Introducing transglycosylation activity into human salivary alpha-amylase (HSA)

Synthesis of 4-nitrophenyl 1-thio-beta-D-maltoside, maltotrioside, and maltotetraoside in yields up to 60% has been achieved by a Tyr151Met (Y151M) mutant of human salivary alpha-amylase. Y151M is capable of transferring maltose and maltotriose residues from a maltotetraose donor onto different p-nitrophenyl glycosides. (1)H and (13)C NMR studies revealed that the mutated enzyme preserved the stereo- and regioselectivity. The glycosylation took place at position 4 of the glycosyl acceptor, forming the alpha(1-4)glycosidic bond, exclusively. [reaction: see text]     

Synthesis and Biological Evaluation of the Forssman Antigen Pentasaccharide and Derivatives by a One‐Pot Glycosylation Procedure

The synthesis and biological evaluation of the Forssman antigen pentasaccharide and derivatives thereof by using a one‐pot glycosylation and polymer‐assisted deprotection is described. The Forssman antigen pentasaccharide, composed of GalNAcα(1,3)GalNAcβ(1,3)Galα(1,4)Galβ(1,4)Glc, was recently identified as a ligand of the lectin SLL‐2 isolated from an octocoral Sinularia lochmodes. The chemo‐ and α‐selective glycosylation of a thiogalactoside with a hemiacetal donor by using a mixture of Tf₂O, TTBP and Ph₂SO, followed by activation of the remaining thioglycoside, provided the trisaccharide at the reducing end in a one‐pot procedure. The pentasaccharide was prepared by the α‐selective glycosylation of the N‐Troc‐protected (Troc=2,2,2‐trichloroethoxycarbonyl) thioglycoside with a 2‐azide‐1‐hydroxyl glycosyl donor, followed by glycosidation of the resulting disaccharide at the C3 hydroxyl group of the trisaccharide acceptor in a one‐pot process. We next applied the one‐pot glycosylation method to the synthesis of pentasaccharides in which the galactosamine units were partially and fully replaced by galactose units. Among the three possible pentasaccharides, Galα(1,3)GalNAc and Galα(1,3)Gal derivatives were successfully prepared by the established method. An assay of the binding of the synthetic oligosaccharides to a fluorescent‐labeled SLL‐2 revealed that the NHAc substituents and the length of the oligosaccharide chain were both important for the binding of the oligosaccharide to SLL‐2. The inhibition effect of the oligosaccharide relative to the morphological changes of Symbiodinium by SLL‐2, was comparable to their binding affinity to SLL‐2. In addition, we fortuitously found that the synthetic Forssman antigen pentasaccharide directly promotes a morphological change in Symbiodinium. These results strongly indicate that the Forssman antigen also functions as a chemical mediator of Symbiodinium.     

Chemoenzymatic synthesis of glycopeptides with PglB, a bacterial oligosaccharyl transferase from Campylobacter jejuni

The gram-negative bacterium Campylobacter jejuni has a general N-linked glycosylation pathway encoded by the pgl gene cluster. One of the proteins in this cluster, PgIB, is thought to be the oligosaccharyl transferase due to its significant homology to Stt3p, a subunit of the yeast oligosaccharyl transferase complex. PgIB has been shown to be involved in catalyzing the transfer of an undecaprenyl-linked heptasaccharide to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Using a synthetic disaccharide glycan donor (GaINAc-alpha1,3-bacillosamine-pyrophosphate-undecaprenyl) and a peptide acceptor substrate (KDFNVSKA), we can observe the oligosaccharyl transferase activity of PgIB in vitro. Furthermore, the preparation of additional undecaprenyl-linked glycan variants reveals the ability of PgIB to transfer a wide variety of saccharides. With the demonstration of PgIB activity in vitro, fundamental questions surrounding the mechanism of N-linked glycosylation can now be addressed.     

Carbohydrate decoration of microporous polypropylene membranes for lectin affinity adsorption: comparison of mono- and disaccharides

Carbohydrates (saccharides) are ubiquitous on the extracellular surface of living cells and mediate a myriad of biological recognition and signaling processes. Carbohydrate decoration of polymer surfaces with covalent attachment of saccharides offers a new realm of opportunities to mimic cellular events such as protein recognition and binding. We describe the carbohydrate decoration (surface glycosylation) of poly(2-hydroxyethyl methacrylate)-grafted microporous polypropylene membranes (poly(HEMA)-g-MPPMs) with mono- and disaccharides. Galactose, lactose, glucose, and maltose were covalently attached on the surfaces of poly(HEMA)-g-MPPMs and were compared in detail. The process was verified by solid state (13)C NMR spectra. Membranes with high binding degree (BD) of saccharide ligands on the surfaces were facilely prepared from poly(HEMA)-g-MPPMs with high grafting degree (GD) of poly(HEMA). For poly(HEMA)-g-MPPM with the same GD of poly(HEMA), the BD of disaccharides is lower than that of monosaccharides and the disaccharide-decorated MPPMs are more hydrophilic than the monosaccharide-decorated ones. The carbohydrate-decorated MPPMs prepared from galactose, lactose, glucose, and maltose (denoted as MPPM-Gal, MPPM-Lac, MPPM-Glc and MPPM-Mal, respectively) recognize and adsorb specifically one of the two lectins, concanavalin A (Con A) and peanut agglutinin (PNA). As the BD of saccharide increases, the "glycoside cluster effect" plays a primary role in lectin adsorption. MPPM-Lac has enhanced affinity to PNA as compared with MPPM-Gal having similar BD of saccharide., on the other hand, MPPM-Mal shows no enhanced affinity to Con A in comparison with MPPM-Glc as the BD of saccharide is above 0.9 μmol/cm(2), where the "glycoside cluster effect" occurs.     

2-(Hydroxycarbonyl)benzyl glycosides: a novel type of glycosyl donors for highly efficient beta-mannopyranosylation and oligosaccharide synthesis by latent-active glycosylation

2-(Benzyloxycarbonyl)benzyl (BCB) glycosides were prepared by coupling of the corresponding tetraacetylglycosyl bromides and benzyl 2-(hydroxymethyl)benzoate. The BCB glycosides were converted almost quantitatively into the corresponding 2-(hydroxycarbonyl)benzyl (HCB) glycosides by selective hydrogenolysis of the benzyl ester functionality without affecting the benzylidene acetal and the benzyl ether. Treatment of the HCB 4,6-O-benzylidenemannopyranoside 4 with triflic anhydride in the presence of di-tert-butylmethylpyridine and subsequent addition of the glycosyl acceptor having a primary hydroxyl group afforded exclusively the disaccharide of the beta-mannopyranosyl linkage. Glycosylation of the compound 4 with secondary and tertiary alcohols also provided beta-mannopyranosides as the major products. Glycosylation of the HCB 4,6-O-cyclohexylidenemannoside 5 with primary alcohols was also highly beta-selective, and the HCB 2,3-O-cyclohexylidenemannoside 6 exhibited the moderate beta-selectivity. On the other hand, unlike the HCB mannosides, the HCB 4,6-O-benzylideneglucoside 7 gave exclusively the disaccharides of the alpha-glycopyranosyl linkage in the glycosylation with primary alcohols. The latent BCB-disaccharide 23, which was obtained from the HCB mannoside 4 as the donor and the BCB glucoside 12 as the acceptor by the present glycosylation method, was converted into the active HCB-disaccharide 39 by selective hydrogenolysis. Repetitive glycosylation of the donor 39 with the same acceptor 12 afforded the BCB-trisaccharide 40. Other BCB-trisaccharides 42 and 46 were also efficiently synthesized by employing the present methodology.     

Understanding the folding process of synthetic polymers by small-molecule folding agents

Two acceptor containing polyimides PDI and NDI carrying pyromellitic diimide units and 1,4,5,8-naphthalene tetracarboxy diimide units, respectively, along with hexa(oxyethylene) (EO6) segments as linkers, were prepared from the corresponding dianhydrides and diamines. These polyimides were made to fold by interaction with specifically designed folding agents containing a dialkoxynaphtha-lene (DAN) donor linked to a carboxylic acid group. The alkali-metal counter-ion of the donor carboxylic acid upon complexation with the EO6 segment brings the DAN unit in the right location to induce a charge-transfer complex formation with acceptor units in the polymer backbone. This two-point interaction between the folding agent and the polymer backbone leads to a folding of the polymer chain, which was readily monitored by NMR titrations. The effect of various parameters, such as structures of the folding agent and polymer, and the solvent composition, on the folding propensities of the polymer was studied.     

Chemoenzymatic Synthesis of Glycopeptides with PglB, a Bacterial Oligosaccharyl Transferase from Campylobacter jejuni

The gram-negative bacterium Campylobacter jejuni has a general N-linked glycosylation pathway encoded by the pgl gene cluster. One of the proteins in this cluster, PglB, is thought to be the oligosaccharyl transferase due to its significant homology to Stt3p, a subunit of the yeast oligosaccharyl transferase complex. PglB has been shown to be involved in catalyzing the transfer of an undecaprenyl-linked heptasaccharide to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Using a synthetic disaccharide glycan donor (GalNAc-α1,3-bacillosamine-pyrophosphate-undecaprenyl) and a peptide acceptor substrate (KDFNVSKA), we can observe the oligosaccharyl transferase activity of PglB in vitro. Furthermore, the preparation of additional undecaprenyl-linked glycan variants reveals the ability of PglB to transfer a wide variety of saccharides. With the demonstration of PglB activity in vitro, fundamental questions surrounding the mechanism of N-linked glycosylation can now be addressed.     

Protection of protein secondary structure by saccharides of different molecular weights during freeze-drying

The protective effects of saccharides with various molecular weights (glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltoheptaose, dextran 1060, dextran 4900, and dextran 10200) against lyophilization-induced structural perturbation of model proteins (BSA, ovalbumin) were studied. Fourier transform infrared (FT-IR) analysis of the proteins in initial solutions and freeze-dried solids indicated that maltose conferred the greatest protection against secondary structure change. The structure-stabilizing effect of maltooligosaccharides decreased in increasing the number of saccharide units. Larger molecules of dextran also showed a smaller structure-stabilizing effect. Increasing the effective saccharide molecular size by a borate-saccharide complexation reduced the protein structure-stabilizing effect of all of the saccharides except glucose. The results indicate that the larger saccharide molecules, and/or the complex formation with borate ion, reduce the free and accessible hydroxyl groups to interact with and stabilize the protein structure by a water-substitution mechanism.     

Incorporating BODIPY fluorophores into tetrakis(arylethynyl)benzenes

Six structural isomers of a tetrakis(arylethynyl)benzene (TAEB) chromophore functionalized with dibutylamine and BODIPY moieties as the corresponding donor and acceptor units were prepared. To evaluate the effectiveness of the donor group, two TAEB molecules and three structurally related bis(arylethynyl)benzene (BAEB) isomers containing only acceptors were also synthesized. The electronic absorption and emission spectra of each series were examined. Additionally, computational studies were employed to corroborate the relative energy levels and gaps present in each series.     

Chemical and chemoenzymatic synthesis of glycosyl-amino acids and glycopeptides related to Trypanosoma cruzi mucins

This study describes the synthesis of the alpha- and beta-linked N-acetyllactosamine (Galp-beta-1,4-GlcNAc; LacNAc) glycosides of threonine (LacNAc-Thr). LacNAc-a-Thr was prepared by direct chemical coupling of a 2-azido-2-deoxy-lactose disaccharide donor to a suitable partially protected threonine unit. In contrast, stepwise chemical generation of beta-linked N-acetylglucosamine followed by enzymatic galactosylation to give LacNAc-beta-Thr proved effective, whereas use of a 2-azido-2-deoxy-lactose donor in acetonitrile failed to give the desired beta-linked disaccharyl glycoside. This study illustrates that it is possible to overcome the inherent stereoselection for 1,2-trans chemical glycosylation with a GlcNAc donor, and that the well-established preference of bovine beta-1,4-galactosyltransferase for beta-linked acceptor substrates can also be overcome. Using this knowledge, short glycopeptide fragments based on T. cruzi mucin sequences, Thr-Thr-[LacNAcThr]-Thr-Thr-Gly, were synthesised. All LacNAc-based compounds outlined were shown to serve as acceptor substrates for sialylation by T. cruzi trans-sialidase.     

Prearranged Glycosides,Part 12, Intramolecular Mannosylations of Glucose Derivatives via Prearranged Glycosides

A series of prearranged glycosides 5 , 17 , 23 , 28 , 37 , and 41 , having a benzyl‐protected 1‐thiomannosyl donor linked through its positions 2, 3, 4, and 6 via succinate and malonate tethers, respectively, to positions 2, 3, and 6 of a benzyl glucopyranoside acceptor, were prepared by condensation of the respective mannosyl succinates and malonates with suitably protected benzyl glucopyranosides. The prearranged glycosides were intramolecularly coupled under various conditions to give the corresponding tethered (1→4)‐linked disaccharides. The yields and anomer ratios of the products of these couplings were interpreted in terms of the thermodynamic stability of the resulting disaccharides. In the case of prearranged glycoside 17 , having positions 3 of both the donor and the acceptor linked by a succinate tether, a strong dependence of the diastereoselectivity of the intramolecular glycosylation on the activation procedure was observed. All other cases did not show a significant dependence of the outcome of the anomeric configuration in intramolecular glycosylation on the activation procedure or the solvent.     

Luminescent copolymers for applications in multicolor‐light‐emitting devices

Light‐emitting diodes based on organic materials [organic light‐emitting diodes (OLEDs)] have attracted much interest over the past decade. Several different attempts have been made to realize multicolor OLEDs. This article describes a new approach based on energy transfer in a donor/acceptor system. A copolymer containing both donor and acceptor compounds as comonomer units is prepared. The polymer consists of a derivative of a luminescent dye [4‐dicyanmethylene‐2‐methyl‐6‐4H‐pyran (DCM); acceptor compound], which is copolymerized with fluorene (donor compound) to combine the properties of an electroactive polymer with a highly luminescent dye. Photochemical processing is achieved by UV irradiation of this copolymer in the presence of gaseous trialkylsilanes. This reagent selectively saturates the C?C bonds in the DCM comonomer units while leaving the fluorene units essentially unaffected. As a result of the photochemical process, the red electroluminescence of the acceptor compound vanishes, and the blue‐green electroluminescence from the polyfluorene units is recovered. Compared with previous approaches based on polymer blends, this copolymer approach avoids problems associated with phase‐separation phenomena in the active layer of OLEDs. © 2006Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 4317–4327, 2006     

A highly α-stereoselective synthesis of oligosaccharide fragments of the Vi antigen from Salmonella typhi and their antigenic activities

In this paper, a convenient approach to the synthesis of the repeating α-(1→4)-linked N-acetyl galactosaminuronic acid units from the capsular polysaccharide of Salmonella typhi is reported. The exclusively α-stereoselective glycosylation reactions were achieved by using oxazolidinone-protected glycosides as building blocks based on a pre-activation protocol. Di-, tri-, and tetrasaccharides were prepared by this short and efficient approach in high yields. The enzyme-linked immunosorbent assay experiments show that our synthetic tri- and tetrasaccharide had much higher antigenic activities than previously reported ones in the inhibition of antibody binding by the native polysaccharide. The results demonstrate that the antigenic activities of saccharides can be strengthened greatly by increasing the number of acetyl groups present.     

Selective protection of 2-azido-lactose and in situ Ferrier rearrangement during glycosylation: synthesis of a dimeric Lewis X fragment

In our efforts to design new anti-cancer vaccines based on the tumor associated carbohydrate antigen dimeric Lex, we have synthesized the fragment GlcNAc-beta-(1-->3)-Gal-beta-(1-->4)-GlcNAc-beta-(1-->O)-Me. Although it is notoriously difficult to chemically protect the primary OH groups in beta-lactoside derivatives, a 6,6'-disilylated intermediate was prepared in 82% yield. It was converted to a glycosyl acceptor free at O-3' that was glycosylated with a 2-deoxy-2-phthalimido trichloroacetimidate glucosyl donor. This glycosylation required large amounts of TMSOTf to proceed. Such conditions led to the formation of a Ferrier rearrangement glycosylation product. Despite these hurdles, the desired trisaccharide was isolated in 53% yield and easily deprotected in four steps.     

Synthesis of two linear PADRE conjugates bearing a deca- or pentadecasaccharide B epitope as potential synthetic vaccines against Shigella flexneri serotype 2a infection

The blockwise synthesis of the 2-aminoethyl glycosides of a deca- and a pentadecasaccharide made of two and three repeating units, respectively, of the Shigella flexneri serotype 2a specific polysaccharide is reported. The strategy relies on trifluoromethanesulfonic acid mediated glycosylation of a pentasaccharide building block acting as a glycosyl donor and a potential glycoside acceptor. Both targets were made available in amounts large enough for their subsequent conversion into glycoconjugates. Indeed, efficient elongation of the spacer through an acetylthioacetyl moiety and subsequent conjugation onto a Pan HLA DR-binding epitope (PADRE) T-cell-universal peptide resulted in two fully synthetic neoglycopeptides, which will be evaluated as potential vaccines against S. flexneri serotype 2a infections.     

Complex oligosaccharide investigations: synthesis of an octasaccharide incorporating the dimeric Le(x) structure of PSGL-1

The synthesis of an octasaccharide containing the dimeric Le(x) oligosaccharide structure found in PSGL-1 carbohydrate chains is reported. Several approaches were investigated employing regioselective and stereoselective glycosylation procedures, and a novel Lewis(x) trisaccharide donor, 7, was prepared and utilized as a key intermediate building block in the scheme developed for the construction of octasaccharide 3. Toward the preparation of 7, investigations into the influence of different protecting groups upon the relative reactivities of disaccharide acceptor moieties, 25 or 26, and the fucosyl donors, 10 and 11, were conducted using similar glycosylating conditions. Dramatic differences were noted between the effects of electron-donating and electron-withdrawing groups upon the reactivity of the acceptor hydroxyl. A similar effect upon the glycosylating capability of the donor molecule was, likewise, observed. The repeat use of donor 7 was instrumental in the synthesis of the desired dimeric octasaccharide structure 3. The structure and purity of 3 and important intermediates were fully characterized by DQF-COSY, TOCSY, ROESY, and ESI mass spectroscopy.     

Quaternary self-organization of porphyrin and fullerene units by clusterization with gold nanoparticles on SnO2 electrodes for organic solar cells

Novel organic solar cells prepared using quaternary self-organization of porphyrin (donor) and fullerene (acceptor) dye units by clusterization with gold nanoparticles on SnO2 electrodes exhibit the remarkable enhancement of the photoelectrochemical properties relative to the reference systems.     

Galacturonic acid lactones in the synthesis of all trisaccharide repeating units of the zwitterionic polysaccharide Sp1

A modular approach toward the synthesis of all possible trimer repeating units of the type 1 capsular polysaccharide of Streptococcus pneumonia, Sp1, is described. This zwitterionic polysaccharide is built up from trisaccharide repeats, which in turn are composed of two galacturonic acid monomers and a 2,4,6-trideoxy-4-amino-2-acetamido-D-galactose moiety. All monomeric constituents are linked through cis-glycosidic bonds. To overcome the difficulty associated with the efficient stereoselective introduction of the α-galacturonic acid bonds, we have employed galacturonic acid-[3,6]-lactone building blocks. Not only did these building blocks perform well when used as donor galactosides, they were also shown to be reactive acceptor glycosides when equipped with a free hydroxyl function. All three frame-shifted trimer repeats were constructed via highly stereoselective glycosylation reactions, with one exception. The epimeric mixture of trisaccharides, formed in the nonselective glycosylation event, could be readily separated after global deprotection using high performance anion exchange chromatography (HPEAC).     

α:β Selectivity in the synthesis of 3-substituted, 4-methyl umbelliferone glycosides of N-acetyl glucosamine and chitobiose

The influence of phenolic acceptor nucleophilicity; for example, 3-substituted, 4-methylumbelliferones, and glycosyl donor electrophilicity; for example, 3- and 4-substituted N-acetylglucosamines, on glycosylation stereochemistry has been evaluated. In a systematic comparison, the stereochemical outcome as well as the reaction yield appeared to be influenced by the 3- and 4-substituents of the donor as well as the 3-substituent of the aryl acceptor. In the context of synthesizing a fluorogenic substrate for oligosaccharyltransferase, an α-glycoside was desired. Although most acceptor–donor pairs led to predominantly or exclusively the β-glycoside, reaction of the most activated (3,4-di-O-benzyl) donor and the least nucleophilic acceptor (3-Br), resulted in a 1:1 ratio of α,β arylglycosides.     

Co2(CO)6-propargyl cation mediates glycosylation reaction by using thioglycoside

We discovered that the cobalt-propargyl cation can mediate the glycosylation reaction by activating the thioglycoside donor. The glyco-oxacarbenium cation was formed by transferring the thio-aglycone to the cobalt-propargyl cation that was generated from the cobalt-propargylated acceptor in situ via the activating with Lewis acid. The reactivity of the donor (Armed or dis-armed) and the amount of the Lewis acid control the releasing rate of the cobalt-propargyl group.