Synthesis of β-1, 3-Glucan and β-1, 3 and β-1, 4-Mixed Glucan from UDP-α-d-Glucose

A particulate enzyme preparation from Phaseolus aureus (mung bean) seedlings catalyzed the synthesis of a water insoluble β-1,3-glucan from UDP-α-d-glucose (UDPG) at high concentrations (0.4~20 mm) and an alkaline insoluble β-1,3 and β-1,4-mixed glucan from UDPG at a low concentration (8.5 µm).

Furthermore, the two kinds of β-glucan synthetases which were investigated with two reaction systems at high and low concentrations of UDPG had different properties in optimal pH, stability of enzyme activity, and metallic ion requirement.     

Synthesis of methyl α- and β-maltotriosides and aryl β-maltotriosides

Reaction of β-maltotriose hendecaacetate with phosphorus pentachloride gave 2′,2″,3,3′,3″,4″,6,6′,6″,-nona-O-acetyl-(2)-O-trichloroacetyl-β-maltotriosyl chloride (2) which was isomerized into the corresponding α anomer (8). Selective ammonolysis of 2 and 8 afforded the 2-hydroxy derivatives 3 and 9, respectively; 3 was isomerized into the α anomer 9. Methanolysis of 2 and 3 in the presence of pyridine and silver nitrate and subsequent deacetylation gave methyl α-maltotrioside. Likewise, methanolysis and O-deacetylation of 9 gave methyl β-maltotrioside which was identical with the compound prepared by the Koenigs—Knorr reaction of 2,2′,2″,3,3′,3″,4″,6,6′,6″-deca-O-acetyl-α-maltotriosyl bromide (12) with methanol followed by O-deacetylation. Several substituted phenyl β-glycosides of maltotriose were also obtained by condensation of phenols with 12 in an alkaline medium. Alkaline degradation of the o-chlorophenyl β-glycoside decaacetate readily gave a high yield of 1,6-anhydro-β-maltotriose.     

Synthesis and reactions of α- and β-d-glucopyranosyluronic esters of amino acids

The synthesis of the fully benzylated α- and β-d-glucopyranosyluronic esters of 1-benzyl N-benzyloxycarbonyl-l-aspartic and -glutamic acids and N-(tert-butoxycarbonyl)-l-phenylalanine, followed by hydrogenolysis, afforded the respective anomers of the 1-O-acyl-d-glucopyranuronic acids 2, 7, and 12. Esterification of both anomers of the N-acetylated derivatives of 2 and 7 by diazomethane was accompanied by glycosyl-bond cleavage, and, in the case of the α anomers, with concomitant 1→2 acyl migration to give, after O-acetylation, the 2-O-acyl O-acetyl methyl ester derivatives 5 and 10, respectively. Similarly, 12α yielded methyl 1,3,4-tri-O-acetyl-2-O-[N-(tert-butoxycarbonyl)-l-phenylalanyl]-d-glucopyranuronate and an analogue having a furanurono-6,3-lactone structure. Esterification of the C-5 carboxyl group, in 1-O-acyl-α-d-glucopyranuronic acids by methanol in the presence of the BF_3^?-MeOH reagent (1–1.5 equiv.) proceeded without acyl migration. By using this procedure, followed by acetylation, the N-acetylated derivative of 7α afforded methyl 2,3,4-tri-O-acetyl-1-O-(1-methyl N-acetyl-l-glutam-5-oyl)-α-d-glucopyranuronate, and 12α gave methyl 2,3,4-tri-O-acetyl-1-O-(N-acetyl-l-phenylalanyl)-α-d-glucopyranuronate; the formation of the latter involved cleavage of the tert-butoxycarbonyl group by BF₃, followed by N-acetylation in the next step.     

Synthesis of disaccharide derivatives employing β-N-acetyl- d-hexosaminidase, β-d-galactosidase and β-d-glucuronidase

-N-Acetyl-d-hexosaminidase from Aspergillus oryzae catalysed the stereo- and regiospecific formation of the 6-O-benzylated disaccharide derivatives GalNAc1-3(6- OBn)Gal-SEt and GlcNAc1-3(6-OBn)Gal-SEt, which were obtained in transglycosylation reactions employing ethyl 6- O-benzyl-1-thio--d-galactopyranoside as acceptor. Preparative amounts of the chitobiose derivative GlcNAc1- 3GlcNAc-OPhNO2-p was prepared as well. - N-Acetyl-d-hexosaminidase from bovine testes catalysed the specific synthesis of GlcNAc1-3(6-OBn)GlcNH2-SEt and GalNAc1-3(6-OBn)GlcNH2-SEt, employing ethyl 2-amino-6-O-benzyl-2-deoxy-1-thio--d-glucopyranoside as acceptor. -d-Glucuronidase from E. coli was found to catalyse the formation of GlcA1-3(6-OBn)GlcNH2- SEt employing the same acceptor.     

Synthesis and antiproliferative activity of novel α- and β-dialkoxyphosphoryl isothiocyanates

A series of 15 mostly new dialkoxyphosphoryl alkyl and aralkyl isothiocyanates were synthesized using two alternative strategies, and their in vitro antiproliferative activity against several cancer cell lines (including drug resistant) is here demonstrated. The IC₅₀ values measured for the new compounds are within the range of 6.3-21.5 μM, and they are quite similar to the activity of two best and most extensively investigated natural benzyl isothiocyanate (A) and phenethyl isothiocyanate (B). Preliminary studies utilizing the cell cycle and reduced glutathione level analysis performed on A549 lung cancer cell line using representative compounds revealed important differences in the mechanism of action possibly correlated with their chemical properties. Hydrophobic compounds react mainly with the cytosolic glutathione reduced leading to its depletion, causing an oxidative stress and cell cycle arrest in G0/G1 phase. On the other hand, hydrophilic compounds cause moderate cell cycle arrest and massive cell death associated with moderate reduced glutathione depletion. These suggest that significant changes in the chemical structure of isothiocyanates, which do not lead to the significant changes in antiproliferative activity, but simultaneously cause a differences in the mechanism of action are possible.     

Synthesis and characterization of quaternized β-chitin

Water-soluble 2′-O-hydroxypropyltrimethylammoniumchitin chloride (2′-O-HTACCt) was prepared directly from β-chitin and 3-chloro-2-hydroxypropyltrimethylammonium chloride (CTA) in basic medium. The effect of alkali concentration, reaction temperature, reaction time, and dosage of CTA on yield and degree of substitution (DS) of 2′-O-HTACCt were studied. These quaternized chitin derivatives were characterized by FTIR and ¹H NMR spectroscopy, conductometric titration, and elemental analysis methods. Research results indicate that β-chitin can react directly with CTA to produce a water-soluble 2′-O-HTACCt derivative with a high DS. The optimal preparation conditions were determined to be 35-40 wt % (aq NaOH), 40 °C (reaction temperature), 6 h (reaction time), and 4 (molar ratio of CTA to β-chitin unit).     

Structure and Synthesis of Dopastin,an Inhibitor of Dopamine β-Hydroxylase

Dopastin, an inhibitor of dopamine β-hydroxylase of microbial origin, was shown to be N-[2(S)-nitrosohydroxylamino-3-methylbutyl] crotonamide based on chemical, spectroscopic and synthetic studies. The total synthesis of dopastin was completed in 8 steps starting from l-valinol. N-Nitrosohydroxylamino function was introduced through an oxaziran with retention of the absolute configuration in the final product. Thus, the 2S-configuration of dopastin was proved by the total synthesis. Racemic dopastin was also synthesized from isobutyraldehyde in 7 steps.     

Synthesis,modification, and cytotoxic evaluation of 2,3-secotriterpenic β-ketoesters

A set of β-ketoesters was synthesized from 2,3-seco-18αH-oleanane and 2,3-secolupane bromomethyl ketones. Additionally, hydroxy derivatives with the A-seco- or five-membered A ring were obtained as a result of the reduction or of alkaline hydrolysis of acetic acid β-ketoesters 4, 9. Cytotoxic screening revealed the compound 4 with marked activity (IC₅₀ 3.07–3.61?µM) against the HCT 116, MS, RD TE32 cancer cells. The studies of the cytotoxic mechanism enabled elucidating the fact that treatment of the HCT 116 cells with compound 4 for 18?h leads to induction of apoptosis in a dose-dependent manner. This observation was confirmed by registration of chromatin condensation, by the fluorescence increased during Annexin V-FITC staining, and by appearance of a sub-G0 peak in the cell cycle analysis with DAPI. Compound 4 also inhibited migration of cancer cells in the wound healing assay.     

Synthesis and hydrogenolysis of the methylene,ethylidene, isopropylidene,and diastereoisomeric 1-phenylethylidene acetals of β-l-arabino- and α-l-rhamnopyranoside derivatives

Both diastereoisomers of 1-phenylethylidene acetals (acetophenone acetals) of methyl and benzyl β-l-arabinopyranoside and α-l-rhamnopyranoside were prepared. Acetal-exchange reactions gave only the endo-phenyl isomers; their 2-O- and 4-O-acetyl derivatives were isomerised into the exo-phenyl compounds. ¹H-N.m.r. data were used to determine the absolute configuration at the acetal carbon atom in these compounds. The protons of the methyl group of the exo-phenyl isomers resonate at lower field than those of the endo-phenyl isomers. Hydrogenolysis of various methylene, ethylidene, and isopropylidene derivatives gave axial ethers. The endo-phenyl isomers of the acetophenone derivatives also gave axial 1-phenylethyl ethers in two diastereoisomeric forms. The exo-phenyl isomers of the arabinosides were stable towards the reagent (LiAlH₄AlCl₃), whereas the corresponding rhamnopyranosides gave the 2-(1-phenylethyl) ethers, but cleavage required prolonged reaction time and higher temperature.     

Synthesis and anti-acetylcholinesterase properties of novel β- and γ-substituted alkoxy organophosphonates

Activated organophosphate (OP) insecticides and chemical agents inhibit acetylcholinesterase (AChE) to form OP-AChE adducts. Whereas the structure of the OP correlates with the rate of inhibition, the structure of the OP-AChE adduct influences the rate at which post-inhibitory reactivation or aging phenomena occurs. In this report, we prepared a panel of β-substituted ethoxy and γ-substituted propoxy phosphonoesters of the type p-NO₂PhO-P(X)(R)[(O(CH₂)_nZ] (R = Me, Et; X = O, S; n = 2, 3; Z = halogen, OTs) and examined the inhibition of three AChEs by select structures in the panel. The β-fluoroethoxy methylphosphonate analog (R = Me, Z = F, n = 2) was the most potent anti-AChE compound comparable (k_i ～6 × 10⁶ M^?1 min^?1) to paraoxon against EEAChE. Analogs with Z = Br, I, or OTs were weak inhibitors of the AChEs, and methyl phosphonates (R = Me) were more potent than the corresponding ethyl phosphonates (R = Et). As expected, analogs with a thionate linkage (PS) were poor inhibitors of the AChEs.     

Synthesis of alkyl fucosides through β-glucosidase-catalyzed condensation of fucose and 1-alcohols

-Glucosidase from almond catalyzed condensations of fucose and 1-alcohols with carbon numbers of 6 to 8 produce the corresponding 1-alkyl -d-fucosides. The enzyme also catalyzed the condensation of galactose and 1-hexanol. The conversion for the synthesis of hexyl fucoside was higher than those for the syntheses of hexyl glucoside and galactoside. The effect of initial saccharide concentrations on the conversions to alkyl glycosides was examined. The conversions at day 8 were almost the same at the low initial concentrations of the saccharides. However, the conversion significantly decreased as the concentrations increased. The surfactant properties of the prepared alkyl glycosides were measured. The critical micelle concentrations of the alkyl fucosides were lower than those of the glucosides and galactosides with the same alkyl chains.     

Synthesis of β-Tubanol

Synthesis of β-tubanol (VIa) was achieved by the bromination of dihydro-β-tubanol acetate (VIIb) followed by dehydrobromination and the subsequent hydrolysis. 2,2-Dimethyl-5-hydroxy-chromanone as well as its derivatives (IV) and the corresponding chromanols (V) and chromans (VII) were also prepared.     

Synthesis of α- and β-glycosides containing spin labels,as probes for studies of carbohydrate-protein interaction

Nitroxide spin-labeled α-d-glycopyranosides were synthesized in good yield and in a highly stereoselective manner by reaction of per-O-benzyl-α-d-glycopyranosyl bromides with 2,2,6,6-tetramethyl-4-piperidinol under the bromide ion-catalyzed conditions devised by Lemieux etal. After hydrogenolysis, the deblocked intermidiates were oxidized to give the desired, spin-labeled α-d-glycopyranosides. Nitroxide spin-labeled α-d-glycopyranosides, as well as a β-maltoside, were synthesized by standard methods. The synthesis is also described of 2-amino-2-deoxy-d-glucose and -d-galactose derivatives having a spin label at C-2, and of the spin-labeled compound 1-[4-(β-d-galactopyranosyloxy)phenyl]-3-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl)-2-thiourea.     

Synthesis of methyl α- and β-N-dansyl-d-galactosaminides,probes for the combining sites ofN-acetyl-d-galactosamine-specific lectins

The synthesis of the methyl - and -N-dansyl-d-galactosaminides is described using methyl ,-2-azido-2-deoxy-d-galactopyranoside as starting material. This was reduced to the corresponding methyl ,-2-amino-2-deoxy-d-galactopyranoside and then treated with dansyl chloride to yield a mixture of methyl ,-N-dansyl-d-galactosaminides which was separated into individual anomeric forms by flash chromatography on silica gel. Methyl -N-dansyl-d-galactosaminide was used as a fluorescent indicator ligand in continuous substitution titrations to determine the association constants of nonchromophoric carbohydrates with theN-acetyl-d-galactosamine specific lectin fromErythrina corallodendron.Abbreviations ECorL Erythrina corallodendron lectin - MeGalNDns methyl 2-deoxy-2-(5-dimethylamino-1-naphthalenesulfamido)--d-galactopyranoside - MeGalNDns methyl 2-deoxy-2-(5-dimethylamino-1-naphthalenesulfamido)--d-galactopyranoside Dedicated to Hilde De Boeck (1958–1991).     

Synthesis,docking, and biological studies of phenanthrene β-diketo acids as novel HIV-1 integrase inhibitors

In the present study we report the synthesis of halogen-substituted phenanthrene β-diketo acids as new HIV-1 integrase inhibitors. The target phenanthrenes were obtained using both standard thermal- and microwave-assisted synthesis. 4-(6-Chlorophenanthren-2-yl)-2,4-dioxobutanoic acid (18) was the most active compound of the series, inhibiting both 3′-end processing (3′-P) and strand transfer (ST) with IC₅₀ values of 5 and 1.3 μM, respectively. Docking studies revealed two predominant binding modes that were distinct from the binding modes of raltegravir and elvitegravir, and suggest a novel binding region in the IN active site. Moreover, these compounds are predicted not to interact significantly with some of the key amino acids (Q148 and N155) implicated in viral resistance. Therefore, this series of compounds can further be investigated for a possible chemotype to circumvent resistance to clinical HIV-1 IN inhibitors.     

Purification and Some Properties of β-1, 3-Glucanase of Suspension-cultured Tobacco Cells

A laminaran-hydrolyzing enzyme was purified from the homogenate of suspension-cultured tobacco ceils by the treatment with ion-exchangers and gel filtration. The purified enzyme was homogemous in disc-electrophoresis and was a basic protein. The optimal pH of the enzyme was 5.0. The enzyme was stable at temperature below 40°C. The inhibitory effect of Hg²⁺ Cu²⁺ and Ag⁺ was observed. Investigation of the hydrolysis product revealed that the enzyme attacked laminaran endo-wise to form laminari-tetraose, -triose, -biose and glucose.     

Synthesis and assembly of soybean β-conglycinin in vitro

The construction of SP6-derived expression plasmids that encode normal and modified -conglycinin subunits is described. With the exception of an additional methionine at their NH₂-terminal ends and the lack of glycans, the normal subunits synthesized at the direction of these plasmids coresponded to mature and subunits isolated from soybean seeds. The subunits assembled into trimers in vitro that were equivalent in size to those formed in vivo. This result shows that the glycans are not required either for protein folding or oligomer assembly. Subunits produced from other plasmids, which had modifications in a highly conserved hydrophobic region in the COOH-terminal end of the subunits, either did not assemble or assembled at an extremely low rate compared to unmodified subunits. Structural changes at the more hydrophilic NH₂-terminal end had mixed effects. Several subunits modified in this region assembled into trimers at rates that were either equal or greater than those for normal subunits. Others assembled less completely than the normal subunits. Our results indicate that the in vitro synthesis and assembly assay will be useful in evaluating structure-function relationships in modified -conglycinin subunits. The results also show that structural changes at the NH₂-terminal end of the subunits are tolerated to a greater extent than modifications in the hydrophobic conserved region in the COOH-terminal half of the subunits, and this information will be useful in efforts to improve soybean quality.