Over the past decade, bioorthogonal chemistry has emerged as powerful tools used for tracking biomolecules within living systems. Despite the vast number of organic transformations in the literature, only select few reactions meet the stringent requirements of bioorthogonality. There is increasing demands to develop biocompatible reactions that display high specificity and exquisitely fast kinetics under physiological conditions. With the goal of increasing reaction rates as a means for reducing the concentrations of labelling reagents used for bioconjugation, we have developed metal-catalyzed and metal-free alkyne-nitrone cycloadditions as alternatives to azide-alkyne cycloadditions and demonstrate their applications for imaging cell surface proteins. The copper(I)-catalyzed alkyne-nitrone cycloaddition, also known as the Kinugasa reaction, is typically conducted with a Cu(I) catalyst in the absence of air. We have developed highly efficient micelle promoted multicomponent Kinugasa reactions in aqueous media to make the reaction faster and more efficient. Despite good product yields, the slow kinetics, limited substrate scope and competing side-reaction pathways precludes its practical applicability for biological labelling. We have designed and synthesized β-lactam alkyne probes obtained from these reactions for activity-based protein profiling of the activities of membrane proteins. Additionally, we report that alkyne tethered β-lactams serve as surface enhanced Raman spectroscopy (SERS) reporters bound to silver nanoparticles, and demonstrated that alkyne bound silver nanoparticles can be used for SERS imaging cell surface proteins. The strain-promoted alkyne-nitrone cycloaddition (SPANC) was also explored as a rapid alternative bioorthogonal reaction. We found that the reaction proceeded in high yield within aqueous media, and displayed rate enhancements that were 1-2 orders of magnitude faster than analogous reactions involving azides. The scope and kinetics of SPANC was evaluated in model reactions of various nitrones (acyclic and cyclic) with cyclooctynes, with the purpose of identifying stable nitrones that displayed intrinsically faster kinetics than azides in strain-promoted cycloadditions with cyclooctynes. Cyclic nitrones displayed good stability and exceptionally fast reactivity in these reactions. The SPANC reaction exhibited high selectivity in the presence of biological nucleophilic amino acid side chains and the presence of biological media did not adversely affect the reaction. We have utilized SPANC for highly specific labelling of proteins in vitro and for imaging ligand-receptor interactions on the surfaces of live cancer cells. The high selectivity, fast reaction rate, and aqueous compatibility of SPANC makes the reaction suitable for a variety of in vivo biological imaging applications.

A very interesting class of nanoparticles extensively used for bio-applications is that of hydrogel particles, also called nanogels. There is an increasing interest in the design of hydrogel nanoparticles that have biofunctionality for applications in cell targeting, drug delivery, and biomedicine. The dissertation focuses on developing synthetic strategies for making diverse hydrogel nanoparticles customized to have desirable properties for various bio-applications. We have also investigated the potential of such nanoparticles as coatings for biomedical implants. Chapter 1 gives a brief introduction to hydrogel nanoparticles and the properties that make them attractive for various applications. The details of the syntheses of well defined, stable nanoparticles, commonly used in literature, are described in Chapter 2. Chapter 3 describes our synthesis of hollow sub-50 nm nanogels, which are otherwise difficult to synthesize based on the strategy discussed in Chapter 2. Chapter 4 also demonstrates how simple strategies borrowed from organic chemistry help in producing nanogels with multiple functionalities that are otherwise difficult to obtain, which also is an important advance over the synthetic methods discussed in Chapter 2. Chapter 5 describes how a general strategy based on photoaffinity labeling can yield materials with many applications ranging from optical materials, drug delivery, to biosensing. The latter part of the dissertation describes applications of various nanogels in biology especially as coatings that can control inflammation caused by biomaterials. Chapter 6 describes a method to functionalize flexible biomaterials with the nanogels, thus enabling in vivo investigations of the nanogels as potential coatings for controlling inflammation. Chapter 7 describes the biological studies performed (in collaboration with Garcia Group in the School of Mechanical Engineering at Georgia Tech) on various nanogels, aimed towards obtaining the most functional and efficient materials for implant applications. Chapter 8 describes application of hollow nanogels for covalently immobilizing biomolecules. This chapter also demonstrates how simple non-functional materials can be made unique and functional by means of traditional organic reactions. Finally, in order to broaden the applications of nanogel based materials.

Over the past decade, bioorthogonal chemistry has emerged as powerful tools used for tracking biomolecules within living systems. Despite the vast number of organic transformations in the literature, only select few reactions meet the stringent requirements of bioorthogonality. There is increasing demands to develop biocompatible reactions that display high specificity and exquisitely fast kinetics under physiological conditions. With the goal of increasing reaction rates as a means for reducing the concentrations of labelling reagents used for bioconjugation, we have developed metal-catalyzed and metal-free alkyne-nitrone cycloadditions as alternatives to azide-alkyne cycloadditions and demonstrate their applications for imaging cell surface proteins. The copper(I)-catalyzed alkyne-nitrone cycloaddition, also known as the Kinugasa reaction, is typically conducted with a Cu(I) catalyst in the absence of air. We have developed highly efficient micelle promoted multicomponent Kinugasa reactions in aqueous media to make the reaction faster and more efficient. Despite good product yields, the slow kinetics, limited substrate scope and competing side-reaction pathways precludes its practical applicability for biological labelling. We have designed and synthesized β-lactam alkyne probes obtained from these reactions for activity-based protein profiling of the activities of membrane proteins. Additionally, we report that alkyne tethered β-lactams serve as surface enhanced Raman spectroscopy (SERS) reporters bound to silver nanoparticles, and demonstrated that alkyne bound silver nanoparticles can be used for SERS imaging cell surface proteins. The strain-promoted alkyne-nitrone cycloaddition (SPANC) was also explored as a rapid alternative bioorthogonal reaction. We found that the reaction proceeded in high yield within aqueous media, and displayed rate enhancements that were 1-2 orders of magnitude faster than analogous reactions involving azides. The scope and kinetics of SPANC was evaluated in model reactions of various nitrones (acyclic and cyclic) with cyclooctynes, with the purpose of identifying stable nitrones that displayed intrinsically faster kinetics than azides in strain-promoted cycloadditions with cyclooctynes. Cyclic nitrones displayed good stability and exceptionally fast reactivity in these reactions. The SPANC reaction exhibited high selectivity in the presence of biological nucleophilic amino acid side chains and the presence of biological media did not adversely affect the reaction. We have utilized SPANC for highly specific labelling of proteins in vitro and for imaging ligand-receptor interactions on the surfaces of live cancer cells. The high selectivity, fast reaction rate, and aqueous compatibility of SPANC makes the reaction suitable for a variety of in vivo biological imaging applications.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22465号 / 工博第4726号 / 新制||工||1738(附属図書館) / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 関 修平, 教授 水落 憲和, 准教授 菅瀬 謙治, 教授 梶 弘典 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Epithelial ovarian cancer is the fifth leading cause of cancer-related deaths in the United States. However, the mortality rate is relatively high, due to in part to the cancer being in an advance stage at diagnosis, since it is often asymptomatic at the early-stage with a ~94% of five-year survival rate if it is diagnosed at a localized stage (stage 1). Early detection of cancer would likely improve the survival rate. Scientists are searching for novel promising methods to detect ovarian cancer at an asymptomatic early stage; also, the method is cheap and user-friendly despite there are various techniques for ovarian cancer detection. Cancer antigen 125 (CA125), a type of serum biomarker that elevates ~50% of women with early-stage and ~80% of women with advanced-stage, is used mostly for screening epithelial ovarian cancer. However, the lack of sensitivity and specificity are known to be the main drawback of CA125. Finding new potential biomarkers that diagnose cancer at a localized stage will significantly reduce the mortality rate. Human epididymis protein 4 (HE4) is such a biomarker that has a higher sensitivity and specificity compared to all other known biomarkers, and recently it has been approved by food and drug administration (FDA) for clinical applications.In this project, we developed sandwich-type micro particles immunoassay for sensitive detection of HE4 biomarker in plasma. Here, we cross-link elemental particles to a specific functional group of the targeted biomolecules based on a covalent and non-covalent linking chemistry to improve the sensitivity and the selectivity of biomarker detection in which Fe3O4 and SiO2 microparticles were used to conjugate and purify the antibody-antigen in a media. The purified assay with the microparticles was analyzed with laser-induced breakdown spectroscopy (LIBS) for detection and quantization analysis of the HE4 biomarker. Furthermore, along with LIBS, Raman, Fourier transform infrared (FT-IR), and UV- Vis spectroscopic techniques were utilized to understand the conjugation dynamic and confirm the conjugation process.

Thesis advisor: Jianmin Gao / Besides their broad applications in organic chemistry, boronic acids are also increasingly seen in a variety of biological applications. For instance, they have been used in therapeutic drugs for chemotherapy or probes for the detection of saccharides. One unique feature of boronic acids is that they are capable of forming reversible complexes with sugars or even amino acids. Importantly, they are known to have low inherent toxicity to human. In this work, we have focused on two important reactions involving boronic acids: boronate ester formation, in which boronic acids react with diols and iminoboronate formation in which boronic acids form dative bonds with neighboring amino groups. We have demonstrated the potential of these reactions in bacteria targeting or protein modification. We envisioned that boronic acids could be used as a great warhead to be included in the development of novel antibiotics to counter antibiotic resistance of bacteria, which has emerged to be a serious clinical problem. Different from conventional antibiotics, we decided to utilize reversible covalent chemistries in the design of bacterium binding probes. Inspired by the diol-rich environment on bacterial surface, the first strategy took advantage of the reaction between boronic acid and diols to form boronate esters. We have rationally designed a linear peptide containing five copies of the ‘Wulff-type’ boronic acids or bicyclic amphiphilic peptides with two copies of boronic acids. We examined their capability of binding to E. coli cells or their bactericidal activity against S. aureus. Furthermore, we established a synthetic peptide library (OBTC) incorporating the 2-acetylphenyl boronic acid (2-APBA) warhead to form iminoboronate with the target-lipid II, a precursor for the biosynthesis of peptidoglycan. Although this library failed to generate any potent peptide hits, it provided useful information regarding the development of a synthetic library as well as the screening process. As an extension of the iminoboronate chemistry, thiazolidinoboronate (TzB) attracted our attention for its unique reaction mechanism, superior kinetics and excellent selectivity towards N-terminal cysteine residues. In this work, we have proposed an additional acyl transfer following TzB formation to turn this reaction into an irreversible conjugation. The new reaction inherits the fast kinetics and outstanding selectivity from the TzB chemistry. Excitingly, the product of TzB mediated acyl transfer survived complex conditions such as SDS-PAGE and LC/MS. This reaction was also applied to modify two recombinant proteins with N-terminal cysteine residues or to create a C5C phage library with two distinct modifications. We have further extended the substrate from cysteine to diaminopropionic acid (Dap), serine and tris base. We were delighted to observe imidazolidino boronate (IzB) formation and oxazolidino boronate (OzB) formation, which led to the design of cysteineresponsive probes or peptides that can be spontaneously cyclized in neutral aqueous conditions. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Au cours de la dernière décennie, les anticorps conjugués à des médicaments cytotoxiques ou des oligonucléotides ont acquis une grande attention dans la communauté scientifique en raison des propriétés uniques des anticorps, tels que leur long temps de circulation dans le sérum et leur sélectivité élevée par rapport à leur cible. Par exemple, les conjugués d'anticorps (ACs) sont de plus en plus appliqués en thérapie ciblée contre le cancer ou en bioimagerie. Par conséquent, le développement de méthodologies fiables pour la préparation des AC est actuellement en pleine expansion. Cependant, la conjugaison et la préparation contrôlables des ACs avec une structure définie rencontrent encore de nombreux obstacles en raison de l'excès élevé et de la variété des groupes réactifs dans la structure des anticorps, qui sont accessibles pour la conjugaison. En outre, les technologies de liaison actuelles sont basées sur la réaction de maléimide-thiol, produisant des adduits, qui sont instables dans le sang. Ce travail se concentre sur les approches chimiques pour la fonctionnalisation fiable des anticorps, qui permettent la préparation d'ACs stables présentant un ratio anticorps/principe actif bien défini. La première partie est consacrée à la conception et au développement du réactif maléimide-dioxane, solution auto-hydrolysable et stable dans le sérum, comme alternative à la chimie classique du maléimide. La deuxième partie est consacrée à l'évaluation de la réactivité sélective des différents acides aminés portés par les anticorps par spectrométrie de masse native à haute résolution. Finalement, une nouvelle technologie permettant d’obtenir des ACs stables avec un ratio anticorps/principe actif contrôlé est présentée au lecteur dans une 3ème partie. / Within the last decade, antibodies conjugated to cytotoxic drugs or oligonucleotides have gained a great attention in scientific community owing to the unique properties of the antibodies, such as their long circulation time in serum and high selectivity against their target. For instance, antibody conjugates (ACs) are increasingly applied for targeted cancer therapy or bioimaging. Consequently, the development of reliable methodologies for ACs preparation is currently of high demand. However, the controllable conjugation and preparation of ACs with defined structure are still challenging due to high excess and variety of reactive groups in antibody structure, which are accessible for conjugation. Moreover, current linker technologies are based on the maleimide-thiol reaction, yielding adducts, which are unstable during circulation in blood.This work is focused on chemical approaches for the reliable antibody functionalisation, which enable the preparation of stable ACs with well-defined payload to antibody ratios. The first part is devoted to design and development of maleimide-dioxane reagents as self-hydrolysable and serum-stable alternative to classical maleimide chemistry. The second part is dedicated to a screening approach for evaluation of residue-selective functionalities in reactions with an antibody using high resolution native mass spectrometry. Finally, in the third part the reader is introduced with a novel technology, which enables efficient preparation of stable ACs with a defined degree of conjugation and particularly mono-functionalisation of antibodies.

Résumé: Le travail rapporté dans cette thèse concerne le développement de nouvelles réactions organométalliques pour la création de liaisons C-S dans des systèmes complexes.Dans Le premier chapitre, nous rapportons une méthode originale de bioconjugation de peptides et protéines dans l'eau à température ambiante. La méthode a été appliquée avec succès à la bioconjugation de l'anticorp Trastuzumab.Le deuxième chapitre est consacré à l'étude de la réactivité des alpha ou béta thiosucres dans le couplage de Buchwald-Hartwig-Mitiga avec différent partenaires électrophiles.Dans un premier sous chapitre, nous rapportons une réaction inattendue de couplage de thiosucres avec le précatalyseur G3-Xanthphos pour conduire à de 2-aminobiphenyl glycosides bifonctionnels. La reaction a lieu à température ambiante et dans l'eau et produit des atropidiastéréomères stables.Dans une seconde partie de ce travail, nous décrivons une méthode originale d'accès à benzothiapinones et benzoxathiapinones glycosylées.Cette méthode fait intervenir un couplage de thiosucres avec des aryls iodés ayant un ester en ortho, suivi d'une lactonisation (ou lactimisation) pour conduire aux produits cyclisés.L'avant dernière partie de ce travail, concerne la synthèse de polysaccharides via une réaction de couplage de 2-iodo glycals avec des thiosucres de configuration définie.Ce travail se termine par l'application de couplage de thiosucres à la synthèse de Carbènes N-Hétérocycliques thioglycosylés (NHCs) hydrosolubles. / Abstract: The work reported in this thesis concerns the development of new organometallic reactions for the creation of C-S bonds in complex systems.In the first chapter, we report an original method of bioconjugation of peptides and proteins in water at room temperature. The method has been successfully applied to the bioconjugation of the antibody Trastuzumab.The second chapter is devoted to the study of the reactivity of alpha or beta-thiosugars in the Buchwald-Hartwig-Mitiga coupling with different electrophilic partners.In a first sub-chapter, we report an unexpected reaction of thiosugar coupling with the G3-Xanthphos precatalyst to yield bifunctional 2-aminobiphenyl glycosides. The reaction takes place at room temperature and in water and produces stable atropidiastereomers.In a second part of this work, we describe an original method of access to glycosylated benzothiapinones (and benzoxathiapinones).This method involves thiosugar coupling with iodinated aryls having an ortho ester, followed by lactonization (or lactimization) to yield cyclized products.The last part of this work concerns the synthesis of polysaccharides via a coupling reaction of 2-iodo glycals with thiosugars of defined configuration.This work ends with the application of thiosugar coupling to the synthesis of water-soluble thioglycosylated N-Heterocyclic Carbenes (NHCs) .

Doctor of Philosophy / Department of Chemistry / Stefan Bossmann / Estrogen-derived DNA adducts in human could be the initiating step of breast and prostate cancer, as the scientific literature suggests. Previous studies demonstrated that 4-hydroxy-estrone (estradiol)-1-N3Adenine and 4-hydroxy-estrone (estradiol)-1-N7Guanine were the most abundant adducts found in urine of human subjects. Sensitive detection of these adducts in urine samples could lead to better breast and prostate cancer risk assessment. The standard adducts were synthesized and characterized by NMR and mass spectrometry. Since these adducts are not fluorescent at room temperature an aminomethyl (-CH2NH2) linker was introduced at the C-17 position for derivatization with fluorescence label. This linker allowed to attach highly fluorescent water soluble quantum dots (QDs) for indirect adduct detection. A direct gram-scale synthesis of highly fluorescent, photostable water soluble QDs was executed by developing a new class of 4,4’-bipyridinium salt based twin ligands with 85% and 15% of carboxylic acid and maleimide termini, respectively. These ligands not only stabilized the QDs in water but also provided versatile linkers for two labeling strategies. The twin ligands were afforded by a facile synthesis through SN2 nucleophilic substitution reaction. Labeling of adducts was achieved via a covalent coupling between the (-CH2NH2) linker and the carboxyl (-COOH) terminal ligand on the QDs. However, ELISA experiments utilizing an IgM antibody didn’t reveal any measurable signal from adduct-QD complexes suggesting that one QD is bound to a large number of adducts through –COOH terminal ligands present on QD surface. To explore the binding capabilities of QDs in more detail, a maleimide terminal ligand (a twin partner on the QDs) was synthesized to explore the thiol (-SH) functionality of thiopyrene. Preliminary ELISA showed that these QDs gave detectable fluorescent signal originating from the [pyrene-S-QD] ̶ 8E11 monoclonal antibody (mAb) complex when QD was selectively excited at 470 nm. This clearly indicates that it is necessary to develop a strategy for a distinct 1:1 labeling procedure between QD and the adduct of interest. In addition, IgG (instead of IgM) antibodies should be developed for biosensor application. The latter could afford binding of mAb in upright position, leading to an increase in surface density of mAb and better detection limit.

<p>We have harnessed two reactions catalyzed by the enzyme sortase A and applied them to generate new methods for the purification and site-selective modification of recombinant protein therapeutics. </p><p>We utilized native peptide ligation —a well-known function of sortase A— to attach a small molecule drug specifically to the carboxy-terminus of a recombinant protein. By combining this reaction with the unique phase behavior of elastin-like polypeptides, we developed a protocol that produces homogenously-labeled protein-small molecule conjugates using only centrifugation. The same reaction can be used to produce unmodified therapeutic proteins simply by substituting a single reactant. The isolated proteins or protein-small molecule conjugates do not have any exogenous purification tags, eliminating the potential influence of these tags on bioactivity. Because both unmodified and modified proteins are produced by a general process that is the same for any protein of interest and does not require any chromatography, the time, effort, and cost associated with protein purification and modification is greatly reduced.</p><p>We also developed an innovative and unique method that attaches a tunable number of drug molecules to any recombinant protein of interest in a site-specific manner. Although the ability of sortase A to carry out native peptide ligation is widely used, we demonstrated that Sortase A is also capable of attaching small molecules to proteins through an isopeptide bond at lysine side chains within a unique amino acid sequence. This reaction —isopeptide ligation— is a new site-specific conjugation method that is orthogonal to all available protein-small conjugation technologies and is the first site-specific conjugation method that attaches the payload to lysine residues. We show that isopeptide ligation can be applied broadly to peptides, proteins, and antibodies using a variety of small molecule cargoes to efficiently generate stable conjugates. We thoroughly assessed the site-selectivity of this reaction using a variety of analytical methods and showed that in many cases the reaction is site-specific for lysines in flexible, disordered regions of the substrate proteins. Finally, we showed that isopeptide ligation can be used to create clinically-relevant antibody-drug conjugates that have potent cytotoxicity towards cancerous cells</p> / Dissertation