Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Control Student: Kristína Prokešová Supervisor: doc. PharmDr. Miroslav Miletín, PhD. Consultant: Dr. Richard Wombacher Title: Synthesis of "Chemical Tags" and their application for selective protein labelling This diploma thesis is aimed on the synthesis and application of chemical tags. In theoretical part, protein labelling in general is discussed and fluorescent proteins, as routinely used technique for protein tracking, are shortly presented. The greatest attention is dedicated to chemical tags which consist of genetically encoded protein or peptide tag fused to the protein of interest (POI) and from a small fluorescent molecule which labels the POI-tag fusion. Particular representatives with their advantageous and disadvantageous properties are mentioned and super-resolution microscopy and calcium imaging, as applications of chemical tags, are explained. Experimental part is divided into chemical synthesis and biological methods. In synthetic part, four precursors of chemical tags have been prepared - two precursors of TMP-tag small molecule and two precursors of a chemical calcium dye. These precursors can be connected to create a new TMP-tag applicable for calcium imaging. One...

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Protein modification is an essential tool in Chemical Biology, allowing a functional biomolecule to be equipped with a small molecule tag or label. However, as proteins are constructed from a limited palette of around 20 canonical amino acids, achieving selective modification can be problematic. Previously reported methods for protein modification will be discussed in Chapter 1; these often rely on alteration of the protein sequence to introduce a uniquely reactive (often non-canonical) amino acid which may then be covalently modified in a bioorthogonal manner. An alternative approach is to identify a uniquely reactive site within the native protein sequence, such as the protein N-terminus or the reactive side chain of an amino acid with low frequency, and modify this using selective chemistry. In this project, modification of a native sequence protein was achieved by targeting a low abundance residue, tyrosine (Tyr), in a selective manner. Tyr was identified as the ideal candidate as it displays only ~3% frequency in the proteome and, due to its electron-rich aryl ring, it can be selectively modified by electrophilic aromatic substitution. Using a diazonium salt as the tuned electrophile, modification results in formation of an azobenzene motif which may be orthogonally cleaved under mild reducing conditions. The resulting cleavage product bears an o-aminophenol modification on the Tyr side chain, which can then be conjugated to a fluorescent label using established chemistry. This system has been developed on a solid-phase platform to give further control over the extent of modification achieved. In Chapter 2, the component parts of this method are developed through reactions performed in-solution on small molecule substrates. In Chapter 3, this work is then moved onto a solid-phase resin in order to 'catch-and-release' small molecule and peptide substrates. Finally in Chapter 4, the resin-based catch-and-release system is optimised for use in protein modification, and analysis of the modification site is explored.

Immune recruiters are small molecule immunotherapeutics which redirect endogenous components of the immune system to target cells to elicit anti-cancer responses. Current immune recruiters made in the Rullo Lab are heterobispecific molecules which bind receptors on cancer cells and ligand-specific antibodies. Upon antibody binding, a proximity-induced covalent reaction with nearby nucleophilic residues installs a targeting ligand onto the protein. The resultant antibody conjugate then facilitates cancer killing through immune cell recruitment. Covalency circumvents limited binding affinity of the ligand•antibody complex, however antibody•immune receptor affinity remains an issue. This thesis presents an alternative immune recruiting strategy through direct engagement of effector immune cells; monocyte covalent immune recruiters (mCIRs). mCIRs utilize a monocyte specific peptide (cp33) to bind CD64, an activating receptor on monocytes. By incorporating a sulfonyl fluoride electrophile onto the N-terminus of cp33, selective covalent labelling of CD64 was achieved within 24 h. Furthermore, mCIRs demonstrated enhanced monocyte function relative to antibody recruiting platforms. However, these constructs have demonstrated that the order of addition to the target receptor then to CD64 is critical for bridging the two species. As a result, the effect of covalency on complex simplification and monocyte function has yet to be determined. Despite this, mCIRs represent a covalent immune recruitment strategy with the potential to address shortcomings of antibody-based therapeutics. / Thesis / Master of Science (MSc)

Visualizing proteins in living cells without perturbing biological function remains a key challenge in chemical biology. A chemical approach to this problem is the synthesis of small molecule fluorophores that react specifically with a protein of interest (POI). We have developed a site-specific labelling method based on a Fluorogenic Addition Reaction (FlARe). The FlARe probe’s fluorescence is quenched until it undergoes thiol addition with a small, genetically encoded dicysteine peptide tag fused to the POI. Recent blue coumarin probes were shown to be highly selective for target proteins over other cellular thiols; however, fluorogens that can label in the red and green channels of the fluorescence microscope are more desirable for cellular imaging, as red light is lower in energy and therefore less photo-toxic. In the work presented herein, we use DFT calculations to guide the design of red-shifted, PeT-quenched BODIPY based dimaleimide fluorogens. Driven by the preliminary results of a FlARe probe (YC29) that emitted in the red channel, we attempted to prepare the hit compound through a new synthetic approach to further evaluate kinetics and in cellulo labelling. Given the time available, this compound was unable to be synthesized through an SNAr or Pd-catalyzed approach. Alternatives probes lacking the red-shifting substituent were synthesized and evaluated in vitro and in cellulo. The fluorescent enhancement and reaction kinetics of these probes were evaluated in detail, in order to determine the suitability of their application to cellular labelling. A green-BODIPY fluorogen was synthesized that exhibits suitable kinetics for labelling and a dramatic fluorescent enhancement of ~800-fold upon tagging. This probe was successfully applied to the specific, fluorescent labelling of a nuclear histone protein in cellulo.

Development of enzymatic labelling methods has been driven by the importance of studying molecular structures and interactions to comprehend cellular processes. Methyltransferases (MTases), which regulate genetic expression by transferring a methyl group from the cofactor S-adenosyl-L-methionine (SAM) to DNA, histones and various proteins, have been shown to accept SAM analogues with an alternative alkyl group on the sulfonium centre. These alkyl groups can be transferred to the substrate, and with a further reaction can be selectively functionalized. Thus, MTases together with SAM analogues have emerged as novel labelling tools. The project aims to use MTases to obtain an orthogonal system that can selectively use a SAM cofactor analogue to transfer functional chains to proteins with a specific motif. To achieve selectivity of the system, the SAM analogue cofactor was modified on the ribose ring; to obtain a new transferase activity of the system, the transferable methyl on the sulfonium centre was changed to a different substituent. SAM analogues were produced enzymatically with hMAT2A by using 3'-deoxy-ATP and methionine or ethionine. Mutants of SET8 and novel substrates were designed to have modifications at residues in the active site, within the vicinity of the ribose ring of SAM, and were assessed for selective activity with the new analogue cofactor. The results showed that the new cofactor 3'-deoxy-S-adenosyl-L-methionine (3'dSAM) was efficient in the mono-methylation of the substrate peptide RFRKVL, and that the mutant SET8 C270V exhibited over 13 fold MTase activity in presence of 3'dSAM and the RFRKVL substrate, in comparison with the activity with the WT sequence RHRKVL and the SAM cofactor. In addition, glutathione S-transferase (GST) was used as a model protein to express the motif RFRKVL, to transform it into a potential substrate for SET8. Assessment of the MTase activity of SET8, 3'dSAM and the novel GST substrate indicated mono-methylation of the substrate. Moreover, the motif showed no interference with GST native activity. Based on the observations, a new enzymatic system shows higher selectivity with a new analogue cofactor over SAM to effectively methylate proteins expressing the consensus RFRKVL. Work on substrates, enzymes and cofactors should continue to obtain a functional-chain transferase activity of the enzymatic system.

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.

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.