Synthesis of a Novel Unnatural Amino Acid for Protein Incorporation and Click Mediated Conjugation

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ISBN 13 :
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Book Synopsis Synthesis of a Novel Unnatural Amino Acid for Protein Incorporation and Click Mediated Conjugation by : Christopher A. Farley

Download or read book Synthesis of a Novel Unnatural Amino Acid for Protein Incorporation and Click Mediated Conjugation written by Christopher A. Farley and published by . This book was released on 2014 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Unnatural amino acids (UAAs) contain side chains, or R groups, that are not found in the 20 canonical amino acids. These noncanonical groups afford the capability to incorporate powerful chemical capabilities in proteins that are ordinarily unavailable with the naturally-occurring amino acids. Among the most useful moieties to incorporate into proteins are functional groups that can undergo Huisgen [3+2] cycloadditions, or “click,” reactions. This reaction occurs between azides and alkynes, and its mild conditions and high regioselectivity and reactivity make it an ideal process for bioconjugation. Photoreactivity is another useful characteristic that can be conferred to UAAs. Photolabile caging groups can inhibit the function of a protein until brief irradiation with UV light induces an intramolecular rearrangement and its displacement, reestablishing normal function. In this thesis, we propose a synthesis to incorporate both of these moieties into a single UAA.

Unnatural Amino Acid Incorporation and Click Chemistry

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Publisher : GRIN Verlag
ISBN 13 : 3668663467
Total Pages : 53 pages
Book Rating : 4.6/5 (686 download)

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Book Synopsis Unnatural Amino Acid Incorporation and Click Chemistry by :

Download or read book Unnatural Amino Acid Incorporation and Click Chemistry written by and published by GRIN Verlag. This book was released on 2018-03-19 with total page 53 pages. Available in PDF, EPUB and Kindle. Book excerpt: Seminar paper from the year 2014 in the subject Chemistry - Bio-chemistry, grade: 1,0, LMU Munich (Chemie), language: English, abstract: In the present work a modified version of yellow fluorescent protein containing an unnatural structural homologue of the natural amino acid pyrrolysine with a norbornene moiety was produced by expression in Escherichia coli. The incorporation of the unnatural amino acid was achieved by amber stop codon suppression method. A bio-othogonal click reaction was performed, binding a synthetic fluorescent dye to the modified protein. All steps towards necessary for obtaining the genetically modified organism were performed and documented. The artificial amino acid, as well as the dye used in the click reaction were synthetically prepared. The success of the project was demonstrated by LC/MS studies of the products. Fluorescence spectroscopy of click reaction product and the protein was performed, but no conclusive proof of FRET effects could as yet be made. This point remains of interest for future studies.

Novel Strategies for Producing Proteins with Non-proteinogenic Amino Acids

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ISBN 13 :
Total Pages : 129 pages
Book Rating : 4.:/5 (87 download)

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Book Synopsis Novel Strategies for Producing Proteins with Non-proteinogenic Amino Acids by : Ryan Christopher Nangreave

Download or read book Novel Strategies for Producing Proteins with Non-proteinogenic Amino Acids written by Ryan Christopher Nangreave and published by . This book was released on 2013 with total page 129 pages. Available in PDF, EPUB and Kindle. Book excerpt: The biological and chemical diversity of protein structure and function can be greatly expanded by position-specific incorporation of non-natural amino acids bearing a variety of functional groups. Non-cognate amino acids can be incorporated into proteins at specific sites by using orthogonal aminoacyl-tRNA synthetase/tRNA pairs in conjunction with nonsense, rare, or 4-bp codons. There has been considerable progress in developing new types of amino acids, in identifying novel methods of tRNA aminoacylation, and in expanding the genetic code to direct their position. Chemical aminoacylation of tRNAs is accomplished by acylation and ligation of a dinucleotide (pdCpA) to the 3'-terminus of truncated tRNA. This strategy allows the incorporation of a wide range of natural and unnatural amino acids into pre-determined sites, thereby facilitating the study of structure-function relationships in proteins and allowing the investigation of their biological, biochemical and biophysical properties. Described in Chapter 1 is the current methodology for synthesizing aminoacylated suppressor tRNAs. Aminoacylated suppressor tRNACUAs are typically prepared by linking pre-aminoacylated dinucleotides (aminoacyl-pdCpAs) to 74 nucleotide (nt) truncated tRNAs (tRNA-COH) via a T4 RNA ligase mediated reaction. Alternatively, there is another route outlined in Chapter 1 that utilizes a different pre-aminoacylated dinucleotide, AppA. This dinucleotide has been shown to be a suitable substrate for T4 RNA ligase mediated coupling with abbreviated tRNA-COHs for production of 76 nt aminoacyl-tRNACUAs. The synthesized suppressor tRNAs have been shown to participate in protein synthesis in vitro, in an S30 (E. coli) coupled transcription-translation system in which there is a UAG codon in the mRNA at the position corresponding to Val10. Chapter 2 describes the synthesis of two non-proteinogenic amino acids, L-thiothreonine and L-allo-thiothreonine, and their incorporation into predetermined positions of a catalytically competent dihydrofolate reductase (DHFR) analogue lacking cysteine. Here, the elaborated proteins were site-specifically derivitized with a fluorophore at the thiothreonine residue. The synthesis and incorporation of phosphorotyrosine derivatives into DHFR is illustrated in Chapter 3. Three different phosphorylated tyrosine derivatives were prepared: bis-nitrobenzylphosphoro-L-tyrosine, nitrobenzylphosphoro-L-tyrosine, and phosphoro-L-tyrosine. Their ability to participate in a protein synthesis system was also evaluated.

Synthesis of Novel Unnatural Amino Acids

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ISBN 13 :
Total Pages : 400 pages
Book Rating : 4.:/5 (549 download)

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Book Synopsis Synthesis of Novel Unnatural Amino Acids by : Mark Sabat

Download or read book Synthesis of Novel Unnatural Amino Acids written by Mark Sabat and published by . This book was released on 2001 with total page 400 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Non-Natural Amino Acids

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Publisher : Academic Press
ISBN 13 : 0080921639
Total Pages : 334 pages
Book Rating : 4.0/5 (89 download)

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Book Synopsis Non-Natural Amino Acids by :

Download or read book Non-Natural Amino Acids written by and published by Academic Press. This book was released on 2009-07-24 with total page 334 pages. Available in PDF, EPUB and Kindle. Book excerpt: By combining the tools of organic chemistry with those of physical biochemistry and cell biology, Non-Natural Amino Acids aims to provide fundamental insights into how proteins work within the context of complex biological systems of biomedical interest. The critically acclaimed laboratory standard for 40 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. With more than 400 volumes published, each Methods in Enzymology volume presents material that is relevant in today's labs -- truly an essential publication for researchers in all fields of life sciences. Demonstrates how the tools and principles of chemistry combined with the molecules and processes of living cells can be combined to create molecules with new properties and functions found neither in nature nor in the test tube Presents new insights into the molecular mechanisms of complex biological and chemical systems that can be gained by studying the structure and function of non-natural molecules Provides a "one-stop shop" for tried and tested essential techniques, eliminating the need to wade through untested or unreliable methods

A General Strategy for Site-specific Incorporation of Unnatural Amino Acids Into Proteins

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ISBN 13 :
Total Pages : 676 pages
Book Rating : 4.:/5 (33 download)

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Book Synopsis A General Strategy for Site-specific Incorporation of Unnatural Amino Acids Into Proteins by : Spencer Jay Anthony-Cahill

Download or read book A General Strategy for Site-specific Incorporation of Unnatural Amino Acids Into Proteins written by Spencer Jay Anthony-Cahill and published by . This book was released on 1990 with total page 676 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Synthesis of Unnatural Amino Acids for Protein Labeling and Activation

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ISBN 13 :
Total Pages : 217 pages
Book Rating : 4.:/5 (886 download)

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Book Synopsis Synthesis of Unnatural Amino Acids for Protein Labeling and Activation by : Jessica Torres

Download or read book Synthesis of Unnatural Amino Acids for Protein Labeling and Activation written by Jessica Torres and published by . This book was released on 2014 with total page 217 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Aminoacyl-tRNA Synthetase Production for Unnatural Amino Acid Incorporation and Preservation of Linear Expression Templates in Cell-free Protein Synthesis Reactions

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ISBN 13 :
Total Pages : 63 pages
Book Rating : 4.:/5 (975 download)

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Book Synopsis Aminoacyl-tRNA Synthetase Production for Unnatural Amino Acid Incorporation and Preservation of Linear Expression Templates in Cell-free Protein Synthesis Reactions by : Andrew Broadbent

Download or read book Aminoacyl-tRNA Synthetase Production for Unnatural Amino Acid Incorporation and Preservation of Linear Expression Templates in Cell-free Protein Synthesis Reactions written by Andrew Broadbent and published by . This book was released on 2016 with total page 63 pages. Available in PDF, EPUB and Kindle. Book excerpt: The first idea is the adaptation of CFPS to make proteins containing unnatural amino acids. Unnatural amino acids are not found in natural biological proteins; they are synthesized artificially to possess useful properties which are then conferred upon any protein made with them. However, current methods for incorporating unnatural amino acids do not allow incorporation of more than one type of unnatural amino acid into a single protein. This work helps lay the groundwork for the incorporation of different unnatural amino acid types into proteins. It does this by using modified aminoacyl-tRNA synthetases (aaRSs), which are key components in CFPS, to be compatible with unnatural amino acids.

Genetic Incorporation of Unnatural Amino Acids Into Proteins

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ISBN 13 :
Total Pages : 308 pages
Book Rating : 4.:/5 (845 download)

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Book Synopsis Genetic Incorporation of Unnatural Amino Acids Into Proteins by : Isaac Nnamdi Ugwumba

Download or read book Genetic Incorporation of Unnatural Amino Acids Into Proteins written by Isaac Nnamdi Ugwumba and published by . This book was released on 2010 with total page 308 pages. Available in PDF, EPUB and Kindle. Book excerpt: The introduction of unnatural amino acids with novel properties into proteins is potentially a useful tool for understanding and manipulating their functions. The development of an in vitro method for the incorporation of a fluorescent amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco), into proteins is described here.

Unnatural Amino Acids

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Publisher : Humana Press
ISBN 13 : 9781493958887
Total Pages : 409 pages
Book Rating : 4.9/5 (588 download)

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Book Synopsis Unnatural Amino Acids by : Loredano Pollegioni

Download or read book Unnatural Amino Acids written by Loredano Pollegioni and published by Humana Press. This book was released on 2016-08-23 with total page 409 pages. Available in PDF, EPUB and Kindle. Book excerpt: Even though they are present in nature, non-proteinogenic amino acids are usually defined as unnatural or non-natural. Beside their structural diversity, interest in these compounds is due to their occurrence in nature, their biological properties, the analytical aspects, their use as probes, and their incorporation into peptides and proteins, among other reasons. Divided into five convenient sections, Unnatural Amino Acids: Methods and Protocols deals with enzymatic methods used to produce non-natural amino acids, aspects concerning the presence of unnatural amino acids in peptides with antimicrobial properties, genetic incorporation of unnatural amino acids into proteins (yeast and mammalian cells), and detection and quantification of D-amino acids and related enzymes. Written in the highly successful Methods in Molecular BiologyTM series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and accessible, Unnatural Amino Acids: Methods and Protocols serves as an ideal guide for scientists and contributes to directing the attention of researchers to the many fields of growing scientific interest in non-natural amino acids.

Protein Evolution in the Presence of an Unnatural Amino Acid

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ISBN 13 :
Total Pages : 430 pages
Book Rating : 4.:/5 (871 download)

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Book Synopsis Protein Evolution in the Presence of an Unnatural Amino Acid by : Amrita Singh

Download or read book Protein Evolution in the Presence of an Unnatural Amino Acid written by Amrita Singh and published by . This book was released on 2012 with total page 430 pages. Available in PDF, EPUB and Kindle. Book excerpt: The field of protein engineering has been greatly augmented by the expansion of the genetic code using unnatural amino acids as well as the development of cell-free synthesis systems with high protein yield. Cell-free synthesis systems have improved considerably since they were first described almost 40 years ago. Residue specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an amino acid depleted cell-free protein synthesis system that can be used to study residue specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines high protein expression yields with a high level of analog substitution in the target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid-depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo format. We use this amino acid depleted cell-free synthesis system for the directed evolution of streptavidin, a protein that finds wide application in molecular biology and biotechnology. We evolve streptavidin using in vitro compartmentalization in emulsions to bind to desthiobiotin and find, at the conclusion of our experiment, that our evolved streptavidin variants are capable of binding to both biotin and desthiobiotin equally well. We also discover a set of mutations for streptavidin that are potentially powerful stabilizing mutations that we believe will be of great use to the greater research community.

Site-specific Photo-proteolysis of Proteins and Peptides by Incorporation of Unnatural Amino Acid

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ISBN 13 :
Total Pages : 186 pages
Book Rating : 4.:/5 (131 download)

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Book Synopsis Site-specific Photo-proteolysis of Proteins and Peptides by Incorporation of Unnatural Amino Acid by : Portia Duodu

Download or read book Site-specific Photo-proteolysis of Proteins and Peptides by Incorporation of Unnatural Amino Acid written by Portia Duodu and published by . This book was released on 2010 with total page 186 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Unnatural Amino Acids in Proteins for Development of Novel Biochemical Tools

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ISBN 13 :
Total Pages : 150 pages
Book Rating : 4.:/5 (15 download)

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Book Synopsis Unnatural Amino Acids in Proteins for Development of Novel Biochemical Tools by : Jordan Villa

Download or read book Unnatural Amino Acids in Proteins for Development of Novel Biochemical Tools written by Jordan Villa and published by . This book was released on 2015 with total page 150 pages. Available in PDF, EPUB and Kindle. Book excerpt: Unnatural amino acids (UAAs) permit the incorporation of novel biochemical functionalities into proteins. This expansion of the genetic code has enabled enhanced spatial and temporal control of protein activity and conferred novel protein reactivity. This study examines the incorporation of three UAAs: fluoro-tyrosine, ortho-nitrobenzyl-tyrosine, and propargyloxy-phenylalanine towards various applications. Each UAA was successfully incorporated into a protein of interest (GFP or PRMT1) to facilitate the desired manipulation of protein function. The resulting alterations to GFP fluorescence, PRMT1 activity, or immobilization using Glaser-Hay bioconjugation demonstrate the success and practicality of the utilization of UAAs in the development of novel biochemical tools.

Synthesis of Optically Active [alpha]-amino Acids

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Publisher : Pergamon
ISBN 13 : 9780080359397
Total Pages : 410 pages
Book Rating : 4.3/5 (593 download)

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Book Synopsis Synthesis of Optically Active [alpha]-amino Acids by : Robert Michael Williams

Download or read book Synthesis of Optically Active [alpha]-amino Acids written by Robert Michael Williams and published by Pergamon. This book was released on 1989 with total page 410 pages. Available in PDF, EPUB and Kindle. Book excerpt: The purpose of this book is to review and critically evaluate the best new methods to synthesize alpha-amino acids in optically active form. There is so much new literature on amino acid synthesis that the experimentalist will undoubtedly have difficulty in selecting the most appropriate methodology for constructing the amino acid of immediate interest. This book is a guide for steering the scientist through the maze of existing reports on the subject and contains the most up-to-date critical reviews of methods of asymmetric synthesis of amino acids. In areas that are relatively new conceptually and less studied experimentally, an effort has been made to review the most salient works with an eye towards future development. Over 330 schemes and figures are presented with references for rapid visual retrieval of information. The book will be of great value to academic and industrial organic research chemists, especially those concerned with medicinal and agricultural chemistry, as well as to graduate and post graduate students, biochemists and biologists.

Fluorine-containing Amino Acids

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Publisher : John Wiley & Sons
ISBN 13 :
Total Pages : 432 pages
Book Rating : 4.3/5 (91 download)

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Book Synopsis Fluorine-containing Amino Acids by : Valery P. Kukhar

Download or read book Fluorine-containing Amino Acids written by Valery P. Kukhar and published by John Wiley & Sons. This book was released on 1995-04-06 with total page 432 pages. Available in PDF, EPUB and Kindle. Book excerpt: In recent years, organo-fluorine chemistry has made a marked impact on the design and synthesis of a large variety of biologically active molecules, such as steroids, carbohydrates, amines, amino acids, peptides and other natural products. Naturally occurring amino acids play a pivotal role in living systems, and therefore synthetic fluorine-containing amino acids have been of significant interest to researchers working towards the understanding and modification of physiological processes. Fluorine-containing Amino Acids: is the first volume devoted to the synthesis and properties of fluorine-containing amino acids pays special attention to the preparation of enantiomerically pure acids (which are essential to the modern pharmaceutical industry) deals with a rapidly expanding field of research has been written by experienced researchers who are responsible for many developments in the field highlights the interdisciplinary nature of this topic Fluorine-containing Amino Acids is the only dedicated reference in this subject and will be essential for researchers in synthetic organic, peptide, natural product, and medicinal chemistry and biochemistry.

The DESIGN AND SYNTHESIS OF NOVEL UNNATURAL AMINO ACIDS AND THE DESIGN AND SYNTHESIS OF PEPTIDES & PEPTIDOMIMETICS CONTAINING UNNATURAL AMINO ACIDS FOR THE STUDY OF G-PROTEIN COUPLED RECEPTORS.

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ISBN 13 :
Total Pages : 454 pages
Book Rating : 4.:/5 (659 download)

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Book Synopsis The DESIGN AND SYNTHESIS OF NOVEL UNNATURAL AMINO ACIDS AND THE DESIGN AND SYNTHESIS OF PEPTIDES & PEPTIDOMIMETICS CONTAINING UNNATURAL AMINO ACIDS FOR THE STUDY OF G-PROTEIN COUPLED RECEPTORS. by :

Download or read book The DESIGN AND SYNTHESIS OF NOVEL UNNATURAL AMINO ACIDS AND THE DESIGN AND SYNTHESIS OF PEPTIDES & PEPTIDOMIMETICS CONTAINING UNNATURAL AMINO ACIDS FOR THE STUDY OF G-PROTEIN COUPLED RECEPTORS. written by and published by . This book was released on 2010 with total page 454 pages. Available in PDF, EPUB and Kindle. Book excerpt: Nature has gifted peptides as important modulators in the human body, but these types of molecules often have not been favored when we were looking for therapeutic agents. The poor bioavailability, fast degradation and until recent high manufacturing costs of some bioactive peptides lowered their potential usage in the health industry. Under these circumstances, unnatural amino acids were developed as indispensible tools providing enormous support to peptide science. By incorporating proper unnatural amino acids into a peptide or protein, we now can significantly improve peptide's or protein's half-life, cell permeability, bio-distribution, etc. In addition, their potency and receptor/acceptor selectivity could also be enhanced. Site-specific modifications of peptides and proteins under physiological conditions with the use of unnatural amino acids also have been made easier with the advance of biotechnology. Therefore, my research described in this dissertation contributes to the efforts in the development of novel unnatural amino acids. In particular, I have focused on novel methods in the synthesis of anti beta-functionalized gamma, delta-unsaturated amino acids. These amino acids have special interests in peptide chemistry: they can provide conformational constraints to the peptide 3D structures; the beta-functionalization allows the introduction of pharmaceutically interesting side chain groups; and the terminal double bond which is orthogonal to peptide synthesis provides access to further chemical modifications. Two general methodologies for the synthesis of both racemic and optically active anti beta-functionalized gamma, delta--unsaturated amino acids were developed by using the thio-Claisen rearrangement (TCR) reaction. Excellent diastereoselectivies and enantioselectivities were obtained when C2-symmetric chiral auxiliaries were selected to control the stereochemistry outcome. The mechanism and the scope of the TCR reaction were also studied, showing unique advantages in the preparation of these biological interesting amino acids. Another effort of developing angiotensin II type 1 (AT1) receptor biased peptide ligands is also documented in this dissertation. The AT1 receptor is a 7-transmembrane G-protein coupled receptor, which recent researches have shown could be activated through a beta-arrestins only, but G-protein independent, pathway. We synthesized 12 analogs of Sar1,Ile4,Ile8-AngII (SII), and tested them in biological assays, and obtained valuable information for further "perfect" biased ligands design.

Unnatural Amino Acids Designed for Click Chemistry and Their Application to the Modification of Peptides & Nitrene Transfer Reactions Catalyzed by Metalloporphyrins

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ISBN 13 :
Total Pages : 223 pages
Book Rating : 4.:/5 (466 download)

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Book Synopsis Unnatural Amino Acids Designed for Click Chemistry and Their Application to the Modification of Peptides & Nitrene Transfer Reactions Catalyzed by Metalloporphyrins by :

Download or read book Unnatural Amino Acids Designed for Click Chemistry and Their Application to the Modification of Peptides & Nitrene Transfer Reactions Catalyzed by Metalloporphyrins written by and published by . This book was released on 2009 with total page 223 pages. Available in PDF, EPUB and Kindle. Book excerpt: The field of peptidomimetics has rapidly grown into an area of great interest for the design and synthesis of pharmaceutical drug targets. The large array of natural peptides with biological function as well as the growing understanding of the roles of these peptides in biological events has led to a large interest in these compounds as drug candidates. The majority of peptide and peptide-like molecules have not found widespread pharmaceutical utility; however, due to there lability in biological systems. This major drawback leads to the necessity for the development of peptide-like molecules with increased stability under biological conditions. To this end there has been an increased interest in the development of unnatural amino acids for the synthetic modification of peptides and proteins. Presented in this dissertation is the synthesis of a series of unnatural amino acids designed for applications to [3+2] click cycloaddition reactions. It also covers the introduction of these amino acids into the sequence of peptides for the purpose of labeling the peptide with aryl triazole chromophores in an attempt to analyze the electron transfer capabilities of aryl triazoles. The information from the fluorescent studies of these peptides will provide a basis for the design of fluorophoric peptide probes that can be introduced into a peptide at any time under labile conditions. This methodology provides a powerful tool for the analysis of peptide structure and the analysis of peptide-macromolecular interactions.