Development and Use of Chemical and Enzymatic Methods in Both Solution and Solid Phase for the Synthesis of Glycopeptides, Glycoproteins and Oligosaccharides PDF Download

Author: Krista Leah Witte
Publisher:
ISBN: 9780591826135
Category : Binding sites (Biochemistry)
Languages : en
Pages : 444

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Book Description
Presented in this thesis is work on developing an in vitro chemo-enzymatic methodology for the specific construction of glycopeptides and glycoproteins of known sequence, thus providing a route to investigate the importance of displayed oligosaccharides in cellular and molecular communication. Studies of the dependence of subtilisin stability in organic solvents on the buffer counterion are presented. The synthesis and subsequent use of the known slow-binding inhibitor Boc-Ala-Val-Phe-trifluoroketone to determine the effect organic solvents have on the hydrogen bonding in the active site of subtilisin are also discussed. The use of subtilisin in the ligation of glycopeptides is explored. A systematic study is presented in which the permissiveness of subtilisin toward the glycosyl moiety in the enzyme subsites is investigated. In addition a cleavable linker is synthesized and used which provides a direct route via solid-phase to produce a peptide ester suitable for subtilisin ligation. Using the heterogeneous glycoprotein RNase B as a model compound, several novel glycoforms were synthesized enzymatically. This was done by first removing the glycan except for the inner-most GlcNAc to give a homogeneous starting material. Then glycosyltransferases were used to build the unique N-linked glycan moiety. In addition the RNase derivative containing only a single GlcNAc could be proteolyzed at the known site to give peptide S and GlcNAc-protein S. These could be religated under thermodynamic conditions to form the full GlcNAc-RNase. The new methodology was used to incorporate a heavy metal labeled sialic acid into a new RNase glycoform. Heavy atom doping is often used to help elucidate difficult structures and presently there is very little information of the structure of glycan portions of many important glycoproteins.

Author: Valentin Wittmann
Publisher: Springer Science & Business Media
ISBN: 3540367608
Category : Science
Languages : en
Pages : 271

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Book Description
In the last 50 years molecular biology was dominated by the exploration of proteins and nucleic acids. Beside their role in energy metabolism, oligos- charides,which represent thethirdclass ofbiomacromolecules, have received less attention. Today it is well established that oligosaccharides are involved in many important biologicalregulation and recognition processes fromp- tein folding to cell-cell communication. Glycosylation of proteins is the most complexformofco-andposttranslationalmodi'cation. Thedeterminationof structure-function relationships, however, remains dif'cult due to the mic- heterogeneity of glycoproteins that exist in many different glycoforms. Thus chemical synthesis of glycoproteins and glycopeptides with de'ned glycan structures plays a pivotal role for the detailed determination of the role of protein glycosylation. This topic is covered by the ?rst two chapters of this bookdealingwiththechemicaland enzymatic synthesis ofglycopeptides and glycoproteins. The third chapter describes the construction of glycopeptide andglycoproteinmimetics containingnon-naturalstructuralelements. These so-calledneoglycopeptidesandneoglycoproteins,respectively,canprovide- sight on the importance of distinct structural elements on biological activity andmayhaveimproved propertiessuchasanincreased stability. Theappli- tion of synthetic glycopeptides, in many cases at the clinical level, as vaccines forbothcancerandHIVisthesubjectofthefourthchapter. Glycopeptide antibiotics are glycosylated secondary metabolites of bacteria and fungi that are synthesized by non-ribosomal peptide synthetases. Some of them serve as antibiotics of last resort in the treatment of nosocomial infections with enterococci and methicillin-resistant Staphylococcus aureus (MRSA) strains. Their structure, biosynthesis, and mode of action are summarized in the ?fth chapter. The last chapter covers current methods for the determination of high-resolution structures of glycopeptides and glycoproteins mainly based onNMRspectroscopy, X-raycrystallography,and molecular modeling.

Author: David G. Large
Publisher: CRC Press
ISBN: 9780824795313
Category : Medical
Languages : en
Pages : 786

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Book Description
"Presents state-of-the-art methods for the synthesis, analysis, and conformational investigation of glycoproteins and glycopeptides. Discusses the history of glycopeptide synthesis, therapeutic applications, and the future of research."

Author: Yu-Ying Yang
Publisher:
ISBN: 9780549110033
Category : Glycopeptides
Languages : en
Pages : 400

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Book Description
Based on the same principle of native chemical ligation and auxiliary-assisted ligation, we recently developed the "sugar-assisted ligation" method for glycopeptide and glycoprotein synthesis. In this method, we take the advantage of sugar by anchoring a thiol handle onto the C2 position of sugar moiety to facilitate the transthioesterification followed by S to N acyl transfer. The sugar moiety utilizes its rigid conformation to bring the two reacting groups of thioester intermediate into close proximity to facilitate acyl transfer. The ligation junction in sugar-assisted ligation tolerates a broad spectrum of amino acids with high preference to less hindered residues (Gly, Ala) and those with side chain that can serve as a general base (His, Asp) in the ligation pathway. The utility of this ligation method has been demonstrated in the successful synthesis of [beta]-O-linked glycopeptides, N-linked glycopeptides, [alpha]-O-linked glycopeptide and "mucin-type" [alpha]-O-linked glycoproteins. Enzymatic peptide ligation using subtilisin BPN' 8397 was also exploited here for the synthesis of a-linked glycopeptides. Under the kinetically-controlled conditions, we successfully performed the enzymatic ligations where N-linked, [alpha]-O-linked or [alpha]-S-linked glycopeptides reacted as the acyl donors. In comparison with N-linked glycopeptides, [alpha]-linked glycopeptide, where the sugar moieties located at their P2 positions, showed superior enzymatic ligation efficiencies. On the other hand, to further explore the utility of expressed protein ligation method on the synthesis of glycosylated hIL-2, we continued our previous efforts here to synthesize the native hIL-2 glycoform. We utilized the highly-specific TEV protease to remove the affinity tag from recombinant His-tagged hIL-2, and unmasked the N-terminal cysteine for ligation. Various glycopeptide thioesters, which were synthesized by chemical or chemo-enzymatic methods, were ligated to truncated IL-2 fragment (residues 6-133). The ligation studies suggested that moving the sugar moiety farther away from the ligation junction is able to improve the ligation yield. Besides, in order to isolate the ligation product from other protein mixtures, we developed and tested several affinity chromatography methods to purify glycosylated human IL-2.

Author: Shang-Cheng Hung
Publisher: John Wiley & Sons
ISBN: 1119006473
Category : Science
Languages : en
Pages : 769

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Book Description
This book is a comprehensive and concise review on principles, strategies, and crucial advances in glycochemistry. It focuses on synthesis and practical applications and emphasizes state-of-the-art approaches to the assembly and design of sugars. • Provides detailed discussion on specific topics like oligosaccharide assembly and design of sugars, techniques in glycoconjugate preparation, multivalency, and carbohydrate-based drug design • Uses notable examples, like solution-based one-pot methods and automated methods for sugar assembly, to illustrate important concepts and advances in a rapidly emerging field • Discusses practical applications of carbohydrates, like medicine, therapeutics, drug and vaccine development

Author: Peter H. Seeberger
Publisher: John Wiley & Sons
ISBN: 0471460729
Category : Science
Languages : en
Pages : 321

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Book Description
Solid-phase synthesis of carbohydrates presents unique challenges to synthetic chemists and currently represents one of the hottest areas of research in bioorganic chemistry. Solid Support Oligosaccharide Synthesis and Combinatorial Carbohydrate Libraries addresses the exciting expectation that solid-phase assembly of oligosaccharides will have a fundamental impact on the field of glycobiology. This publication details the methodologies currently investigated for the attachment of carbohydrates to beads, synthesis including coupling strategies, and removal of the product from beads. With chapters written by eminent contributing authors, the material explores different synthetic strategies, glycosylation protocols, the use of solid supports versus soluble polymeric supports, and "on-resin" analytical methods. Tremendous progress in the field has set the stage for Solid Support Oligosaccharide Synthesis and Combinatorial Carbohydrate Libraries to provide a wealth of information on such topics as: Specific oligosaccharide structures used in signal transduction processes Preparation and screening of glycopeptide libraries Solid-phase carbohydrate synthesis

Author: Stephen Hanessian
Publisher: CRC Press
ISBN: 9780824798024
Category : Science
Languages : en
Pages : 668

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Book Description
Detailing commonly used methods and procedures, this reference discusses the reactions and derivative forms of carbohydrates. Preparative Carbohydrate Chemistry covers the formation, cleavage, and reactions of derivatives and illustrates bond-forming reactions of SN2 types, free radicals, chain extensions, and branching. The contents include: sugar derivatives; selected reactions in carbohydrate chemistry; chemical synthesis of oligosaccharides and O-and N -glycosyl compounds; enzymatic synthesis of sialic acid, KDO, and related deoxyulosonic acids, and of oligosaccharides; synthesis of -glycosyl compounds; carbocycles from carbohydrates; and total synthesis of sugars from non-sugars. This authoritative reference offers relevant chapters on reactions and derivative forms of carbohydrates, including commonly used methods as well as new experimental procedures. It also contains insightful chapter commentaries and succinct topic histories.

Author: Stephen M. Dean
Publisher:
ISBN:
Category : Glycopeptides
Languages : en
Pages : 284

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Book Description
The hypothesis that CD2, a glycoprotein that requires glycosylation for folding, could be made to fold by bioorthogonal chemistry was investigated by applying glycosyl iodoacetamides to CD2 variants bearing a thiol handle install by cysteine mutation at the glycosylation site. Unfortunately, folding could not be induced by chemical glycosylation, suggesting that the linkage and proximity of the sugar are crucial to achieving stabilization.

Author: Roslyn M. Bill
Publisher: Springer Science & Business Media
ISBN: 9780792383376
Category : Medical
Languages : en
Pages : 536

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Book Description
Protein Glycosylation provides clear, up-to-date, and integrated coverage of key topics in this field. Particular emphasis is placed on the biosynthetic pathways that result in a wide variety of identified protein-bound oligosaccharides. Protein Glycosylation begins with an overview of the chemical structures of mono- and oligosaccharides, to provide a scientific basis for the later chapters. The book includes discussions on the purification, function, and enzyme kinetics of selected glycosidases and glycotransferases, as well as a review of the roles of oligosaccharides in glycoprotein function and the in vivo role of glycoproteins themselves. Finally, the in vitro synthesis of glycoproteins is presented, together with future directions in glycobiology. Protein Glycosylation serves as an excellent text for upper-level undergraduate and graduate students as well as a reference for those scientists whose training is not in glycobiology but who are moving into this field.

Author: Kwang-Seuk Ko
Publisher:
ISBN:
Category :
Languages : en
Pages : 520

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Book Description
This dissertation focuses on two specific aspects of the chemistry of carbohydrates. The first is the solid- and fluorous-phase synthesis of oligosaccharides and their use in forming microarrays. The solid- and fluorous-phase synthesis of oligosaccharides has emerged as a powerful method for the development of improvements in terms of synthesis speed and efficiency. The advantage of solid phase synthesis can be obtained very high yields by use of excess donors, ease of purification, and synthesis automation. Moreover, there are still unsolvable matters in solid-phase synthesis of oligosaccharides, which have to use a way over excess of amount donor group in each reaction. The fluorou-phase synthesis has developed the new additional overcome method to synthesize oligosaccharide, which allows to use in the solution phase synthesis of oligosaccharides and to use equivalent donor group to the acceptor group. A new fluorous tag-assisted solution phase strategy allows the rapid, and potentially automated, modular synthesis of carbohydrates and their use in forming microarrays. A new simpler concept in microarray formation is based on noncovalent fluorous-based interactions. A fluorous tail is designed not only to aid in saccharide purification but also to allow direct formation of carbohydrate microarrays on fluorous-derivatized glass slides for biological screening with lectins, such as concanavalin A. The noncovalent interactions in the fluorous-based array are even strong enough to withstand the detergents used in assays with the Erythrina crystagalli lectin. Additionally, the utility of benzyl carbonate protecting groups on fucose building blocks for the formation of [Alpha]-linkages is demonstrated. The second topic is the strategies for the chemoenzymatic synthesis of deoxysugar nucleotides and stable activated sugar mimics. The carbaglucose-1-phosphate and deoxyglucose-1-phosphate present evidence that these classes of enzymes can exercise kinetic discrimination in choosing carbohydrates of comparable binding affinity for catalytic turnover. Synthetic 6-deoxy, 4-deoxy, and 3-deoxyglucose-1-phosphate and the natural substrate glucose-1-phosphate were tested with a representative prokaryotic glucose-1-phosphate uridylyltransferase [EC 2.7.7.9] from Escherichia coli, which is also known to accept thymidine triphosphate, and the comparable eukaryotic enzyme from yeast. The results are reported the first synthesis of the carbocyclic version of the most common naturally occurring sugar-1-phosphate, glucose-1-phosphate, and its evaluation with bacterial and eukaryotic sugar nucleotidyltransferases. In contrast to results with the eukaryotic enzyme, the carbaglucose-1-phosphate serves as a substrate for the bacterial enzyme to provide the carbocyclic uridinediphosphoglucose. This result demonstrates the first chemoenzymatic strategy to this class of glycosyltransferase inhibitors.