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1.
Angew Chem Int Ed Engl ; 57(51): 16638-16642, 2018 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-30375138

RESUMEN

For decades, researchers have endeavored to develop a general automated system to synthesize oligosaccharides that is comparable to the preparation of oligonucleotides and oligopeptides by commercially available machines. Inspired by the success of automated oligosaccharide synthesis through chemical glycosylation, a fully automated system is reported for oligosaccharides synthesis through enzymatic glycosylation in aqueous solution. The designed system is based on the use of a thermosensitive polymer and a commercially available peptide synthesizer. This study represents a proof-of-concept demonstration that the enzymatic synthesis of oligosaccharides can be achieved in an automated manner using a commercially available peptide synthesizer.


Asunto(s)
Glicosiltransferasas/metabolismo , Oligosacáridos/biosíntesis , Péptidos/metabolismo , Automatización , Glicosilación , Glicosiltransferasas/química , Estructura Molecular , Oligosacáridos/química , Péptidos/química
2.
Chem Rev ; 118(17): 8151-8187, 2018 09 12.
Artículo en Inglés | MEDLINE | ID: mdl-30011195

RESUMEN

Oligosaccharides together with oligonucleotides and oligopeptides comprise the three major classes of natural biopolymers. Automated systems for oligonucleotide and oligopeptide synthesis have significantly advanced developments in biological science by allowing nonspecialists to rapidly and easily access these biopolymers. Researchers have endeavored for decades to develop a comparable general automated system to synthesize oligosaccharides. Such a system would have a revolutionary impact on the understanding of the roles of glycans in biological systems. The main challenge to achieving automated synthesis is the lack of general synthetic methods for routine synthesis of glycans. Currently, the two main methods to access homogeneous glycans and glycoconjugates are chemical synthesis and enzymatic synthesis. Enzymatic glycosylation can proceed stereo- and regiospecifically without protecting group manipulations. Moreover, the reaction conditions of enzyme-catalyzed glycosylations are extremely mild when compared to chemical glycosylations. Over the past few years methodology toward the automated chemical synthesis of oligosaccharides has been developed. Conversely, while automated enzymatic synthesis is conceptually possible, it is not as well developed. The goal of this survey is to provide a foundation on which continued technological advancements can be made to promote the automated enzymatic synthesis of oligosaccharides.


Asunto(s)
Automatización , Técnicas de Química Sintética/métodos , Glicoconjugados/síntesis química , Glicosiltransferasas/química , Oligosacáridos/síntesis química , Secuencia de Carbohidratos , Catálisis , Glicoconjugados/química , Glicosilación , Oligosacáridos/química , Estereoisomerismo
3.
Bioorg Med Chem Lett ; 27(18): 4285-4287, 2017 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-28844388

RESUMEN

A cation exchange assisted binding-elution (BE) strategy for enzymatic synthesis of human milk oligosaccharides (HMOs) was developed. An amino linker was used to provide the cation ion under acidic condition which can be readily bound to cation exchange resin and then eluted off by saturated ammonium bicarbonate. Ammonium bicarbonate in the collections was easily removed by vacuum evaporation. This strategy circumvented the incompatible issue between glycosyltransferases and solid support or large polymers, and no purification was needed for intermediate products. With current approach, polyLacNAc backbones of HMOs and fucosylated HMOs were synthesized smoothly.


Asunto(s)
Glicosiltransferasas/metabolismo , Leche Humana/química , Oligosacáridos/biosíntesis , Bicarbonatos/química , Bicarbonatos/metabolismo , Cationes/química , Cationes/metabolismo , Relación Dosis-Respuesta a Droga , Glicosiltransferasas/química , Humanos , Leche Humana/metabolismo , Estructura Molecular , Oligosacáridos/química , Relación Estructura-Actividad
4.
Anal Chim Acta ; 962: 32-40, 2017 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-28231878

RESUMEN

Reductive amination is an indispensable method for glycomic analysis, as it tremendously facilitates glycan characterization and quantification by coupling functional tags at the reducing ends of glycans. However, traditional in-solution derivatization based approach for the preparation of reductively aminated glycans is quite tedious and time-consuming. Here, a simpler and more efficient strategy termed solid-phase reductive amination was investigated. The general concept underlying this new approach is to streamline glycan extraction, derivatization, and purification on non-porous graphitized carbon sorbents. Neutral and sialylated standard glycans were utilized to test the feasibility of the solid-phase method. As results, almost complete labeling of those glycans with four common labels of aniline, 2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA) and 2-amino-N-(2-aminoethyl)-benzamide (AEAB) was obtained, and negligible desialylation occurred during sample preparation. The labeled glycans derived from glycoproteins showed excellent reproducibility in high performance liquid chromatography (HPLC) and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Direct comparisons based on fluorescent absorbance and relative quantification using isotopic labeling demonstrated that the solid-phase strategy enabled 20-30% increase in sample recovery. In short, the solid-phase strategy is simple, reproducible, efficient, and sensitive for glycan analysis. This method was also successfully applied for N-glycan profiling of HEK 293 cells with MALDI-TOF MS, showing its attractive application in the high-throughput analysis of mammalian glycome.


Asunto(s)
Glicómica/métodos , Aminación , Células HEK293 , Humanos , Metilación , Oxidación-Reducción , Polisacáridos/química , Polisacáridos/metabolismo
5.
J Proteomics ; 146: 90-8, 2016 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-27282921

RESUMEN

UNLABELLED: Core-fucosylation (CF) plays important roles in regulating biological processes in eukaryotes. Alterations of CF-glycosites or CF-glycans in bodily fluids correlate with cancer development. Therefore, global research of protein core-fucosylation with an emphasis on proteomics can explain pathogenic and metastasis mechanisms and aid in the discovery of new potential biomarkers for early clinical diagnosis. In this study, a precise and high throughput method was established to identify CF-glycosites from human plasma. We found that alternating HCD and ETD fragmentation (AHEF) can provide a complementary method to discover CF-glycosites. A total of 407 CF-glycosites among 267 CF-glycoproteins were identified in a mixed sample made from six normal human plasma samples. Among the 407 CF-glycosites, 10 are without the N-X-S/T/C consensus motif, representing 2.5% of the total number identified. All identified CF-glycopeptide results from HCD and ETD fragmentation were filtered with neutral loss peaks and characteristic ions of GlcNAc from HCD spectra, which assured the credibility of the results. This study provides an effective method for CF-glycosites identification and a valuable biomarker reference for clinical research. BIOLOGICAL SIGNIFICANCE: CF-glycosytion plays an important role in regulating biological processes in eukaryotes. Alterations of the glycosites and attached CF-glycans are frequently observed in various types of cancers. Thus, it is crucial to develop a strategy for mapping human CF-glycosylation. Here, we developed a complementary method via alternating HCD and ETD fragmentation (AHEF) to analyze CF-glycoproteins. This strategy reveals an excellent complementarity of HCD and ETD in the analysis of CF-glycoproteins, and provides a valuable biomarker reference for clinical research.


Asunto(s)
Proteínas Sanguíneas/análisis , Fucosa/metabolismo , Glicopéptidos/análisis , Proteómica/métodos , Secuencias de Aminoácidos , Proteínas Sanguíneas/química , Secuencia de Consenso , Glicosilación , Humanos , Fragmentos de Péptidos/análisis
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