Use of phenyl 2-alpha-selenoglycosides of N-acetylneuraminic acid as a glycosyl donor for the glycosylation reactions

Phenyl 2-alpha-selenoglycosides of Neu5Ac were successfully prepared from the corresponding peracetylated chloro derivative of Neu5Ac 1 and phenylselenol in the presence of N,N-di-isopropylethylamine in excellent yields. The reaction of with various alcohols was effectively catalyzed by NIS/TfOH or DMTST to produce a variety of glycosides in moderate yields. Selective activation of over phenyl 2-alpha-thioglycoside of Neu5Ac with AgOTf/K(2)CO(3) was also achieved.     

Glycoprotein synthesis in lysolecithin-treated cells using sugar nucleotides as glycosyl donors

The 3T3 cells were treated with 50 mu g/ml lysolecithin (LL) followed by the addition of exogenously supplied radiolabelled sugar nucleotides to serve as direct glycosyl donors. These were found to be 1.5 to 3.0 times more active than untreated cells in their glycosyl transferase activities depending on the particular sugar nucleotide used. Mannosyl transferase activity was not inhibited by 2-deoxyglucose or mannose-1-phosphate, indicating that the sugar nucleotide remained intact throughout the assay period. Preincubation of the cells with tunicamycin caused an 85% decrease in mannosyl transfer, which suggested that the normal pathway of glycosylation via lipid intermediates was still operable in the treated cells. Fractionation of control and LL-treated cells after incubation with UDP[3H]galactose revealed that only microsomal and cytosolic proteins from the treated cells were radioactive. Thus, intracellular labelling of permeabilized cells was allowed. About 80% of the radiolabeled product was glycoprotein in nature, based upon its solubilization with pronase.     

Glycoprotein synthesis in lysolecithin-treated cells using sugar nucleotides as glycosyl donors

Summary The 3T3 cells were treated with 50 μg/ml lysolecithin (LL) followed by the addition of exogenously supplied radiolabelled sugar nucleotides to serve as direct glycosyl donors. These were found to be 1.5 to 3.0 times more active than untreated cells in their glycosyl transferase activities depending on the particular sugar nucleotide used. Mannosyl transferase activity was not inhibited by 2-deoxyglucose or mannose-1-phosphate, indicating that the sugar nucleotide remained intact throughouth the assay period. Preincubation of the cells with tunicamycin caused an 85% decrease in mannosyl transfer, which suggested that the normal pathway of glycosylation via lipid intermediates was still operable in the treated cells. Fractionation of control and LL-treated cells after incubation with UDP[³H]galactose revealed that only microsomal and cytosolic proteins from the treated cells were radioactive. Thus, intracellular labelling of permeabilized cells was allowed. About 80% of the radiolabeled product was glycoprotein in nature, based upon its solubilization with pronase. This work was supported by institutional funds granted by Texas Woman's University.     

Evaluation of thioglycosides of Kdo as glycosyl donors

The use of Kdo thioglycosides as glycosyl donors using DMTST, IBr/AgOTf and NIS/AgOTf as promoters has been evaluated. Activation at low temperature allowed to escape the formation of 2,3-glycal byproducts to give glycosides in high yield and with good beta-anomeric selectivity. The use of diethyl ether as solvent and (especially) isopropylidene acetals as protecting groups improved the alpha-anomeric selectivity. NIS/AgOTf as promoter surprisingly yielded the 3-iodo-product via the glycal intermediate.     

Novel biological function of sialic acid (N-acetylneuraminic acid) as a hydrogen peroxide scavenger

We have found that N-acetylneuraminic acid (NANA) consumes toxic hydrogen peroxide (H(2)O(2)) under physiological conditions. Close investigation of this finding revealed that NANA was oxidized by an equimolar amount of H(2)O(2) to provide its decarboxylated product, 4-(acetylamino)-2,4-dideoxy-D-glycero-D-galacto-octonic acid (ADOA). To date, there have been little data on this reaction, and its physiological significance has not been discussed. Examining the detoxification of H(2)O(2) in cultured cells with NANA, we were able to confirm that the cell death caused by H(2)O(2) was suppressed by NANA in a dose-dependent manner. These results revealed a novel role for NANA as a reactive oxygen scavenger. It is known that terminal NANA residues are removed by neuraminidase and that free NANA molecules are recycled or degraded by enzymes. We propose that released monomeric NANA is the potent defense molecule against oxidative damage.     

Synthesis and evaluation of C-9 modified N-acetylneuraminic acid derivatives as substrates for N-acetylneuraminic acid aldolase

Several C-9 modified N-acetylneuraminic acid derivatives have been synthesised and evaluated as substrates of N-acetylneuraminic acid aldolase. Simple C-9 acyl or ether modified derivatives of N-acetylneuraminic acid were found to be accepted as substrates by the enzyme, albeit being transformed more slowly than Neu5Ac itself. 1H NMR spectroscopy was used to evaluate the extent of the enzyme catalysed transformation of these compounds. Interestingly, the chain-extended Neu5Ac derivative 16 is not a substrate for N-acetylneuraminate lyase and behaves as an inhibitor of the enzyme.     

Directed evolution of N-acetylneuraminic acid aldolase to catalyze enantiomeric aldol reactions

Expanding the scope of substrate specificity and stereoselectivity is of current interest in enzyme catalysis. Using error-prone PCR for in vitro directed evolution, the Neu5Ac aldolase from Escherichia coli has been altered to improve its catalytic activity toward enantiomeric substrates including N-acetyl-L-mannosamine and L-arabinose to produce L-sialic acid and L-KDO, the mirror-image sugars of the corresponding naturally occurring D-sugars. The first generation variant containing two mutations (Tyr98His and Phe115Leu) outside the (alpha,beta)(8)-barrel active site exhibits an inversion of enantioselectivity toward KDO and the second generation variant contains an additional amino acid change Val251Ile outside the alpha,beta-barrel active site that improves the enantiomeric formation of L-sialic acid and L-KDO. The X-ray structure of the triple mutant epNanA.2.5 at 2.3A resolution showed no significant difference between the wild-type and the mutant enzymes. We probed the potential structural 'hot spot' of enantioselectivity with saturation mutagenesis at Val251, the mutated residue most proximal to the Schiff base forming Lys165. The selected variant had an increase in k(cat) via replacement with another hydrophobic residue, leucine. Further sampling of a larger sequence space with error-prone PCR selected a third generation variant with significant improvement in L-KDO catalysis and a complete reversal of enantioselectivity.     

Production of cytidine 5'-monophosphate N-acetylneuraminic acid using recombinant Escherichia coli as a biocatalyst

An Escherichia coli strain expressing three recombinant enzymes, i.e., cytidine 5'-monophosphate (CMP) kinase, sialic acid aldolase and cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase, was utilized as a biocatalyst for the production of CMP-NeuAc. Both recombinant E. coli extract and whole cells catalyzed the production of CMP-NeuAc from CMP (20 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetylphosphate (60 mM), resulting in 90% conversion yield based on initial CMP concentration used. It was confirmed that endogenous acetate kinase can catalyze not only the ATP regeneration in the conversion of CMP to CDP but also the conversion of CDP to CTP. On the other hand, endogenous pyruvate kinase and polyphosphate kinase could not regenerate ATP efficiently. The addition of exogenous acetate kinase to the reaction mixture containing the cell extract increased the conversion rate of CMP to CMP-NeuAc by about 1.5-fold, but the addition of exogenous inorganic pyrophosphatase had no influence on the reaction. This E. coli strain could also be employed as an enzyme source for in situ regeneration of CMP-NeuAc in a sialyltransferase catalyzed reaction. About 90% conversion yield of alpha2,3-sialyl-N-acetyllactosamine was obtained from N-acetyllactosamine (20 mM), CMP (2 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetyl phosphate (80 mM) using the recombinant E. coli extract and alpha2,3-sialyltransferase.     

Novel glycosylation of the nitroxyl radicals with peracetylated glycosyl fluorides using a combination of BF(3) x OEt(2) and an amine base as promoters

Glycosylation of the nitroxyl radicals, 4-acetoxy-2,2,6,6-tetramethylpiperidin-1-oxyl (4-acetoxy-TEMPO) and 3-carbamoyl-2,2,5,5-tetramethylpyrollin-1-oxyl (3-carbamoyl-PROXYL) with peracetylglycosyl fluoride as the glycosyl donor, in the presence of boron trifluoride diethyl etherate (BF(3) x OEt(2)) and an amine base afforded the corresponding hydroxylamine-O-glycosides in 25-100% yields.     

Synthesis of the methyl thioglycosides of deoxy-N-acetyl-neuraminic acids for use as glycosyl donors.

Methyl 2-thioglycoside derivatives of 4-, 7-, 8-, and 9-deoxy-N-acetylneuraminic acids have been prepared as glycosyl donors for the synthesis of sialoglycoconjates. Reduction of a (phenoxy)thiocarbonyl group, selectively introduced at the 4 position of methyl [2-(trimethylsilyl)ethyl 5-acetamido-3,5-dideoxy-8,9-O-isopropylidene-D- glycero-alpha-D-galacto-2-nonulopyranosid]onate (1), gave the 4-deoxy compound, which was transformed via O-deisopropylidenation, acetylation, selective removal of the 2-(trimethylsilyl)ethyl group, subsequent acetylation, and displacement of the 2-acetoxy group by a methylthio group, into methyl (methyl 5-acetamido-7,8,9-tri-O-acetyl-3,4,5-trideoxy-2-thio-D-manno-2- nonulopyranosid)onate (17). Methyl [2-(trimethylsilyl)ethyl 5-acetamido-8,9-di-O-acetyl-4-O-benzoyl- 3,5,7-trideoxy-alpha-D-galacto-2-nonulopyranosid]onate, prepared from 1 in five steps, and methyl [2-(trimethylsilyl)ethyl 5-acetamido-4,7,9-tri-O-acetyl-3,5,8-trideoxy-alpha-D-galacto-2- nonulopyranosid]onate, prepared from 1 in six steps, were converted via selective removal of the 2-(trimethylsilyl)ethyl group, O-acetylation, and displacement of the 2-acetoxy group by a methylthio group as described for 17, into the corresponding methyl 7- and 8-deoxy-2-thioglycosides. Reductive dechlorination of methyl [2-(trimethylsilyl)ethyl 5-acetamido-4,7-di-O-benzoyl-9-chloro-3,5,9-trideoxy-D-glycero-alpha-D-g alacto- 2-nonulopyranosid]onate, prepared from methyl [2-(trimethylsilyl)ethyl 5-acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosid++ +]onate by selective 9-O-tert-butyldimethylsilylation, benzoylation, removal of the 9-silyl group, and selective chlorination, gave a 9-deoxy compound. This was transformed, via O-debenzoylation, O-acetylation, selective removal of the 2(trimethylsilyl)ethyl group, 2-O-acetylation, 2-chlorination, displacement with potassium thioacetate, selective S-deacetylation, and S-methylation, into the methyl 2-thio-alpha-glycoside of 9-deoxy-N-acetylneuraminic acid.     

Sialylation reactions with 5-N,7-O-carbonyl-protected sialyl donors: unusual stereoselectivity with nitrile solvent assistance

A 5-N,7-O-carbonyl-protected sialyl donor was synthesized, and, unexpectedly, this donor showed beta-selectivity (alpha/beta = 1/2.4-1/20) on coupling with sugar acceptors in acetonitrile upon treatment with various promoter systems. For the coupling reaction in dichloromethane, a modified Ellervik's method (IBr and AgClO(4).H(2)O) was highly effective in activating the 5-N,7-O-carbonyl donor, providing moderate alpha-selectivity (alpha/beta = ~1.8/1).     

Lipase-catalyzed acetylation of N-acetylneuraminic acid derivative

A facile preparation of triacetylated derivative of 2-phenylthioglycoside of N-acetylneuraminic acid (4) was achieved by treatment with lipase PS in vinyl acetate. The major product 4 has a free hydroxyl group at C-7. Results of time-course HPLC analysis indicate that the reactivity of the hydroxyl groups under this condition is in the following order; C-9 > C-4 > C-8 > C-7.     

Synthesis of the methyl thioglycosides of deoxy-N-acetyl-neuraminic acids for use as glycosyl donors

Methyl 2-thioglycoside derivatives of 4-, 7-, 8-, and 9-deoxy-N-acetylneuraminic acids have been prepared as glycosyl donors for the synthesis of sialoglycoconjates. Reduction of a (phenoxy)thiocarbonyl group, selectively introduced at the 4 position of methyl [2-(trimethylsilyl)ethyl 5-acetamido-3,5-dideoxy-8,9-O-isopropylidene-d-glycero- α-d-galacto-2-nonulopyranosid]onate (1), gave the 4-deoxy compound, which was transformed via O-deisopropylidenation, acetylation, selective removal of the 2-(trimethylsilyl)ethyl group, subsequent acetylation, and displacement of the 2-acetoxy group by a methylthio group, into methyl (methyl 5-acetamido-7,8,9-tri-O-acetyl-3,4,5-trideoxy-2-thio-d-manno-2-nonulopyranosid)onate (17). Methyl [2-(trimethylsilyl)ethyl 5-acetamido-8,9-di-O-acetyl-4-O-benzoyl-3,5,7-trideoxy-α-d-galacto-2- nonulopyranosid]onate, prepared from 1 in five steps, and methyl [2-(trimethylsilyl)ethyl 5-acetamido-4,7,9-tri-O-acetyl-3,5,8-trideoxy-α-d-galacto-2-nonulopyranosid]onate, prepared from 1 in six steps, were converted via selective removal of the 2-(trimethylsilyl)ethyl group, O-acetylation, and displacement of the 2-acetoxy group by a methylthio group as described for 17, into the corresponding methyl 7- and 8-deoxy-2-thioglycosides. Reductive dechlorination of methyl [2-(trimethylsilyl)ethyl 5-acetamido-4,7-di-O-benzoyl-9-chloro-3,5,9-trideoxy-d-glycero-α-d-galacto-2-nonulopyranosid]onate, prepared from methyl [2-(trimethylsilyl)ethyl 5-acetamido-3,5-dideoxy-d-glycero-α-d-galacto-2-nonulopyranosid]onate by selective 9-O-tert-butyldimethylsilylation, benzoylation, removal of the 9-silyl group, and selective chlorination, gave a 9-deoxy compound. This was transformed, via O-debenzoylation, O-acetylation, selective removal of the 2-(trimethylsilyl)ethyl group, 2-O-acetylation, 2-chlorination, displacement with potassium thioacetate, selective S-deacetylation, and S-methylation, into the methyl 2-thio-α-glycoside of 9-deoxy-N-acetylneuraminic acid.     

Chemical evaluation of compounds as nitric oxide or peroxynitrite donors using the reactions with serotonin

The aim of this work was to assess the capacities of some ^·NO-donors to release ^·NO, and consequently NOx in aerobic medium, or to give peroxynitrite. The method was based on the differential reactivity of serotonin (5-HT) with either NO_x or peroxynitrite, leading in phosphate-buffered solutions to 4-nitroso- and 4-nitro-5-HT formation, respectively. Yields and formation rates of 5-HT derivatives with ^·NO-donor were compared to those obtained with authentic ^·NO or peroxynitrite in similar conditions. Aside from the capacity of diazenium diolates (SPER/NO and DEA/NO) to release ^·NO spontaneously, converting 5-HT exclusively to 4-nitroso-5-HT, all other ^·NO donors must undergo redox reactions to produce ^·NO. S-nitrosoglutathione (GSNO) and sodium nitroprus-side (SNP) modified 5-HT only in the presence of Cu²⁺, GSNO yielding 6 times more 4-nitroso-5-HT than SNP. Furthermore, in the presence of Cu⁺, the yield of ^·NO-release from GSNO was 45%. The molsidomine metabolite (SIN-1), which was presumed to release both ^·NO and O2/·- at pH 7.4, reacted with 5-HT differently, depending on the presence of reductant or oxidant. Under aerobic conditions, SIN-1 acted predominantly as a 5-HT oxidant and also as a poor ^·NO and peroxynitrite donor (15% yield of ^·NO-release and 14 % yield of peroxynitrite formation). The strong oxidant Cu²⁺, even in the presence of air oxygen, accelerated oxidation and increased ^·NO release from SIN-1 up to 86%. Only a small part of SIN-1 gave simultaneously ^·NO and O2/·- able to link together to give peroxynitrite, but other oxidants could enhance ^·NO release from SIN-1.     

Chemical evaluation of compounds as nitric oxide or peroxynitrite donors using the reactions with serotonin

It has been suggested that diabetes induces an increase in oxidative stress; the increased expression of heme-oxygenase 1 (HO-1) in liver is believed to be a sensitive marker of the stress response. The aim of this study was to examine whether diabetes is able to induce HO-1 expression in liver. The specific mRNA was amplified by RT/PCR and calibrated with amplified β-actin mRNA.

The mRNA HO-1 levels in the liver of spontaneously diabetic rats were increased by 1.8 fold compared with non diabetics; this supports the hypothesis of weak but significant oxidative damage due to chronic hyperglycaemia. This work represents the first in vivo study exploring the semi-quantitative expression of HO-1 in the liver of spontaneously diabetic rats.     

Synthesis of sialyl Lewis x mimetics as selectin inhibitors by enzymatic aldol condensation reactions

Several D-mannosyl phosphate/phosphonate derivatives have been enzymatically prepared as sialyl Lewis x tetrasaccharide mimics, which showed strong-to-moderate inhibition against E-, P-, and L-selectins. The synthesis of these mimics is very straightforward; mannosyl aldehyde derivatives are condensed with dihydroxyacetone phosphate (DHAP) in the presence of a DHAP-dependent aldolase to provide mannosyl phosphates.