Synthesis of an octamannosyled glycan chain, the key oligosaccharide structure in ER-associated degradation

The high-mannose type decasaccharide (Man(8)GlcNAc(2)), the proposed ligand of ER residing mannosidase-like proteins (MLP), and its monoglycosylated homologue (alpha-Glc(1)Man(8)GlcNAc(2)) were synthesized. The oligosaccharide assembly was performed in a convergent and stereoselective manner, using three oligosaccharide components, a core trisaccharide having a beta-mannoside bond, a liner mannotriose, and a branched mannotetraose.     

Synthesis,structure, and biological properties of some 8α analogues of steroid estrogens with fluorine in position 2

A total synthesis of 8α analogues of steroid estrogens with fluorine in position 2 was achieved. Structural features of these compounds were studied by the example of 17β-acetoxy-2-fluoro-3-methoxy-8α-estra-1,3,5(10)-triene. It was shown that the 8α analogues of 2-fluorosubstituted steroid estrogens have a low uterotropic activity and retain the osteoprotective and hypocholesterolemic activities.     

N-linked oligosaccharide processing,but not association with calnexin/calreticulin is highly correlated with endoplasmic reticulum-associated degradation of antithrombin Glu313-deleted mutant

Previously we showed that two antithrombin mutants were degraded through an endoplasmic reticulum (ER)-associated degradation (ERAD) pathway [F. Tokunaga et al., FEBS Lett. 412 (1997) 65]. Here, we examined the combined effects of inhibitors of glycosidases, protein synthesis, proteasome, and tyrosine phosphatase on ERAD of a Glu313-deleted (DeltaGlu) mutant of antithrombin. We found that kifunensine, an ER mannosidase I inhibitor, suppressed ERAD, indicating that specific mannose trimming plays a critical role. Cycloheximide and puromycin, inhibitors of protein synthesis, also suppressed ERAD, the effects being cancelled by pretreatment with castanospermine. In contrast, kifunensine suppressed ERAD even in castanospermine-treated cells, suggesting that suppression of ERAD does not always require the binding of lectin-like ER chaperones-like calnexin and/or calreticulin. These results indicate that, besides proteasome inhibitors, inhibitors of ER mannosidase I and protein synthesis suppress ERAD of the antithrombin deltaGlu mutant at different stages, and processing of N-linked oligosaccharides highly correlated with the efficiency of ERAD.     

Purification and characterization of the MUC1 mucin-type glycoprotein, epitectin, from human urine: structures of the major oligosaccharide alditols

The MUC1 glycoprotein, epitectin, a component of the human bladder epithelium, was purified from human urine. Sedimentation equilibrium analysis and gel filtration using polysaccharide or protein standards revealed a polydisperse preparation with molecular weights ranging from about 0.9 to 1.3×106. This suggests that in the native state epitectin exists as aggregates of three or four monomer units of 350–400 kDa. Epitectin was found to have significant affinity to hexyl-, octyl- or phenyl agarose indicating that hydrophobic interactions and possibly carbohydrate-carbohydrate interactions may be responsible for the self-association. Chemical and enzymic deglycosylation of [125I]-labeled urine epitectin and metabolically labeled H.Ep.2 epitectin resulted in extremely polydisperse products. The buoyant densities of epitectin purified from urine and H.Ep.2 cells were found to be 1.39–1.40 g ml–1, suggesting that the total carbohydrate content of these preparations is not significantly different. The O-linked saccharides of epitectin were fractionated by HPLC and analyzed by permethylation and FAB-MS. The neutral saccharides from both sources 001contain three common structures, namely Gal13GalNAc, GlcNAc16 (Gal13) GalNAc and Gal14 GlcNAc6 (Gal13)GalNAc. The sialic acid of urine epitectin consisted entirely of N-acetylneuraminic acid. The two sources of epitectin, in vitro labeled on sialic acid, were found to have the same sialyl oligosaccharides but in different proportions. Metabolic labeling and N-glycanase susceptibility experiments firmly established the presence of N-linked saccharides in epitectin as minor components. The remarkable similarities in the total carbohydrate content, the carbohydrate composition and structures of saccharides between epitectin from urine, a non-malignant source, and H.Ep.2 cells is surprising in view of the prevailing view that MUC1 glycoproteins of cancer cells are underglycosylated compared to those produced by non-malignant cells.     

Delay of tomato fruit ripening by an oligosaccharide N-glycan. Interactions with IAA, galactose and lectins

The unconjugated N-glycans Manα1→6(Manα→3)Manα1→6(Manα1→3)-Manβ1→4GlcNAc (Man₅GlcNAc) and Manα1→6(Manα1→3)(Xy1β1→2)-Manβ1→4GlcNAcβ1→4(Fucα1→3)GlcNAc were shown to stimulate and delay ripening of mature green tomato fruit (Lycopersicon esculentum Mill. cv. Rutgers) at 1 and 10 ng (g fresh weight)⁻¹, respectively (Priem and Gross 1992, Plant Physiol. 98: 399–401). Also, the occurrence and structure of 10 unconjugated N-glycans, including Man₅GlcNAc, in tomato fruit were recently reported (Priem et al. 1993, Plant Physiol. 102: 445–458). In this work, we studied the potential interaction between Man₅GlcNAc and several compounds by using a system that allowed pericarp discs to be kept up to 14 days after excision. Studies were performed to determine the effect of Man₅GlcNAc, indole-3-acetic acid (IAA), concanavalin A and tomato lectin on ripening as defined by red coloration of the skin. Ripening in pericarp discs, unlike that in intact fruit, was unaffected by 1 ng (g fresh weight)⁻¹ Man₅GlcNAc. However, discs showed delayed ripening with 10 ng (g fresh weight)⁻¹, and the delay of coloration was galactose dependent. Man₅GlcNAc at 10 ng (g fresh weight)⁻¹ inhibited the stimulation of coloration induced by 100 μ IAA in the presence of 40 μg (g fresh weight)⁻¹ galactose. Man₅GlcNAc lost biological activity in the presence of the two lectins. These results support the possible interaction of IAA and various oligosaccharides during plant growth and development, and for the first time suggest a physiological significance for tomato lectin.     

Synthesis of a terphenyl substituted 4-aza-2,3-didehydropodophyllotoxin analogues as inhibitors of tubulin polymerization and apoptosis inducers

A series of terphenyl based 4-aza-2,3-didehydropodophyllotoxin conjugates (8a–r) were synthesized by a straightforward one-step multicomponent synthesis that demonstrated anticancer activity against five human cancer cell lines (lung, colon, renal, prostate and cervical). All the tested compounds showed potent anticancer activity with IC₅₀ values ranging from 0.87 to 16.59 μM. Among them compounds 8n and 8p showed significant anticancer activity in lung cancer cells with IC₅₀ values 0.91 and 0.87 μM, respectively. Flow cytometric analysis revealed that these compounds induced cell cycle arrest in G₂/M phase in A549 cell line leading to caspase-3 dependent apoptotic cell death. The tubulin polymerization assay and immunofluorescence analysis showed that these compounds effectively inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Further, Hoechst staining, DNA fragmentation analysis also suggested that these compounds induced cell death by apoptosis. Overall, the current study demonstrated that the synthesis of terphenyl based 4-aza-2,3-didehydropodophyllotoxin conjugates as promising anticancer agents with G₂/M cell cycle arrest and apoptotic-inducing activities via targeting tubulin.     

Racemic and optically active tocopherol analogues with a modified side chain: Synthesis and biological activity (Review)

Information on the synthesis and biological activity of natural and synthetic analogues of α-tocopherol with a modified side chain is systematized. These compounds are of interest as vitamin E metabolites, hydrophilic antioxidants, and precursors of drugs with combined pharmacological properties useful in therapy of pathological disorders caused by oxidative stress.     

Synthesis and degranulation-inhibiting activities of the proposed apteniols B,C, and G

The synthesis of compounds with the structures proposed for the oxyneolignan apteniols B, C, and G is described. The diphenyl ether skeletons of the proposed apteniols were formed via Ullmann ether synthesis. In particular, the spectral data for the synthesized apteniols B, C, and G did not agree with those previously reported for the isolated compounds. Furthermore, the synthesized proposed apteniol B did not show degranulation-inhibiting activity, while the prepared proposed apteniols C and G exhibited activities considerably weaker than that of the methyl ester of proposed apteniol A.     

Synthesis, and structural and biological studies of efrapeptin C analogues

A series of analogues of efrapeptin C (1), with variations in the central tripeptide epitope (positions 6-8), were prepared by a combination of solid- and solution-phase peptide syntheses. The conformations of the modified compounds 2-6 were investigated by circular-dichroism (CD) spectroscopy to differentiate between 3(10)- and alpha-helical secondary structures. The inhibitory activities of the new compounds towards F(1)-ATPase from E. coli were determined. The modified congeners 3-5 were less active by one order of magnitude compared to 1 (K(i) 10 microM), and 6 was completely inactive. Our experiments demonstrate that the flexible, central tripeptide epitope, comprising positions 6-8 in 1, is crucial for molecular recognition, even slight sequence modifications being hardly tolerated.     

Synthesis and hemocompatibity evaluation of segmented polyurethane end-capped with both a fluorine tail and phosphatidylcholine polar headgroups

To improve the hemocompatibility of polyurethanes, an amine monomer containing a long fluorine tail and phosphatidylcholine polar headgroups, 2-amino-3-oxo-3-(2-(2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctan amido) ethyl amino) propyl phosphorylcholine (FASPC) was firstly synthesized and characterized. Then four kinds of fluorinated phosphatidylcholine end-capped polyurethanes with different chemical structures were prepared. The surface properties of these prepared polyurethanes were characterized using X-ray photoelectron spectroscopic analysis (XPS) and water contact angle measurements. The results indicated that the phosphatidylcholine (PC) polar headgroups along with the fluorine tail could be easily enriched on the top surfaces, and the PC groups could be highly oriented on the outmost surface when the polymer film was in contact with water for only 30 s at room temperature. The evaluation of hemocompatibity was carried out via fibrinogen adsorption and platelet adhesion. Fibrinogen adsorption (37°C for 90 min) decreased by 98% to 87% compared to that on ordinary polyurethane surfaces, and almost no platelet adhesion and activation was observed at 37°C for 2 h.     

Effects of active site cleft residues on oligosaccharide binding,hydrolysis, and glycosynthase activities of rice BGlu1 and its mutants

Rice BGlu1 (Os3BGlu7) is a glycoside hydrolase family 1 β‐glucosidase that hydrolyzes cellooligosaccharides with increasing efficiency as the degree of polymerization (DP) increases from 2 to 6, indicating six subsites for glucosyl residue binding. Five subsites have been identified in X‐ray crystal structures of cellooligosaccharide complexes with its E176Q acid‐base and E386G nucleophile mutants. X‐ray crystal structures indicate that cellotetraose binds in a similar mode in BGlu1 E176Q and E386G, but in a different mode in the BGlu1 E386G/Y341A variant, in which glucosyl residue 4 (Glc4) interacts with Q187 instead of the eliminated phenolic group of Y341. Here, we found that the Q187A mutation has little effect on BGlu1 cellooligosaccharide hydrolysis activity or oligosaccharide binding in BGlu1 E176Q, and only slight effects on BGlu1 E386G glycosynthase activity. X‐ray crystal structures showed that cellotetraose binds in a different position in BGlu1 E176Q/Y341A, in which it interacts directly with R178 and W337, and the Q187A mutation had little effect on cellotetraose binding. Mutations of R178 and W337 to A had significant and nonadditive effects on oligosaccharide hydrolysis by BGlu1, pNPGlc cleavage and cellooligosaccharide inhibition of BGlu1 E176Q and BGlu1 E386G glycosynthase activity. Hydrolysis activity was partially rescued by Y341 for longer substrates, suggesting stacking of Glc4 on Y341 stabilizes binding of cellooligosaccharides in the optimal position for hydrolysis. This analysis indicates that complex interactions between active site cleft residues modulate substrate binding and hydrolysis.     

Synthesis, separation, and characterization of amphiphilic sulfated oligosaccharides enabled by reversed-phase ion pairing LC and LC-MS methods

Synthesis of amphiphilic oligosaccharides is problematic because traditional methods for separating and purifying oligosaccharides, including sulfated oligosaccharides, are generally not applicable to working with amphiphilic sugars. We report here RPIP-LC and LC–MS methods that enable the synthesis, separation, and characterization of amphiphilic N-arylacyl O-sulfonated aminoglycosides, which are being pursued as small-molecule glycosaminoglycan mimics. The methods described in this work for separating and characterizing these amphiphilic saccharides are further applied to a number of uses: monitoring the progression of sulfonation reactions with analytical RP-HPLC, characterizing sulfate content for individual molecules with ESI-MS, determining the degree of sulfation for products having mixed degrees of sulfation with HPLC and LC–MS, and purifying products with benchtop C18 column chromatography. We believe that the methods described here will be broadly applicable to enabling the synthesis, separation, and characterization of amphiphilic, sulfated, and phosphorylated oligosaccharides and other types of molecules substituted to varying degrees with both anionic and hydrophobic groups.     

Synthesis and antimalarial activity of new chloroquine analogues carrying a multifunctional linear side chain

We report the synthesis and in vitro antimalarial activity of several new 4-amino- and 4-alkoxy-7-chloroquinolines carrying a linear dibasic side chain. Many of these chloroquine analogues have submicromolar antimalarial activity versus HB3 (chloroquine sensitive) and Dd2 (chloroquine resistant strain of Plasmodium falciparum) and low resistance indices were obtained in most cases. Importantly, compounds 11–15 and 24 proved to be more potent against Dd2 than chloroquine. Branching of the side chain structure proved detrimental to the activity against the CQR strain.     

A sensitive mapping strategy for monitoring the reproducibility of glycan processing in an HIV vaccine, RGP-160, expressed in a mammalian cell line

The external envelope glycoprotein (gp 160) of HIV-1 is a candidate for vaccines against AIDS. Most of the surface of the molecule is shielded by carbohydrate and the structures and locations of these glycans may be important in defining the immunogenicity of the viral coat. Here we report a sensitive mapping strategy for profiling and analysing the N-glycosylation of gp160, based on chemical release of glycans, fluorescent labelling and HPLC analysis. This approach has been validated in terms of establishing the reproducibility of all steps in the analytical procedure and on overall reproducibility on a run-to-run and day-to-day basis. The validated analysis technique was used to monitor the consistency of N-glycosylation of one rgp 160 vaccine candidate produced in bovine hamster kidney (BHK) cell culture. It was demonstrated that the variation in the glycan profiles of 6 different lots was not statistically significant.     

Synthesis,biophysical and functional studies of two BP100 analogues modified by a hydrophobic chain and a cyclic peptide

Antimicrobial peptides (AMPs) work as a primary defense against pathogenic microorganisms. BP100, (KKLFKKILKYL-NH₂), a rationally designed short, highly cationic AMP, acts against many bacteria, displaying low toxicity to eukaryotic cells. Previously we found that its mechanism of action depends on membrane surface charge and on peptide-to-lipid ratio. Here we present the synthesis of two BP100 analogs: BP100‑alanyl‑hexadecyl‑1‑amine (BP100-Ala-NH-C₁₆H₃₃) and cyclo(1‑4)‑d‑Cys¹, Ile², Leu³, Cys⁴-BP100 (Cyclo(1‑4)‑cILC-BP100). We examined their binding to large unilamellar vesicles (LUV), conformational and functional properties, and compared with those of BP100. The analogs bound to membranes with higher affinity and a lesser dependence on electrostatic forces than BP100. In the presence of LUV, BP100 and BP100-Ala-NH-C₁₆H₃₃ acquired α-helical conformation, while Cyclo(1‑4)‑cILC-BP100) was partly α-helical and partly β-turn. Taking in conjunction: 1. particle sizes and zeta potential, 2. effects on lipid flip-flop, 3. leakage of LUVs internal contents, and 4. optical microscopy of giant unilamellar vesicles, we concluded that at high concentrations, all three peptides acted by a carpet mechanism, while at low concentrations the peptides acted by disorganizing the lipid bilayer, probably causing membrane thinning. The higher activity and lesser membrane surface charge dependence of the analogs was probably due to their greater hydrophobicity. The MIC values of both analogs towards Gram-positive and Gram-negative bacteria were similar to those of BP100 but both analogues were more hemolytic. Confocal microscopy showed Gram-positive B. subtilis killing with concomitant extensive membrane damage suggestive of lipid clustering, or peptide-lipid aggregation. These results were in agreement with those found in model membranes.     

Synthesis of Lewis X trisaccharide analogues in which glucose and rhamnose replace N-acetylglucosamine and fucose,respectively

Two analogues of the Le(x) trisaccharide, alpha-L-Fucp-(1-->3)-[beta-D-Galp-(1-->4)]-D-Glcp were synthesized as allyl glycosides. In these derivatives either only the N-acetylglucosamine is replaced by glucose or both the N-acetylglucosamine and the fucosyl residue are replaced by glucose and rhamnose, respectively. Our synthetic scheme used armed beta-thiophenyl fuco- and rhamnoside glycosyl donors that were prepared anomerically pure from the corresponding alpha-glycosyl bromides. The protecting groups were chosen to allow access to the fully deprotected trisaccharides without reduction of the allyl glycosidic group. These analogues will be used as soluble antigens in binding experiments with anti-Le(x) antibodies and can also be conjugated to a carrier protein and used as immunogens. In the course of this synthetic work, we also describe the use of reversed-phase HPLC to purify key protected trisaccharide intermediates prior to their deprotection.     

Synthesis and biological activities of peptidomimetic analogues of compound A71623, a potent and selective CCK-A receptor agonist

The involvement of cholecystokinin receptors in thephenomenon of satiety has been the impetus forsignificant research efforts, leading to the designand synthesis of CCK-A selective agonists for thepossible treatment of obesity. The Abbott laboratorieshave described a novel series of pseudotetrapeptidesrepresented by compound A71623, a highly potent andselective peripheral receptor agonist, but possessingvery poor bioavailability. Starting from thestructural requirements of this series of compounds,a peptidomimetic study was investigated, especiallyfocusing on the N-terminal part of A71623. Usingstandard coupling methods, introduction of unnaturalaromatic amino acids bearing a 2-carboxyethyl sidechain on their -amino group, along withbackbone length modulation, afforded selective analogues,presenting a highly modified peptidic backbone. Fromour two lead compounds, further optimization is underdevelopment, tending towards nonpeptidic structures.     

Synthesis and biological activity of brassinolide analogues, 26,27-bisnorbrassinolide and its 6-oxo analogue

Two hitherto unknown brassinolide analogues, (22R,23R)-2α,3α,22,23-tetrahydroxy-B-homo-7-oxa-24-nor-5α-cholestan-6-one (9b) and (22R,23R)-2α,3α,22,23-tetrahydroxy-24-nor-5α-cholestan-6-one (8a), were stereoselectively synthesized. In both the Raphanus and rice-lamina inclination tests, 9b exhibited almost the same activity as brassinolide (1) and 8a also showed ca 10–50% of the activity of 1.     

Convenient enzymatic synthesis of a p-nitrophenyl oligosaccharide series of sialyl N-acetyllactosamine, sialyl Le x and relevant compounds

From the beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p (1) prepared by the transglycosylation of beta-galactosidase from Bacillus circulans, alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p (9) and alpha-D-Neu5Ac-(2-->6)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p (10) were effectively synthesized with an equimolar ratio of CMP-Neu5Ac by recombinant rat alpha-(2-->3)-N-sialyltransferase and rat liver alpha-(2-->6)-N-sialyltransferase, respectively. The former enzyme also transferred effectively the Neu5Ac residue from CMP-Neu5Ac to the location of OH-3 in the non-reducing terminal of beta-D-Gal-(1-->4)-beta-D-Gal-OC6H4NO2-p or beta-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p, while the latter enzyme did not. In the case of equimolar ratio of GDP-Fuc/acceptor, 1 and 9 were further fucosylated quantitatively to form beta-D-Gal-(1-->4)-beta-D-(alpha-l-Fuc-(1-->3)-)-GlcNAc-OC6H4NO2-p (14) and alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-beta-D-(alpha-l-Fuc-(1-->3)-)-GlcNAc-OC6H4NO2-p (13) by recombinant human alpha-(1-->3)-fucosyltransferase VII, respectively.     

Synthesis and characterization of a N-salicylaldimine ligand and its vanadium(V) complex

The reduction of 2-nitro-1,3-di(pyridin-2-yl)-1,3-di(tert -butyldimethylsilyloxy)propane 1 with sodium borohydride affords 2-amino-1,3-di(pyridin-2-yl)-1,3-di(tert-butyldimethylsilyloxy)propane 2 which was subsequently reacted with salicyl aldehyde yielding rac-((2,2,3,3,9,9,10,10-octamethyl-5,7-di(pyridin-2-yl)-4,8-dioxa-3,9-disilaundecan-6-ylimino)methyl)phenol (Proligand 3 = HL(SiMe₂tBu)₂), with excellent yield. Reaction of 3 with vanadyl acetylacetonate followed by aerial oxidation diastereoselectively led to the octahedral coordinated vanadium(V) complex 4([VO(OMe)L(SiMe₂t Bu)]). Compound 3 together with vanadyl acetylacetonate as well as with molybdenyl acetylacetonate shows catalytic activity in the sulfoxidation of (methylsulfanyl)benzene I, which was followed by NMR spectroscopy. The vanadium complex 4 was also able to catalyze the sulfoxidation but was considerably slower. All three tested catalytic systems lead to almost quantitative formation of the sulfoxide with only minor formation of the respective sulfone.