The field of bioorthogonal chemistry uses click chemistry as a tool to further understand cellular function. The tools of bioorthogonal chemistry include cell and metabolic probes, fluorescent probes with varying bioorthogonal handles that can be used for cell labelling, and gauging metabolic function. This is done by using non-toxic, orthogonal, and selective chemical probes that do not interfere with the complex environment of the cell (termed bioorthogonal). An example of a bioorthogonal reaction is the tetrazine ligation, which occurs via an Inverse Electron Demand Diels Alder (IEDDA) reaction with the 4π and 2π orbitals of a tetrazine and an alkyne/alkene, respectively. This reaction is considered bioorthogonal due to the reagents' high selectivity, quick kinetic rates of reaction, and the reaction's high yield, all of which do not disturb the native cellular environment. The key advantage of this reaction is its extremely fast rate, with second-order rate constants ranging from 10³ to 10⁶ M⁻¹s⁻¹ with the exergonic release of N₂ (g) as the sole by-product which causes the reaction to be irreversible. However, tetrazines are easily hydrolyzed and are redox-sensitive. In this work, we describe herein an alternative bioorthogonal reagent to tetrazine in an IEDDA reaction with cyclooctynes or trans-cyclooctene (TCO). Currently, the tetrazine ligation, with an alkene/alkyne is the fastest bioorthogonal reaction known. Based on this, we investigated the synthesis of diazapyrones as an alternative heterocycle to tetrazine. We also investigated the synthesis of trans-cyclooctenes using a photochemical flow reactor developed by Dr. Fox et al. From this we aimed to develop two cycloaddition reactions, the first one using bicyclononyne (BCN) and the second using strained trans-cyclooctenes (sTCO). Both of these reagents act as the dienophiles. Evaluation of reactions between diazapyrones, cyclooctynes, and trans-cyclooctenes was done through kinetic studies using UV-Vis spectroscopy under pseudo-first-order conditions. Different diazapyrones were tested accordingly with BCN and sTCO dienophiles. The effects of varying substituents on the diazapyrones were also studied. From the results, it was determined that electron-poor diazapyrones react the fastest with BCN and sTCO. Kinetic studies also indicated that diazapyrones are unstable in aqueous environments and are prone to hydrolysis. However, this primarily poses issues for electron poor diazapyrone. The reactions between diazapyrones and cyclooctynes or trans-cyclooctenes are slower than the established tetrazine ligation (k2 ~ 1 - 10⁴ M⁻¹s⁻¹). The k2 values are 10⁻² M⁻¹s⁻¹ and 10 M⁻¹s⁻¹ respectively. However, reaction rates are comparable to other established bioorthogonal reactions such as SPACC (k~ 10⁻² - 10 M⁻¹s⁻¹) or SPANC (k~ 10 - 60 M⁻¹s⁻¹). Thus, this reaction is a useful addition to the bioorthogonal toolbox and can be applied in cell labelling experiments.

No description available.

Dengue virus (DENV) is a mosquito-transmitted flavivirus that threatens approximately half of the world's population. In this dissertation, the use of bioorthogonal chemistry as a tool for researching emerging viral diseases, including DENV is explored. To this end, a bioorthogonally-modified amino acid was successfully installed within the proteome of DENV, which was used for the pull down of a known virus-protein interaction. This technology is intended to be broadly used for the determination of any virus-host interaction, through the installment of a non-perturbing modification that 1) does not hinder viral infectivity and 2) can be selectively discriminated by any complimentary probe. En route to using this technology, a new viral purification strategy was developed for DENV that reduces the overall purification time by 10 hours, and improves retention of virion infectivity. This method and a survey of other viral purification methods used with DENV is contained herein. Furthermore, a chemical scaffold that was repurposed for exploration of protein-protein crosslinking, namely for release of a reactive chemical warhead under acidic conditions, was used for the surface modification of DENV. This triazabutadiene probe was found to be activated by light. In this dissertation is reported the first time aryl diazonium ions for protein crosslinking have been generated on a protein or viral surface through UV-irradiation. The advantages and limitations of this chemistry are presented herein.

Sensitive and specific visualisation of biomolecules in living models is highly challenging because of the complexity of cellular systems. Raman spectroscopy provides chemical contrast based upon molecular vibrations within a sample. It is therefore a powerful approach in biomedicine for disease diagnosis, owing to its potential to provide a spectroscopic fingerprint of biological species. However, Raman scattering is a weak process, and therefore novel approaches are required in order to improve the detection sensitivity for biomolecular imaging in living systems. Here, novel approaches for biomolecular visualisation based upon stimulated Raman scattering (SRS) microscopy are reported. Spectroscopically bioorthogonal functional groups, those which produce spectrally isolated Raman peaks distinct from endogenous cellular Raman peaks, are utilised as a general strategy for small-molecule visualisation. A key issue in the pharmaceutical industry is the lack of available techniques which can probe drug uptake and retention in living cells. Spontaneous Raman spectroscopy and SRS imaging are used for the selective intracellular visualisation of small-molecule inhibitors using a spectroscopically bioorthogonal approach in Chapter 2. Ponatinib and erlotinib are tyrosine kinase inhibitors used in clinical cancer treatment. The spectroscopically bioorthogonal alkyne group present within each drug is used as a specific marker to visualise the uptake and distribution of these two compounds in relevant cellular models. However, only a limited number (~2%) of regulatory approved drugs contain spectroscopically bioorthogonal Raman functional groups. Hence, a general strategy for the design of novel bioorthogonal Raman labels would allow SRS microscopy to be fully exploited in the drug discovery process. Therefore, density functional theory (DFT) is utilised as an in silico screening technique for the design of novel Raman labels, which is presented in Chapter 3. A library of bioorthogonal Raman-labelled analogues of the natural product, anisomycin, have been synthesised in order to validate the DFT screening approach. The effects of labelling upon the biological activity of anisomycin is also assessed. Spectroscopically bioorthogonal Raman imaging of several biomolecules was performed using SRS imaging and the results of which are reported in Chapter 4. The intracellular detection of de novo DNA synthesis was investigated using SRS imaging through metabolic incorporation of alkyne-containing nucleosides. Secondly, the intracellular detection of the Raman-labelled anisomycin analogues is reported using both spontaneous Raman spectroscopy and SRS imaging. This approach has enabled the rate of drug uptake to be assessed in real-time. Furthermore, combining multi-colour SRS imaging and fluorescence imaging in a dual-modality approach, enabled the analysis of drug uptake to be visualised across intracellular structures, and to be correlated with markers of cell-cycle status. These studies represent novel approaches for the direct intracellular visualisation of the uptake and retention of small-molecule inhibitors in live cells.

The bioorthogonal copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction exhibits complex but well-defined kinetics in aqueous and organic solution for soluble azides, alkynes, and ligand-bound copper(I). The kinetic profile in two dimensions, however, for CuAAC systems within a lipid bilayer membrane, has yet to be defined. The effect of triazole formation with lipid membrane-bound components on membrane properties such as fluidity and permeability is also of interest. Azide- and alkyne-functionalized lysolipids were synthesized and incorporated into non-fluid vesicles, which were then subject to CuAAC. The rate order for membrane-bound lipid substrates in non-fluid vesicles was observed to be comperable to that of the reaction in solution. Reactions between vesicles showed evidence of lipid transfer between non-fluid membranes, which has not been previously reported. For intervesicular and intravesicular reactions in non-fluid membranes, the observed reactivity was found to be opposite that of previously published reactions between nucleophiles and electrophiles in fluid lipid systems. Applications of this work include the potential for novel symmetric membrane leaflet labeling, bioorthogonal manipulation of cell and tissue function, and the creation of membranes with precisely controlled properties that may not be available in naturally-occurring membranes.

Transition metals have been used to mediate bioorthogonal reactions within a biological environment. In particular, applications of biocompatible palladium catalysis currently range from biomolecules modification to the in cellulo synthesis or activation of drugs. Here, the scope of palladium-mediated chemistry in living systems has been further extended with the development of a new homogenous palladium catalyst. This water-soluble, biocompatible, and traceable catalysts is based on a palladium-carbene complex coupled to a fluorescent labelled homing peptide for targeted delivery inside cells. This “SMART” catalyst is designed to activate both caged fluorophores and drugs through the cleavage of protecting groups or cross-coupling reactions. A second strategy for targeted delivery of a biocompatible palladium catalysis involves metal nanoparticles loaded onto a heterogeneous solid support. This “modular” catalyst can be implanted in vivo at the desired site of action, e.g. a tumour, and locally activate biomolecules. These two catalytic systems will allow us to selectively activate pro-drugs in vivo, with spatial control, thus minimising the side effects of the treatment on the whole body.

Bioorthogonal reactions, due to its excellent selectivity and time-efficiency, have emerged as a popular tool for protein and cell probing. Among all the bioorthogonal reactions, the inverse electron-demand Diels-Alder reaction (IEDDA) reaction has its advantage of bearing the fastest kinetics. Although the IEDDA reaction drew considerable attention in chemical biology in the last decade, challenges lie in finding the suitable dienophiles. Strained dienophiles, for example, trans-cyclooctene derivatives, can undergo ultrafast IEDDA reactions and therefore have been extensively developed. Unstrained alkenes and alkynes, however, have not been well investigated as IEDDA handles. In general, unstrained dienophiles are more straightforward to synthesise compared with strained dienophiles, therefore they are more accessible to researchers. In addition, the absence of a highly reactive bond makes unstrained dienophiles inert to biological nucleophiles, which allows effectively cellular labelling. In this dissertation, I described three different unstrained dienophiles for different biological purposes. Allyl handle is thiol-stable and non-toxic, which was utilised to label apoptotic cells in a pre-targeting manner. Enol ethers can react with tetrazines to decage protected amino acids and prodrugs. Potassium arylethynyltrifluoroborate, as a novel dienophile, was shown to react fast with pyridyl tetrazines controllably and this new IEDDA was applied to label proteins site-selectively and to fluorescently label two proteins orthogonally. In addition to IEDDA reactions, other bioorthogonal reactions were also developed using these versatile unstrained handles. Allyl-bearing amino acids and proteins can undergo an acetophenone-mediated hetero-[2+2] photocycloaddition with maleimide derivatives, expanding the toolbox of photo-triggered chemistry for protein modification. The potassium arylethynyltrifluoroborate handle was also found reactive in copper(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC) and showcased the huge potential for protein labelling and multicolour cellular labelling.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15407号 / 工博第3286号 / 新制||工||1495(附属図書館) / 27885 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 森 泰生, 教授 白川 昌宏 / 学位規則第4条第1項該当

Chemical transformations that join two molecular components together rapidly while remaining highly efficient and selective are valued for their elegant simplicity and effectiveness in a myriad of applications. By applying the principles of ‘click’ chemistry to biology, information about molecular interactions in vivo can therefore be gained from minimally perturbing bioorthogonal coupling reactions. Developing bioorthogonal ‘click’ reactions – reactions that do not cross-react with biological components – provides new ways to accurately study biological systems at the molecular level. This thesis describes the development of such tools. Strain-promoted alkyne-nitrone cycloadditions (SPANC) represent rapid, efficient, selective, and tunable conjugation strategies that are applicable to biomolecular labelling experiments. Herein, SPANC reactions with bicyclo[6.1.0]nonyne are examined using physical organic methods to determine the stereoelectronic factors governing SPANC reactivity. Second-order rate constants (k2) of up to 1.49 M-1s-1 were measured and the resulting cycloadditions are applied to the design and synthesis of nitrone-based molecular probes. The first example of SPANC-mediated metabolic labelling in live-cell bacteria is reported, establishing SPANC as an efficient and bioorthogonal metabolic labelling strategy for cellular labelling.

Bioorthogonal chemistry is a rapidly growing field that enables spatiotemporal monitoring of biomolecules using targeted probes. The development of bioorthogonal reactions therefore requires several criteria to be met. Reactions need to be selective, and fast enough so lower concentrations of reagents are used to mitigate toxicity, and they need to be stable in biological environments. 3-oxidopyridiniums are a class of water stable six-membered heteroaromatic latent dipoles, and have been previously reported to undergo [3+2] cycloadditions with electron deficient dipolarophiles, but have yet to be investigated for bioorthogonal use. To test their applicability as a bioorthogonal reagent, a series of N-methyl-3-oxidopyridiniums were synthesized with varying substituents on the 5 position and were reacted with DIBO (dibenzocyclooctyne). Electron donating 5-substituents have been shown to significantly increase the rate of the reaction, with bimolecular rate constants ranging from 3.31 x 10⁻⁴ with 5-trifluoromethyl-N-methyl-3-oxidopyridinium to 1.07 M⁻¹ s⁻¹ with 5-amino-N-methyl-3-oxidopyridinium, putting the faster reactions on par with commonly used bioorthogonal reactions for cell labelling. Strain-promoted alkyne-nitrone cycloadditions (SPANC) are a class of [3+2] cycloadditions that are commonly used for bioorthogonal reactions. In comparison to their predecessor SPAAC (strain promoted azide alkyne cycloadditions), SPANC offers much better reaction tunability. icSHAPE (in vivo click selective hydroxyl acylation analyzed by primer extension) is a labelling technique used to determine RNA structure. This is done by selectively targeting the 2' hydroxyl on the ribose sugar of RNA that is structurally available in regions of RNA that are single stranded, and the use of SPAAC allows for an improved signal to noise ratio. Herein, DMImO (1-[(p-methoxycarbonylphenyl)methyl]-2,2-dimethyl-5-oxo-3-imidazolin-3-ium-3-olate), a previously used nitrone in SPANC reactions, has been modified to include an electron deficient carbonyl imidazole to allow for a nucleophilic attack from the 2' hydroxyl of the RNA. Hydrolysis of the nitrone probe is on par with previous SHAPE reagents that are used for in vivo labelling and is able to label 5S rRNA for structure determination as effectively as previously used SHAPE reagents.