Combinatorial enzymatic synthesis of heparan sulfate

Escherichia coli K5 heparosan was enzymatically modified by Chen and colleagues to construct a library of heparan sulfate polysaccharides for evaluation, leading to the discovery that a 2-O-sulfoiduronic acid residue is not essential for antithrombin-mediated anticoagulant activity in larger oligosaccharide and polysaccharide structures.     

Formation of lactones from sialylated MUC1 glycopeptides

The tumor-associated carbohydrate antigens TN, T, sialyl TN and sialyl T are expressed on mucins in several epithelial cancers. This has stimulated studies directed towards development of glycopeptide-based anticancer vaccines. Formation of intramolecular lactones involving sialic acid residues and suitably positioned hydroxyl groups in neighboring saccharide moieties is known to occur for glycolipids such as gangliosides. It has been suggested that these lactones are more immunogenic and tumor-specific than their native counterparts and that they might find use as cancer vaccines. We have now investigated if lactonization also occurs for the sialyl TN and T antigens of mucins. It was found that the model compound sialyl T benzyl glycoside , and the glycopeptide Ala-Pro-Asp-Thr-Arg-Pro-Ala from the tandem repeat of the mucin MUC1, in which Thr stands for the 2,3-sialyl-T antigen, lactonized during treatment with glacial acetic acid. Compound gave the 1'--> 2' lactone as the major product and the corresponding 1'--> 4' lactone as the minor product. For glycopeptide the 1'--> 4' lactone constitued the major product, whereas the 1'--> 2' lactone was the minor one. When lactonized was dissolved in water the 1'--> 4' lactone underwent slow hydrolysis, whereas the 1'--> 2' remained stable even after a 30 days incubation. In contrast the corresponding 2,6-sialyl-TN glycopeptide did not lactonize in glacial acetic acid.     

Construction of highly glycosylated mucin-type glycopeptides based on microwave-assisted solid-phase syntheses and enzymatic modifications

A MUC1-related glycopeptide having five core-2 hexasaccharide branches (C330H527N46O207, MW = 8450.9) was synthesized by a new strategy using a combination of microwave-assisted solid-phase synthesis (MA-SPGS) and enzymatic sugar elongation. Synthesis of a key glycopeptide intermediate was best achieved in a combination of PEGA [poly(ethylene glycol)-poly-(N,N-dimethylacrylamide) copolymer] resin and MA-SPGS using glycosylated amino acid building blocks with high speed and high purity. Deprotection of the glycopeptide intermediate and subsequent glycosyltransferase-catalyzed sugar elongations were performed for generation of the additional diversities with the sugar moieties of glycopeptides using beta1,4-galactosyltransferase (beta1,4-GalT) and two kinds of alpha2,3-sialyltransferases [ST3Gal III; alpha2,3-(N)-SiaT and ST3Gal II; alpha2,3-(O)-SiaT]. These reactions proceeded successfully in the presence of 0.2% Triton X-100 to convert the chemically synthesized trisaccharide glycans to disialylated hexasaccharide.     

A combined chemical and enzymatic procedure for the synthesis of 1-deoxynojirimycin and 1-deoxymannojirimycin

1-Deoxynojirimycin and 1-deoxymannojirimycin have been prepared via fructose diphosphate aldolase catalyzed condensation followed by catalytic intramolecular reductive amination.     

Artificial Golgi apparatus: globular protein-like dendrimer facilitates fully automated enzymatic glycan synthesis

Despite the growing importance of synthetic glycans as tools for biological studies and drug discovery, a lack of common methods for the routine synthesis remains a major obstacle. We have developed a new method for automated glycan synthesis that employs the enzymatic approach and a dendrimer as an ideal support within the chemical process. Recovery tests using a hollow fiber ultrafiltration module have revealed that monodisperse G6 (MW = 58 kDa) and G7 (MW = 116 kDa) poly(amidoamine) dendrimers exhibit a similar profile to BSA (MW = 66 kDa). Characteristics of the globular protein-like G7 dendrimer with high solubility and low viscosity in water greatly enhanced throughput and efficiency in automated synthesis while random polyacrylamide-based supports entail significant loss during the repetitive reaction/separation step. The present protocol allowed for the fully automated enzymatic synthesis of sialyl Lewis X tetrasaccharide derivatives over a period of 4 days in 16% overall yield from a simple N-acetyl-d-glucosamine linked to an aminooxy-functionalized G7 dendrimer.     

Automated high-throughput synthesis of artificial glycopeptides. Small-molecule probes for chemical glycobiology

A fully automated method for the synthesis of artificial glycopeptides having two (similar or different) carbon-linked glycosyl moieties on a dipeptide scaffold has been developed. By use of this approach that combines the diversity of peptide/pseudopeptide and glycosides, different glycoside moieties can be incorporated onto the peptide/pseudopeptide backbone in a highly controlled manner. The approach utilizes a stepwise reductive amination with glycoside aldehyde derivatives (model 1) or (ii) glycoside reductive amination followed by glycoside amide bond formation (model 2). Further, an automated method has been utilized in the high-throughput library synthesis of 4 x 96 artificial glycopeptides. These libraries were tested as chemical probes/inhibitors of enzyme systems that convert a glucose moiety into rhamnose prior to incorporation of the rhamnose unit and the conversion of UDP-galactopyranose to UDP-galactofuranose via UDP-galactopyranose mutase enzyme during the biosynthesis of the mycobacterium cell wall.     

Identification of substituted sites on MUC5AC mucin motif peptides after enzymatic O-glycosylation combining beta-elimination and fixed-charge derivatization.

A strategy for determination of O-glycosylation site(s) in glycopeptides has been developed using model compounds obtained by enzymatic glycosylation (by human GaNTase-T2 isoform) on peptides derived from the human MUC5AC mucin tandem repeat motif. The beta-elimination-addition reaction (using dimethylamine and concomitantly ethanethiol) on the formerly glycosylated sites through a Michael-type condensation produced efficient deglycosylation with appropriate chemical modification. After N-terminal derivatization by a phosphonium group, peptide sequencing was then carried out by nanospray tandem mass spectrometry experiments. The highly predictable fragmentation pathways of these fixed-charge phosphonium derivatives enable straightforward recognition of glycosylation site(s) based on the mass increment of +44 Da for originally glycosylated threonine compared to the mass of fragments containing nonglycosylated residues.     

Synthesis of zwitterionic polymer brushes hybrid silica nanoparticles via controlled polymerization for highly efficient enrichment of glycopeptides

Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) materials have been increasingly attractive in glycopeptide enrichment. However, the traditional ZIC-HILIC materials are modified with monolayer zwitterionic molecules on the surface, therefore, the hydrophilicity, detection sensitivity and loading capacity are limited. In this work, we synthesized novel silica nanoparticles with uniform poly(2-(methacryloyloxy)ethyl)dimethyl-(3-sul-fopropyl)ammonium hydroxide (PMSA) brushes grafted onto the surface via reversible addition-fragmentation chain transfer (RAFT) polymerization (denoted as SiO₂-RAFT@PMSA). The resulting SiO₂-RAFT@PMSA nanoparticles demonstrated low detection limit (10 fmol) and high recovery yield (over 88%) for glycopeptide enrichment from tryptic digest of human IgG. The SiO₂-RAFT@PMSA nanoparticles were further applied for the analysis of mouse liver glycoproteome, a total number of 303 unique N-glycosylation sites corresponding to 185 glycoproteins was reliably profiled in three replicate nano-LC–MS/MS runs. Significantly, more glycopeptides were identified than those of nanoparticles, monolayer MSA molecules modified SiO₂@single-MSA and nonuniform multi-layer PMSA brushes coated SiO₂@PMSA, as well as commercial ZIC@HILIC beads and Click Maltose beads. The excellent performance of SiO₂-RAFT@PMSA nanoparticles results from the non-fouling property, a large quantity of functional molecules and suitable link arms provided by uniform PMSA brushes, as well as efficient interaction between glycopeptides and uniform PMSA brushes. It is concluded that the synthesized SiO₂-RAFT@PMSA nanoparticles exhibit great potential in glycoproteome analysis. Moreover, this strategy to modify nanopaticles with uniform polymer brushes via RAFT polymerization can also be explored to design other types of materials for bioseparation application.     

Glycosylation of glycopeptides: a comparison of chemoenzymatic and chemical methods

Glycosylating complex molecules remains a major synthetic challenge, making it hard to explore carbohydrate diversity in biologically active glycosylated natural products and their derivatives. In this paper we compare the efficiency of chemical and enzymatic methods for glycosylation of glycopeptides related to vancomycin and describe the parameters that should be considered in designing synthetic approaches to glycosylated natural product derivatives.     

Combinatorial synthesis of carbon nanotubes by the catalytic chemical vapor deposition method

Summary Bimetallic (Fe-Co) catalyst samples prepared from different precursors over various supports were tested in carbon nanotube (CNT) production. In order to quicken the evaluation of the performance of the catalysts a combinatorial arrangement was used.</o:p>     

Microwave-assisted synthesis of highly functionalized guanidines on soluble polymer support

An efficient method for the N,N′-di(Boc)-protected guanidines containing piperazine and homopiperazine scaffolds has been developed under multi-step microwave irradiation. Followed by alkylation of carbamate-protected guanidines with various alkyl halides is also explored. This protocol proceeds via deprotonation of the acidic N-carbamate hydrogen of the guanidine by sodium hydride on soluble polymer support. In this manner, highly functionalized guanidines were obtained after cleavage from the support. The reaction is tolerant of a wide range of functional groups on both the alkyl halide and guanidine components. In addition, the reaction is sufficiently simple workup by precipitation in each step to yield the substituted guanidines in high purity. In conjunction with microwave irradiation and soluble polymer support, this method provides an efficient route to access highly functionalized guanidines.     

Chemical synthesis of normal and transformed PSA glycopeptides

Chemical syntheses are reported for prostate specific antigen (PSA) N-linked glycopeptide fragments consisting of an uneicosapeptide (residues 27-47 of PSA) with di-, tri-, and tetrabranched N-acetyllactosamine-type glycans. The syntheses involve simultaneous, multiple glycosylations of the corresponding pentasaccharide acceptors prepared from a common trisaccharide precursor. Globally deprotected glycans are aminated and then aspartylated with a hexapeptide, which is then extended using native chemical ligation (NCL). The glycopeptides will be used for the generation of antibodies that may form the basis for a new prostate cancer diagnostic assay.     

An improved chemo-enzymatic synthesis of 1-beta-O-acyl glucuronides: highly chemoselective enzymatic removal of protecting groups from corresponding methyl acetyl derivatives

An improved and widely applicable chemo-enzymatic method for the synthesis of a series of 1-beta-O-acyl glucuronides 5a-f has been developed from the corresponding methyl acetyl derivatives 3a-f, which were stereospecifically synthesized from cesium salts of carboxylic acids 1a-f and methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucopyranuronate (2). Chemoselectivity of lipase AS Amano (LAS) in the hydrolytic removal of O-acetyl groups of 3a-f to provide methyl esters 4a-f was influenced by the nature of their 1-beta-O-acyl groups; high selectivity was evident only for 3b and 3f. Carboxylesterase from Streptomyces rochei (CSR), newly screened as an alternative to LAS, showed much greater chemoselectivity toward the O-acetyl groups than LAS; 3a, 3d, and 3e were chemoselectively hydrolyzed only by CSR. The combination of CSR with LAS yielded better results in the hydrolysis of 3c and 3f than did single usage of CSR. Final deprotection of the methyl ester groups of 4a-f to provide 5a-f was chemoselectively achieved by using lipase from Candida antarctica type B (CAL-B) as well as esterase from porcine liver (PLE), although CAL-B possessed higher chemoselectivity and catalytic efficiency than did PLE. CSR also exhibited high chemoselectivity in the synthesis of (S)-naproxen 1-beta-O-acyl glucopyranoside (7) from its 2,3,4,6-tetra-O-acetyl derivative 6.     

Soap-free synthesis of highly monodisperse magnetic polymer particles with amphoteric initiator

A method applying soap-free emulsion polymerization with an amphoteric initiator, 2,2′-azobis[N-(2-carboxyethyl)-2-2-methyl-propionamidine], is proposed for synthesis of highly monodisperse particles composed of magnetic nanoparticles (Fe₃O₄/γ–Fe₂O₃) and polystyrene. The magnetic nanoparticles were pretreated by surface modification for introducing double bonds onto the particles. In the polymerization, magnetic nanoparticles were continuously supplied to the system for a certain period after the initiation of polymerization at various pH. Dissociation degrees of ionizable groups in the initiator molecules were controlled through pH by changing NH₃ concentrations at a constant NH₄Cl concentration. Selection of suitable pH in the polymerization could produce polymer particles that perfectly incorporated the supplied magnetic nanoparticles. The magnetic polymer particles had a coefficient of variation of size distribution as low as 4.3% with an average diameter of 515 nm and a saturation magnetization of 7.3 emu/g-sample. Electrophoresis measurements indicated that the magnetic polymer particles had an isoelectric point of pH 4.1.     

3-Mercaptopropanol as a traceless linker for chemical and enzymatic synthesis of oligosaccharides

The reducing end of protected carbohydrates can be equipped with a series of aglycones via the photochemical installation of a 3-mercaptoethanol linker. This linker is stable during chemical and enzymatic glycosylation reactions but can be activated readily and efficiently to couple oligosaccharides with different nucleophiles. This approach provides straightforward access to a range of molecules that serve as probes for carbohydrate modifying enzymes. [reaction: see text].     

Combinatorial synthesis of heterocycles: solid-phase synthesis of 2-amino-4(1H)-quinazolinone derivatives

A new solid-phase synthesis of various substituted 2-amino-4(1H)-quinazolinones from a resin bound amine component is described. The amine was readily converted to the corresponding polymer bound S-methylthiopseudourea. Condensation with different substituted isatoic anhydrides afforded 2-amino-4(1H)-quinazolinone derivatives. The method is amenable for combinatorial library generation.