Post-translational insertion of boron in proteins to probe and modulate function

Boron is absent in proteins, yet is a micronutrient. It possesses unique bonding that could expand biological function including modes of Lewis acidity not available to typical elements of life. Here we show that post-translational Cβ–Bγ bond formation provides mild, direct, site-selective access to the minimally sized residue boronoalanine (Bal) in proteins. Precise anchoring of boron within complex biomolecular systems allows dative bond-mediated, site-dependent protein Lewis acid–base-pairing (LABP) by Bal. Dynamic protein-LABP creates tunable inter- and intramolecular ligand–host interactions, while reactive protein-LABP reveals reactively accessible sites through migratory boron-to-oxygen Cβ–Oγ covalent bond formation. These modes of dative bonding can also generate de novo function, such as control of thermo- and proteolytic stability in a target protein, or observation of transient structural features via chemical exchange. These results indicate that controlled insertion of boron facilitates stability modulation, structure determination, de novo binding activities and redox-responsive ‘mutation’

Post-translational insertion of boron in proteins to probe and modulate function

Boron is absent in proteins, yet is a micronutrient. It possesses unique bonding that could expand biological function including modes of Lewis acidity not available to typical elements of life. Here we show that post-translational Cβ–Bγ bond formation provides mild, direct, site-selective access to the minimally sized residue boronoalanine (Bal) in proteins. Precise anchoring of boron within complex biomolecular systems allows dative bond-mediated, site-dependent protein Lewis acid–base-pairing (LABP) by Bal. Dynamic protein-LABP creates tunable inter- and intramolecular ligand–host interactions, while reactive protein-LABP reveals reactively accessible sites through migratory boron-to-oxygen Cβ–Oγ covalent bond formation. These modes of dative bonding can also generate de novo function, such as control of thermo- and proteolytic stability in a target protein, or observation of transient structural features via chemical exchange. These results indicate that controlled insertion of boron facilitates stability modulation, structure determination, de novo binding activities and redox-responsive ‘mutation’

Concepts of catalysis in site-selective protein modifications

The manipulation and modulation of biomolecules has the potential to herald new modes of Biology and Medicine through chemical "editing". Key to the success of such processes will be the selectivities, reactivities and efficiencies that may be brought to bear in bond-formation and bond-cleavage in a benign manner. In this Perspective, we use select examples, primarily from our own research, to examine the current opportunities, limitations and the particular potential of metal-mediated processes as exemplars of possible alternative catalytic modes and manifolds to those already found in nature

Concepts of catalysis in site-selective protein modifications

The manipulation and modulation of biomolecules has the potential to herald new modes of Biology and Medicine through chemical "editing". Key to the success of such processes will be the selectivities, reactivities and efficiencies that may be brought to bear in bond-formation and bond-cleavage in a benign manner. In this Perspective, we use select examples, primarily from our own research, to examine the current opportunities, limitations and the particular potential of metal-mediated processes as exemplars of possible alternative catalytic modes and manifolds to those already found in nature

Residue-selective protein C-formylation via sequential difluoroalkylation-hydrolysis

The carbonyl group is now a widely useful, nonproteinogenic functional group in chemical biology, yet methods for its generation in proteins have relied upon either cotranslational incorporation of unnatural amino acids bearing carbonyls or oxidative conversion (chemical or enzymatic) of existing natural amino acids. If available, alternative strategies for directly adding the C=O group through C-C bond-forming C-carbonylation, particularly at currently inaccessible amino acid sites, would provide a powerful method for adding valuable reactivity and expanding possible function in proteins. Here, following a survey of methods for HCF<sub>2</sub>· generation, we show that reductive photoredox catalysis enables mild radical-mediated difluoromethylation-hydrolysis of native protein residues as an effective method for carbonylation. Inherent selectivity of HCF<sub>2</sub>· allowed preferential modification of Trp residues. The resulting C-2-difluoromethylated Trp undergoes Reimer-Tiemann-type dehalogenation providing highly effective spontaneous hydrolytic collapse in proteins to carbonylated HC(O)-Trp (<i>C</i>-formyl-Trp = CfW) residues. This new, unnatural protein residue CfW not only was found to be effective in bioconjugation, ligation, and labeling reactions but also displayed strong "red-shifting" of its absorption and fluorescent emission maxima, allowing direct use of Trp sites as UV-visualized fluorophores in proteins and even cells. In this way, this method for the effective generation of masked formyl-radical "HC(O)·" equivalents enables first examples of C-C bond-forming carbonylation in proteins, thereby expanding the chemical reactivity and spectroscopic function that may be selectively and post-translationally "edited" into biology

Synthesis of fluorinated alkyl aryl ethers by palladium-catalyzed C–O cross-coupling

Herein, we report a highly effective protocol for the cross-coupling of (hetero)aryl bromides with fluorinated alcohols using the commercially available precatalyst tBuBrettPhos Pd G3 and Cs2CO3 in toluene. This Pd-catalyzed coupling features a short reaction time, excellent functional group tolerance, and compatibility with electron-rich and -poor (hetero)arenes. The method provides access to 18F-labeled trifluoroethyl ethers by cross-coupling with [18F]trifluoroethanol

Radiosynthesis of [18F]ArylSCF2H using aryl boronic acids, S-(chlorofluoromethyl)benzenesulfonothioate and [18F]fluoride

Herein, we report a mild and practical protocol for the copper-catalyzed chlorofluoromethylthiolation of (hetero)aryl boronic acids with the novel reagent PhSO2SCFClH. The resulting products are amenable to halogen exchange 18F-fluorination with cyclotron-produced [18F]fluoride affording [18F]ArSCF2H. This process highlights the combined value of reagent development and (hetero)aryl boron precursors for radiochemistry by adding the [18F]SCF2H group to the list of 18F-motifs within reach for positron emission tomography studies

Precise Probing of Residue Roles by Post-Translational β,γ-C,N Aza-Michael Mutagenesis in Enzyme Active Sites.

Biomimicry valuably allows the understanding of the essential chemical components required to recapitulate biological function, yet direct strategies for evaluating the roles of amino acids in proteins can be limited by access to suitable, subtly-altered unnatural variants. Here we describe a strategy for dissecting the role of histidine residues in enzyme active sites using unprecedented, chemical, post-translational side-chain-β,γ C-N bond formation. Installation of dehydroalanine (as a "tag") allowed the testing of nitrogen conjugate nucleophiles in "aza-Michael"-1,4-additions (to "modify"). This allowed the creation of a regioisomer of His (iso-His, Hisiso) linked instead through its pros-Nπ atom rather than naturally linked via C4, as well as an aza-altered variant aza-Hisiso. The site-selective generation of these unnatural amino acids was successfully applied to probe the contributing roles (e.g., size, H-bonding) of His residues toward activity in the model enzymes subtilisin protease from Bacillus lentus and Mycobacterium tuberculosis pantothenate synthetase

Imaging of translocator protein upregulation is selective for pro‐inflammatory polarized astrocytes and microglia

Translocator protein (TSPO) expression is increased in activated glia, and has been used as a marker of neuroinflammation in PET imaging. However, the extent to which TSPO upregulation reflects a pro‐ or anti‐inflammatory phenotype remains unclear. Our aim was to determine whether TSPO upregulation in astrocytes and microglia/macrophages is limited to a specific inflammatory phenotype. TSPO upregulation was assessed by flow cytometry in cultured astrocytes, microglia, and macrophages stimulated with lipopolysaccharide (LPS), tumor necrosis factor (TNF), or interleukin‐4 (Il‐4). Subsequently, mice were injected intracerebrally with either a TNF‐inducing adenovirus (AdTNF) or IL‐4. Glial expression of TSPO and pro‐/anti‐inflammatory markers was assessed by immunohistochemistry/fluorescence and flow cytometry. Finally, AdTNF or IL‐4 injected mice underwent PET imaging with injection of the TSPO radioligandandnbsp;18F‐DPA‐713, followed by ex vivo autoradiography. TSPO expression was significantly increased in pro‐inflammatory microglia/macrophages and astrocytes both in vitro, and in vivo after AdTNF injection (pandthinsp;andlt;andthinsp;.001 vs. control hemisphere), determined both histologically and by FACS. Both PET imaging and autoradiography revealed a significant (pandthinsp;andlt;andthinsp;.001) increase inandnbsp;18F‐DPA‐713 binding in the ipsilateral hemisphere of AdTNF‐injected mice. In contrast, no increase in either TSPO expression assessed histologically and by FACS, or ligand binding by PET/autoradiography was observed after IL‐4 injection. Taken together, these results suggest that TSPO imaging specifically reveals the pro‐inflammatory population of activated glial cells in the brain in response to inflammatory stimuli. Since the inflammatory phenotype of glial cells is critical to their role in neurological disease, these findings may enhance the utility and application of TSPO imaging.</p

Imaging of translocator protein upregulation is selective for pro‐inflammatory polarized astrocytes and microglia

Translocator protein (TSPO) expression is increased in activated glia, and has been used as a marker of neuroinflammation in PET imaging. However, the extent to which TSPO upregulation reflects a pro‐ or anti‐inflammatory phenotype remains unclear. Our aim was to determine whether TSPO upregulation in astrocytes and microglia/macrophages is limited to a specific inflammatory phenotype. TSPO upregulation was assessed by flow cytometry in cultured astrocytes, microglia, and macrophages stimulated with lipopolysaccharide (LPS), tumor necrosis factor (TNF), or interleukin‐4 (Il‐4). Subsequently, mice were injected intracerebrally with either a TNF‐inducing adenovirus (AdTNF) or IL‐4. Glial expression of TSPO and pro‐/anti‐inflammatory markers was assessed by immunohistochemistry/fluorescence and flow cytometry. Finally, AdTNF or IL‐4 injected mice underwent PET imaging with injection of the TSPO radioligand 18F‐DPA‐713, followed by ex vivo autoradiography. TSPO expression was significantly increased in pro‐inflammatory microglia/macrophages and astrocytes both in vitro, and in vivo after AdTNF injection (p &lt; .001 vs. control hemisphere), determined both histologically and by FACS. Both PET imaging and autoradiography revealed a significant (p &lt; .001) increase in 18F‐DPA‐713 binding in the ipsilateral hemisphere of AdTNF‐injected mice. In contrast, no increase in either TSPO expression assessed histologically and by FACS, or ligand binding by PET/autoradiography was observed after IL‐4 injection. Taken together, these results suggest that TSPO imaging specifically reveals the pro‐inflammatory population of activated glial cells in the brain in response to inflammatory stimuli. Since the inflammatory phenotype of glial cells is critical to their role in neurological disease, these findings may enhance the utility and application of TSPO imaging.</p