Synthesis of 6-formyluridine 5'-monophosphate: 6-formyl-UMP

A synthesis of 6-formyluridine 5'-monophosphate (6-formylUMP) in 6 steps starting from uridine is described. This approach should be applicable to the preparation of other O5'-phosphorylated 6-formylUrds such as 6-formylUDP and 6-formylUTP.     

SYNTHESIS OF 6-FORMYLURIDINE 5′-MONOPHOSPHATE: 6-FORMYL-UMP

A synthesis of 6-formyluridine 5′-monophosphate (6-formylUMP) in 6 steps starting from uridine is described. This approach should be applicable to the preparation of other O5′-phosphorylated 6-formylUrds such as 6-formylUDP and 6-formylUTP.     

Mechanisms of inactivation of human S-adenosylhomocysteine hydrolase by 5',5',6',6'-tetradehydro-6'-deoxy-6'-halohomoadenosines

In an effort to design more specific and potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, we investigated the mechanisms by which 5',5',6', 6'-tetradehydro-6'-deoxy-6'-halohomoadenosines (X = Cl, Br, I) inactivated this enzyme. The 6'-chloro (a) and 6'-bromo (b) acetylenic nucleoside analogues produced partial ( approximately 50%) loss of enzyme activity with a concomitant ( approximately 50%) reduction of E-NAD(+) to E-NADH. In addition, Ade and halide ions were released from the inhibitors in amounts suggestive of a process involving enzyme catalysis. AdoHcy hydrolase, which was inactivated with compound a, was shown to contain 2 mol of the inhibitor covalently bound to Lys318 of two subunits of the homotetramer. These data suggest that the enzyme-mediated water addition at the 5' position of compound a or b produces an alpha-halomethyl ketone intermediate, which is then attacked by a proximal nucleophile (i.e., Lys318) to form the enzyme-inhibitor covalent adduct (lethal event); in a parallel pathway (nonlethal event), addition of water at the 6' position produces an acyl halide, which is released into solution and chemically degrades into Ade, halide ion, and sugar-derived products. In contrast, compound c completely inactivated AdoHcy hydrolase by converting 2 equiv of E-NAD(+) to E-NADH and causing the release of 2 equiv of E-NAD(+) into solution. Four moles of the inhibitor was shown to be tightly bound to the tetrameric enzyme. These data suggest that compound c inactivates AdoHcy hydrolase by a mechanism similar to the acetylenic analogue of Ado described previously by Parry et al. [(1991) Biochemistry 30, 9988-9997].     

A convenient isomerization of 6-oxo-3 alpha,5-cyclo-5 alpha-steroids to 6-oxo-delta 2-5 alpha-steroids

Direct isomerization of 6-oxo-3 alpha,5-cyclo-5 alpha-steroids to 6-oxo-delta 2-5 alpha-steroids was accomplished by pyridinium hydrobromide in dimethyl-formamide.     

Isolation and chemical conversion of two novel prostaglandin endoperoxides: 5(6)-epoxy-PGG1 and 5(6)-epoxy-PGH1

The possible molecular heterogeneity of human transferrin receptors was analyzed using two murine monoclonal antibodies, Tü15 and Tü67. Both reagents precipitated from lysates of 125I-labeled HL-60 cells a major component of 88 kDa which could be identified as the transferrin receptor by comparison with the proteins detected by monoclonal antibody OKT9. Although sequential immunoprecipitations appeared to demonstrate molecular heterogeneity of transferrin receptors, since the Tü15-reactive species were fully included in the Tü67-positive population, but not vice versa, the possible association of Tü15-reactive molecules with transferrin receptor is also discussed.     

The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex

Stabilization and processing of stalled replication forks is critical for cell survival and genomic integrity. We characterize a novel DNA repair heterodimer of Nse5 and Nse6, which are nonessential nuclear proteins critical for chromosome segregation in fission yeast. The Nse5/6 dimer facilitates DNA repair as part of the Smc5-Smc6 holocomplex (Smc5/6), the basic architecture of which we define. Nse5-Nse6 [corrected] (Nse5 and Nse6) [corrected] mutants display a high level of spontaneous DNA damage and mitotic catastrophe in the absence of the master checkpoint regulator Rad3 (hATR). Nse5/6 mutants are required for the response to genotoxic agents that block the progression of replication forks, acting in a pathway that allows the tolerance of irreparable UV lesions. Interestingly, the UV sensitivity of Nse5/6 [corrected] is suppressed by concomitant deletion of the homologous recombination repair factor, Rhp51 (Rad51). Further, the viability of Nse5/6 mutants depends on Mus81 and Rqh1, factors that resolve or prevent the formation of Holliday junctions. Consistently, the UV sensitivity of cells lacking Nse5/6 can be partially suppressed by overexpressing the bacterial resolvase RusA. We propose a role for Nse5/6 mutants in suppressing recombination that results in Holliday junction formation or in Holliday junction resolution.     

Thiosugar nucleotide analogs: synthesis of 5'-(2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-alpha-D-galactopyranosyl diphosphate)

The synthesis of a novel analog of uridine diphosphate galactose (UDP-Gal) is described. A sulfur atom was inserted into the 6-position of galactose to give uridine 5'-(2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-alpha-D-galactopyranosyl diphosphate). This peracetylated thiol analogue of UDP-Gal has been synthesized in nine steps starting from methyl alpha-D-galactopyranoside in an overall yield of 3%.     

Assessment of essential fatty acid and omega3-fatty acid status by measurement of erythrocyte 20:3omega9 (Mead acid), 22:5omega6/20:4omega6 and 22:5omega6/22:6omega3

BACKGROUND: Early suspicion of essential fatty acid deficiency (EFAD) or omega3-deficiency may rather focus on polyunsaturated fatty acid (PUFA) or long-chain PUFA (LCP) analyses than clinical symptoms. We determined cut-off values for biochemical EFAD, omega3-and omega3/22:6omega3 [docosahexaenoic acid (DHA)]-deficiency by measurement of erythrocyte 20:3omega9 (Mead acid), 22:5omega6/20:4omega6 and 22:5omega6/22:6omega3, respectively. METHODS: Cut-off values, based on 97.5 percentiles, derived from an apparently healthy omnivorous group (six Dominica breast-fed newborns, 32 breast-fed and 27 formula+LCP-fed Dutch low-birth-weight infants, 31 Jerusalem infants, 33 Dutch 3.5-year-old infants, 69 omnivorous Dutch adults and seven Dominica mothers) and an apparently healthy group with low dietary LCP intake (81 formula-fed Dutch low-birth-weight infants, 12 Dutch vegans). Cut-off values were evaluated by their application in an EFAD suspected group of 108, mostly malnourished, Pakistani children, three pediatric patients with chronic fat-malabsorption (abetal-ipoproteinemia, congenital jejunal and biliary atresia) and one patient with a peroxisomal beta-oxidation disorder. RESULTS: Erythrocyte 20:3omega9, 22:5omega6/20:4omega6 and 22:5omega6/22:6omega3 proved age-dependent up to 0.2 years. Cut-off values for ages above 0.2 years were: 0.46mol% 20:3omega9 for EFAD, 0.068mol/mol 22:5omega6/20:4omega6 for omega3-deficiency, 0.22mol/mol 22:5omega6/22:6omega3 for omega3/DHA-marginality and 0.48mol/mol 22:5omega6/22:6omega3 for omega3/DHA-deficiency. Use of RBC 20:3omega9 and 22:5omega6/20:4omega6 cut-off values identified 20.4% of the Pakistani subjects as EFAD+omega3-deficient, 12.9% as EFAD+omega3-sufficient, 38.9% as EFA-sufficient+omega3-deficient and 27.8% as EFA-sufficient+omega3-sufficient. The patient with the peroxisomal disorder was classified as EFA-sufficient, omega3-sufficient (based on RBC 22:5omega6/20:4omega6) and omega3/DHA-deficient (based on RBC 22:5omega6/22:6omega3). The three other pediatric patients were classified as EFAD, omega3-deficient and omega3/DHA-deficient. CONCLUSION: Use of the combination of the present cut-off values for EFA, omega3 and omega3/DHA status assessment, as based on 97.5 percentiles, may serve for PUFA supplement intervention until better concepts have emerged.     

EphA5 and EphA6: regulation of neuronal and spine morphology

Background

The Eph family of receptor tyrosine kinases plays important roles in neural development. Previous studies have implicated Eph receptors and their ligands, the ephrins, in neuronal migration, axon bundling and guidance to specific targets, dendritic spine formation and neural plasticity. However, specific contributions of EphA5 and EphA6 receptors to the regulation of neuronal cell morphology have not been well studied.

Results

Here we show that deletion of EphA5 and EphA6 results in abnormal Golgi staining patterns of cells in the brain, and abnormal spine morphology.

Conclusion

These observations suggest novel functions of these Eph receptors in the regulation of neuronal and spine structure in brain development and function.

     

Antioxidant activity of 5-alkoxymethyl-6-chromanols

The 5-alkoxymethyl-2,2,7,8-tetramethyl-6-chromanols (II) are excellent antioxidants against autoxidising safflower oil (ASO), although not as good as 2,2,5,7,8-pentamethyl-6-chromanol (I), the model compound of alpha-tocopherol. The aim of this work was to determine whether the rate of reaction of (II) with the radicals diphenylpicrylhydrazyl (DPP*) and galvinoxyl (ArO*) was directly proportional to their antioxidant activity against ASO. Compounds (II) reacted faster with DPP* than with ArO* but, in each case, slower than compound (I). The rates of reaction of I and II with both radicals followed the order I > II (R = H) > II (R = CH3) > II (R = other alkyls) and were directly proportional to their antioxidant activity against ASO.     

Antioxidant activity of 5-alkoxymethyl-6-chromanols

Abstract

The 5-alkoxymethyl-2,2,7,8-tetramethyl-6-chromanols (II) are excellent antioxidants against autoxidising safflower oil (ASO), although not as good as 2,2,5,7,8-pentamethyl-6-chromanol (I), the model compound of -tocopherol. The aim of this work was to determine whether the rate of reaction of (II) with the radicals diphenylpicrylhydrazyl (DPP^·) and galvinoxyl (ArO^·) was directly proportional to their antioxidant activity against ASO. Compounds (II) reacted faster with DPP^·. than with ArO^·. but, in each case, slower than compound (I). The rates of reaction of I and II with both radicals followed the order I > II (R = H) > II (R = CH₃) > II (R = other alkyls) and were directly proportional to their antioxidant activity against ASO.     

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.