-glucopyranosylbromide afforded the corresponding alpha and beta anomeric products, which could be resolved by silica gel chromatography. (3) Deprotection of these by hydrolysis using an anion exchange resin gave 1D- and 1L-1-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol 13 and 15 and the corresponding beta-coupled anomers 14 and 16. Only 13, and to a much lesser extent 15, were used by enzymes present in an ammonium sulphate fraction of a cellfree extract of Mycobacterium smegmatis for the enzymatic synthesis of mycothiol. In the absence of acetyl-SCoA, the immediate biosynthetic precursor of 1, desacetylmycothiol, was the major product.     

The preparation and phosphorylation of 2,5- and 1D-2,6-di-O-benzyl-myo-inositol.

1,3,4,6-Tetra-O-allyl-myo-inositol was converted into the 2,5-di-O-benzyl- and 2,5-di-O-p-methoxybenzyl ethers, and the products were deallylated to give the 2,5-di-O-benzyl (and p-methoxybenzyl) ethers of myo-inositol, which were converted into the mono-O-isopropylidene derivatives. Both the 2,5-di-O-benzyl ether and its mono-O-isopropylidene derivative were converted into the crystalline octa(2-cyanoethyl) ester of 2,5-di-O-benzyl-myo-inositol 1,3,4,6-tetrakisphosphate. (+-)-1,3,4,5-Tetra-O-allyl-myo-inositol was converted into (+-)-2,4-di-O-benzyl-myo-inositol which gave a separable mixture of the 1,6- and 5,6-O-isopropylidene derivatives. The 1,6-O-isopropylidene derivative was resolved via (-)- and (+)-omega-camphanates and was also converted into (+-)-2,6-di-O-benzyl-1,5-di-O-p-methoxybenzyl-myo-inositol, which was resolved via the (-)-omega-camphanates. The 5,6-O-isopropylidene derivative and 1,3-di-O-allyl-myo-inositol were converted into (+-)-1,3-di-O-allyl-2,6-di-O-benzyl-myo-inositol, which was resolved as the (-)-omega-camphanates. 1D-1,3,4,5-Tetra-O-allyl-myo-inositol and the above described, relevant diaste reoisomers were converted into 1D-2,6-di-O-benzyl-myo-inositol which gave the syrupy octabenzyl ester of 1D-2,6-di-O-benzyl-myo-inositol 1,3,4,5-tetrakisphosphate.