Author: Gabriela De Almeida Publisher: ISBN: Category : Languages : en Pages : 261
Book Description
The ability to study biomolecules within their naturally occurring context is often complicated by the complexity of biological systems. To overcome this obstacle, methods to selectively tag the biomolecule of interest with a fluorophore or affinity handle have been developed; once tagged, the desired biomolecule has a unique signal that allows it to be identified and studied within the complex biological setting. Ideally, these tags are small and non-perturbing so as not to modify the behavior of the biomolecule. However, while these design rules for bioorthogonal reagents seem simple, translating the rules into useful tools is challenging. In the first chapter of this dissertation, I outline current methods to label biomolecules with small tags. I begin by describing genetically-encoded tags that are useful for protein-based studies. Next, the field of bioorthogonal chemistry and the ability to tag non-proteinaceous biomolecules is introduced. An overview of various chemical reporters used for live cell- and whole organism- studies is presented, and the future requirements for the field are discussed. In Chapter 2, I describe a novel class of molecule for azide-based bioorthogonal tagging, the thiacycloalkyne. By introducing an endocyclic sulfur atom, the strain of the cycloalkynes can be fine-tuned. Taking advantage of this strategy, a previously reported thiacycloheptyne, TMTH, is shown to be exceptionally reactive with azides and to have promising qualities as a bioorthogonal reagent. In Chapter 3, I extend the endocyclic large heteroatom strategy to the design of dibenzoselenacycloheptynes. However, while these compounds can be trapped with azides in situ, they behave as triplet diradicals in the absence of a trap. In Chapter 4, I focus on the development of a new strategy to bioorthogonally label glycans on specific cell-types. By creating trivalent reagents that comprise of a targeting aptamer, an azide-reactive cyclooctyne, and a fluorophore, I show that cell-selective glycan labeling can be achieved. These simple-to-use, modular reagents will be helpful in studying the geneses and outcomes of cell-specific glycosylation changes. Finally, in Chapter 5, I present work towards the development of a novel method for single-step, non-toxic protein tagging. This method is based on the reaction between 1,2-aminothiols and cyanobenzothiazole, which has previously been shown to be selective, bio-friendly, and fast. Both phage display and rational peptide design are implemented in attempts to find a short peptide tag that can covalently react with cyanobenzothiazole, but only high-affinity binding and reversible reactions are achieved.
Author: Publisher: Academic Press ISBN: 0128211555 Category : Science Languages : en Pages : 526
Book Description
Chemical Tools for Imaging, Manipulating, and Tracking Biological Systems: Diverse Chemical, Optical and Bioorthogonal Methods, Volume 641 in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. Chapters in this new release include caged cyclopropanes with improved tetrazine ligation kinetics, an analysis of metabolically labeled inositol phosphate messengers by NMR, cell-permeant caged inositol pyrophosphates for probing ß-cells, imaging phospholipase D activity with clickable alcohols via transphosphatidylation, fluorescent biorthogonal labeling of class B GPCRs in live cells, near-infrared photoactivatable nitric oxide donors with integrated photoacoustic monitoring, and much more. Provides the authority and expertise of leading contributors from an international board of authors Presents the latest release in the Methods in Enzymology series Includes the latest information on retinoid signaling pathways
Author: Ellen Sletten Publisher: ISBN: Category : Languages : en Pages : 1408
Book Description
Bioorthogonal is defined as not interfering or interacting with biology. Chemical reactions that are bioorthogonal have recently become valuable tools to visualize biomolecules in their native environments, particularly those that are not amenable to traditional genetic modification. The field of bioorthogonal chemistry is rather young, with the first published account of the term bioorthogonal in 2003, yet it is expanding at a rapid rate. The roots of this unique subset of chemistry are in classic protein modification and subsequent bioconjugation efforts to obtain uniformly and site-specifically functionalized proteins. These studies are highlighted in Chapter 1. Chapter 2 opens with a summary of the bioorthogonal chemical reporter strategy, a two-step approach where a bioorthogonal functional group is installed into a biomolecule of interest, most often using endogenous metabolic machinery, and detected through a secondary covalent reaction with an appropriately functionalized chemical partner. It is this chemical reporter strategy that empowers bioorthogonal chemistry and allows for a wide variety of biological species to be assayed. Chapter 2 proceeds to outline the discovery of the Staudinger ligation, the first chemical reaction developed for use in the bioorthogonal chemical reporter strategy. The Staudinger ligation employed the azide as the chemical reporter group and, since its debut in 2000, many laboratories have capitalized on the exquisite qualities of the azide (small, abiotic, kinetically stable) that make it a versatile chemical reporter group. The success of the azide prompted the development of other bioorthogonal chemistries for this functional group. One of these chemistries, Cu-free click chemistry, is the 1,3-dipolar cycloaddition between cyclooctynes and azides. The cycloaddition is promoted at physiological conditions by the 1̃8 kcal/mol of ring strain contained within cyclooctyne, and further modifications to the cyclooctyne reagents have lead to increased reactivity through augmentation of the ring strain or optimization of orbital overlap. When I began my graduate work, a difluorinated cyclooctyne (DIFO), which was 60-fold more reactive than other existing bioorthogonal chemistries, had just been synthesized and employed for labeling azides on live cells and within living mice. DIFO performed very well on cultured cells, but it was outperformed by the slower Staudinger ligation in the more complex environment of the mouse. We hypothesized that DIFO was too hydrophobic to be effective in mice and designed a more hydrophilic cyclooctyne reagent, a dimethoxyazacyclooctyne (DIMAC). DIMAC was synthesized in nine steps in a 10% overall yield (Chapter 3). As predicted, DIMAC displayed reaction kinetics similar to early generation cyclooctynes, but exhibited improved water-solubility. Consequently, DIMAC labeled cell-surface azides with comparable efficiencies to the early generation cyclooctynes but greater signal-to-noise ratios were achieved due to minimal background staining. Encouraged by these results, we assayed the ability for DIMAC to label azides in living mice and found that DIMAC was able to modify azides in vivo with moderate signal over background. However, the Staudinger ligation was still the superior bioorthogonal reaction for labeling azides in vivo. Our results collectively indicated that both hydrophilicity and reactivity are important qualities when optimizing the cyclooctynes for in vivo reaction with azides (Chapter 4). We were also interested in modifying DIMAC so that it would become fluorescent upon reaction with an azide. Previous work in the lab had established that fluorogenic reagents could be easily created if a functional group was cleaved from the molecule upon reaction with an azide. We envisioned a leaving group could be engineered into the azacyclooctyne scaffold by strategically positioning a labile functional group across the ring from a nitrogen atom. The cyclooctyne structure should be stable, as it is rigid and intramolecular reactions are not favorable. However, upon reaction with an azide, a significant amount of strain is liberated and the intramolecular reaction should readily occur. Efforts toward the synthesis of this modified DIMAC reagent are chronicled in Chapter 5. Chapter 6 is a very short account of our early work to use DIFO-based reagents for proteomics. The results contained in this chapter are preliminary and further endeavors towards this goal are underway by others within the group. Chapters 7, 8 and 9 are devoted to strategies to increase the second-order rate constant of Cu-free click chemistry. In Chapter 7, various routes toward a tetrafluorinated cyclooctyne are outlined, although none of them successfully yielded this putatively highly reactive cyclooctyne. Chapter 8 describes the synthesis of a difluorobenzocyclooctyne (DIFBO), which is more reactive than DIFO, but unstable due to its propensity to form trimer products. However, DIFBO can be kinetically stabilized by encapsulation in beta-cyclodextrin. Only beta-cyclodextrin and not the smaller (alpha) or larger (gamma) cyclodextrins were able to protect DIFBO. We did observe an intriguing result when complexation with the larger gamma-cyclodextrin was attempted. It appears as though two DIFBO molecules can fit inside the gamma-cyclodextrin and dimeric products, which were not apparent in the absence of gamma-cyclodextrin, were observed. We hypothesized that all oligomer products of DIFBO were derived from a common cyclobutadiene intermediate. While DIFBO was chemically interesting, it was not a useful reagent for labeling azides in biological settings. Thus, Chapter 9 is devoted to the modification of DIFBO, with the aim of identifying a reactive yet stable cyclooctyne. The data from Chapter 9 suggest we are rapidly approaching the reactivity/stability limit for cyclooctyne reagents. The results contained within Chapters 7-9 indicated that it was time to explore other bioorthogonal chemistries. When embarking on the development of a new bioorthogonal chemical reaction, we aimed to explore unrepresented reactivity space, such that the new reaction would be orthogonal to existing bioorthogonal chemistries. We became attracted to the highly strained hydrocarbon quadricyclane and performed a screen to find a suitable reactive partner for this potential chemical reporter group (Chapter 10). Through this analysis, we discovered that quadricyclane cleanly reacts with Ni bis(dithiolene) reagents and this transformation appeared to be a good prototype for a new bioorthogonal chemical reaction. After a thorough mechanistic investigation and many rounds of modification to the Ni bis(dithiolene) species, a nickel complex with suitable reaction kinetics, water-solubility, and stability was obtained (Chapter 11). Gratifyingly, this Ni bis(dithiolene) reagent selectively modified quadricyclane-labeled bovine serum albumin, even in the presence of cell lysate (Chapter 12). Other results in Chapter 12 highlight that this new bioorthogonal ligation reaction is indeed orthogonal to Cu-free click chemistry as well as oxime ligation chemistry. Additionally, quadricyclane-dependent labeling is observed on live cells, although further optimization is necessary. The final chapter of this dissertation outlines the current state of the field. There are now many methods to modify biomolecules including several new, although relatively untested, bioorthogonal chemistries. The rapid pace of this field makes it an exciting time to be pursuing bioorthogonal chemistry.
Author: Samantha Jo Boyd Publisher: ISBN: 9781369116120 Category : Languages : en Pages : 177
Book Description
My doctoral research under the advisement of Joseph Fox at the University of Delaware has focused on exploiting the unique reactivity of trans-cyclooctenes (TCOs) in synthetic design, reaction method development, and bioorthogonal applications. Chapter 1 is focused on the importance of trans-cyclooctenes in synthesis and biorthogonal chemistry. I have outlined a concise history of the synthesis of trans-cyclooctene and more complex derivatives. I have also briefly described the more recent development of select bioorthogonal reactions. In this chapter, I have focused on the TCO-tetrazine ligation, a bioorthogonal reaction first described by the Fox group in 2008. This reaction has been utilized in a variety of applications spanning cell imaging, nuclear medicine, and materials science. Synthesizing trans-cyclooctenes has not always been a trivial process. Within the past 10 years, the Fox group has developed a closed-loop flow system to produce multi-gram quantities of a variety of trans-cyclooctene derivatives. The requirement of expansive and non-conventional equipment, however, has limited adoption by a broader audience. I sought to develop a system that could be used globally by the wider scientific community, in particular those involved in the biological applications of these useful molecules. In Chapter 2, I discuss improvements to the current flow chemistry system as well as a novel ‘no-flow’ photochemistry apparatus. The molecules synthesized have been used in a variety of reactions described in Chapter 1. I have also employed UV-Vis stopped-flow kinetics to measure reaction rates of various TCO-tetrazine partners under aqueous conditions. Understanding reaction rates continues to provide the tools necessary to improve molecular design. I also discuss the development of a one-pot diastereoselective reduction used to selectively synthesize axial substituted trans-cyclooctenes. Lastly, I briefly describe utilizing Trolox to prevent trans-cyclooctene isomerization in the presence of thiols. Finally, trans-cyclooctene derivatives have proven to be useful intermediates for target-directed synthesis due to the strain-driven reactivity of the trans alkene. trans-Cyclooctenes possess an inherent sense of planar chirality which I have exploited in two different reaction methodology developments. In Chapter 3, I describe the development of a transannular etherification reaction. This method has shown to be both regio- and diastereoselective, providing efficient access to more complex bicyclic moieties. One complication is accessing the necessary trans-cyclooctene diastereomer in useful quantities. To this end, I have developed a diastereoselective photoisomerization process that uses catalytic chiral sensitizer and takes advantage of the inherent sense of planar chirality. Efforts towards the natural product laureoxanyne are briefly described. I conclude with the development of new trans -cyclooctene reagents used to probe oxidative stress in proteins.
Author: Luca D. D'Andrea Publisher: John Wiley & Sons ISBN: 111904409X Category : Science Languages : en Pages : 584
Book Description
Presenting a wide array of information on chemical ligation – one of the more powerful tools for protein and peptide synthesis – this book helps readers understand key methodologies and applications that protein therapeutic synthesis, drug discovery, and molecular imaging. • Moves from fundamental to applied aspects, so that novice readers can follow the entire book and apply these reactions in the lab • Presents a wide array of information on chemical ligation reactions, otherwise scattered across the literature, into one source • Features comprehensive and multidisciplinary coverage that goes from basics to advanced topics • Helps researchers choose the right chemical ligation technique for their needs
Author: Milan Vrabel Publisher: Springer ISBN: 3319296868 Category : Science Languages : en Pages : 159
Book Description
The series Topics in Current Chemistry Collections presents critical reviews from the journal Topics in Current Chemistry organized in topical volumes. The scope of coverage is all areas of chemical science including the interfaces with related disciplines such as biology, medicine and materials science. The goal of each thematic volume is to give the non-specialist reader, whether in academia or industry, a comprehensive insight into an area where new research is emerging which is of interest to a larger scientific audience. Each review within the volume critically surveys one aspect of that topic and places it within the context of the volume as a whole. The most significant developments of the last 5 to 10 years are presented using selected examples to illustrate the principles discussed. The coverage is not intended to be an exhaustive summary of the field or include large quantities of data, but should rather be conceptual, concentrating on the methodological thinking that will allow the non-specialist reader to understand the information presented. Contributions also offer an outlook on potential future developments in the field.
Author: Xuechen Li Publisher: Humana ISBN: 9781071624913 Category : Science Languages : en Pages : 0
Book Description
This volume provides updated protocols for chemical protein synthesis. Chapters guide readers through development methods, strategies, and applications of protein chemical synthesis. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols. Authoritative and cutting-edge, Chemical Protein Synthesis aims to be a useful and practical guide to new researchers and experts looking to expand their knowledge.
Author: Chelsea Gloria Gordon Publisher: ISBN: Category : Languages : en Pages : 368
Book Description
Bioorthogonal chemistries are reactions that are designed to proceed in living environments without perturbing endogenous biological functionalities. These reactions are valuable tools for labeling and studying biomolecules both in vitro and in vivo, often providing unique insights into dynamic, living processes. For a reaction to be considered bioorthogonal, it must proceed in aqueous solvents at physiological pH and temperature. The reaction must also be rapid and selective, generating a stable, covalent adduct that is not reactive towards biological functionalities. Finally, one of the reaction partners must be capable of installation onto the biomolecule of interest. A major motivator in the development of bioorthogonal chemistries is their potential utility in imaging and studying biomolecules in living animals. Chapter one chronicles advancements in the use of bioorthogonal reactions to tag biomolecules in multicellular organisms, focusing on the most prevalent reactions developed to date -- the Staudinger ligation, copper-click chemistry, copper-free click chemistry, and the tetrazine ligation. Examples are provided to highlight the importance of fast reaction kinetics as well as pharmacokinetics on the success of a ligation in vivo. Chapter one also provides commentary on unmet challenges in the field as well as an outlook on future advancements. The in vivo applications of bioorthogonal chemistry discussed in chapter one serve as motivation for the experimental work presented in chapter two. Here, we describe our efforts to understand the factors that contribute to the kinetic profile of the copper-free click reaction. Copper-free click chemistry is a bioorthogonal 1,3-dipolar cycloaddition between azides and strained cyclooctynes to form triazoles. The reaction has seen widespread use in selectively tagging biomolecules both in vitro and in vivo. These successes have prompted the development of cyclooctyne analogs with improved reactivity toward the azide. However, predicting a cyclooctyne's reactivity is challenging, requiring researchers to design and undertake lengthy syntheses of alkynes that may or may not prove successful bioorthogonal reagents. In chapter two, we discuss our work towards defining and predicting the effects of strain and electronics on the reactivity of a cyclooctyne reagent. Through synthesis of analogs of biarylazacyclooctynone (BARAC), the fastest cyclooctyne developed to date, and subsequent reactivity measurements, we gain new insights into the effects of cyclooctyne strain and electronics on reactivity. As well, through computational modeling of our BARAC analogs we conclude that the distortion/interaction model of 1,3-dipolar cycloaddition kinetics serves as a valuable predictor of cyclooctyne reactivity in the copper-free click reaction. Chapter three describes our motivation to develop new bioorthogonal ligations, highlighting the dearth of mutually orthogonal reactions capable of achieving multiplexed imaging. In addition, we discuss the need for bioorthogonal chemistries with new functional capabilities (i.e. polymerizations, reversible reactions, etc.). We then introduce the quadricyclane (QC) ligation, a new bioorthogonal reaction developed in the Bertozzi lab. The QC ligation is a formal [2s+2s+2p] reaction between QC and nickel bis(dithiolene). The reaction has been shown to fulfill many of the requirements of bioorthogonality, but no method of incorporating the QC functionality into a biomolecule of interest has been demonstrated. In chapter three, we discuss our use of the pyrrolysine synthetase/tRNACUA system for site-specific incorporation of a QC amino acid into a protein and subsequent tagging of this QC functionality with a nickel bis(dithiolene) reagent. In chapter four we discuss efforts to further develop the QC ligation, exploring new chemical transformations accessible through this unique reaction. Specifically, we analyze the photodissociation of the QC/nickel bis(dithiolene) adduct to form nickel bis(dithiolene) and norbornadiene, a transformation that has the potential to make the QC ligation a "click-unclick" reaction. In addition, we have begun to analyze possible secondary reaction partners for the norbornadiene product of the photodissociation. Chapter four chronicles our ongoing work to optimize these unique chemical transformations for reversible tagging of model proteins.
Author: Gabriela De Almeida
Author: 
Author: Ellen Sletten
Author: Samantha Jo Boyd
Author: Luca D. D'Andrea
Author: Yu-Hsuan Tsai
Author: Vinod K. Tiwari
Author: Milan Vrabel
Author: Xuechen Li
Author: Chelsea Gloria Gordon