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Post-translational modifications (PTMs) greatly expand the structures and functions of proteins in nature 1 , 2 . Although synthetic protein functionalization strategies allow mimicry of PTMs 3 , 4 , as well as formation of unnatural protein variants with diverse potential functions, including drug carrying 5 , tracking, imaging 6 and partner crosslinking 7 , the range of functional groups that can be introduced remains limited. Here we describe the visible-light-driven installation of side chains at dehydroalanine residues in proteins through the formation of carbon-centred radicals that allow C–C bond formation in water. Control of the reaction redox allows site-selective modification with good conversions and reduced protein damage. In situ generation of boronic acid catechol ester derivatives generates RH 2 C • radicals that form the native (β-CH 2 –γ-CH 2 ) linkage of natural residues and PTMs, whereas in situ potentiation of pyridylsulfonyl derivatives by Fe( ii ) generates RF 2 C • radicals that form equivalent β-CH 2 –γ-CF 2 linkages bearing difluoromethylene labels. These reactions are chemically tolerant and incorporate a wide range of functionalities (more than 50 unique residues/side chains) into diverse protein scaffolds and sites. Initiation can be applied chemoselectively in the presence of sensitive groups in the radical precursors, enabling installation of previously incompatible side chains. The resulting protein function and reactivity are used to install radical precursors for homolytic on-protein radical generation; to study enzyme function with natural, unnatural and CF 2 -labelled post-translationally modified protein substrates via simultaneous sensing of both chemo- and stereoselectivity; and to create generalized ‘alkylator proteins’ with a spectrum of heterolytic covalent-bond-forming activity (that is, reacting diversely with small molecules at one extreme or selectively with protein targets through good mimicry at the other). Post-translational access to such reactions and chemical groups on proteins could be useful in both revealing and creating protein function. A wide range of side chains are installed into proteins by addition of photogenerated alkyl or difluroalkyl radicals, providing access to new functionality and reactivity in proteins.

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 site-selective formation of boronoalanine in proteins enables applications of boron for binding partner capture, footprinting of interactions with reactive oxygen species, proteolytic control and mapping of transient structures.

Category: Midfoot/Forefoot; Sports Introduction/Purpose: Turf toe, a plantar plate injury of the first metatarsophalangeal joint, commonly occurs in athletes participating in outdoor cutting sports. To the knowledge of the authors, this study reports on clinical and return to sport outcomes after surgical repair of turf toe for the largest cohort of patients presenting with a Grade III turf toe injury. The purpose of this study is to identify risk factors for turf toe and analyze foot function after turf toe injury and surgical repair. This study reports on clinical outcomes and return to sport for athletic patients treated for a turf toe injury at our institution. We hypothesize that patients will have significant improvement in pre- to postoperative clinical outcomes and quickly return to sport after surgical repair. Methods: This was a single-center retrospective study conducted from the institutional review board-approved Foot and Ankle Registry data, and the protocol was approved by the steering committee at the investigators’ institution. Inclusion criteria included athletes of at least high school level competition who underwent plantar plate repair at the first metatarsophalangeal joint between 2016 to 2023 by 3 fellowship-trained foot and ankle orthopedic surgeons. Excluded were patients with histories of ipsilateral forefoot surgeries, rheumatoid arthritis, or gout. Twenty-eight patients were identified. Patient-reported outcomes via PROMIS scores were collected preoperatively and at least one year postoperatively for all patients. Return to sport surveys were distributed to patients at least one year postoperatively. Results: The average time from surgery to final follow-up was 2.8 (range, 1-5.3) years. PROMIS scores were found to significantly improve for Physical Function, Pain Interference, Pain Intensity, and Global Physical Health domains. 21 return to sport surveys were completed from the patient cohort. 19 out of 21 (90.5%) of patients were able to return to pre-injury levels of physical competition with an average time to return to sport was 20.35 (range, 12-32) weeks. Conclusion: This study compared short- to medium-term patient-reported clinical outcomes and return to sport data for athletes after a Grade III turf toe injury and surgical repair. Patients demonstrated significant improvement in patient-reported outcomes and were able to return to sport approximately five months after surgery.

Methods to directly post-translationally modify proteins are perhaps the most straightforward and operationally simple ways to create and study protein post-translational modifications (PTMs). However, precisely altering or constructing the C–C scaffolds pervasive throughout biology is difficult with common two-electron chemical approaches. Recently, there has been a surge of new methods that have utilized single electron/radical chemistry applied to site-specifically “edit” proteins that have started to create this potentialone that in principle could be near free-ranging. This review provides an overview of current methods that install such “edits”, including those that generate function and/or PTMs, through radical C–C bond formation (as well as C–X bond formation via C• where illustrative). These exploit selectivity for either native residues, or preinstalled noncanonical protein side-chains with superior radical generating or accepting abilities. Particular focus will be on the radical generation approach (on-protein or off-protein, use of light and photocatalysts), judging the compatibility of conditions with proteins and cells, and novel chemical biology applications afforded by these methods. While there are still many technical hurdles, radical C–C bond formation on proteins is a promising and rapidly growing area in chemical biology with long-term potential for biological editing.

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In eukaryotes, DNA is packaged into repeating units called nucleosomes, which consist of ~150 base pairs of DNA wrapped around eight proteins called histones. These histones are often laden with post-translational modifications (PTMs), and while many PTMs have been shown to have profound effects on gene expression and chromatin organization, most remain uncharacterized. This thesis strives to elucidate the functions of PTM containing histones. Chapter 1 describes a light-driven reaction that site-specifically inserts sidechains of interest into histones via radical addition to previously installed dehydroalanine residues, with a scope encompassing many native and PTM containing sidechains, as well as several reactive functional groups. Chapter 2 utilizes the reaction from Chapter 1, as well as others from our group, in several independent applications: The enzymatic removal of installed histone PTMs is tested, histone methylation and phosphorylation are assessed in the structural context of a nucleosome, PTM processing on fluorinated histone sidechains is tracked by 19F-NMR, and interaction partners are crosslinked to histones bearing electrophilic sidechains. Chapter 3 studies histones in more complex, living systems. The understudied ability of histones to penetrate cell membranes is investigated, revealing the incorporation of extracellular histones into the chromatin of nearby cells, provoking a strong inflammatory and immune response. Furthermore, several light-driven histone modification reactions are attempted in the translucent model organism, the zebrafish. In conclusion, this thesis describes powerful tools for modifying histones and applies them to relevant problems in epigenetics, revealing unknown roles and functions of histones and their PTMs.

The fluorination of amino acid residues represents a near-isosteric alteration with the potential to report on biological pathways, yet the site-directed editing of carbon–hydrogen (C–H) bonds in complex biomolecules to carbon–fluorine (C–F) bonds is challenging, resulting in its limited exploitation. Here, we describe a protocol for the posttranslational and site-directed alteration of native γCH 2 to γCF 2 in protein sidechains. This alteration allows the installation of difluorinated sidechain analogs of proteinogenic amino acids, in both native and modified states. This chemical editing is robust, mild, fast and highly efficient, exploiting photochemical- and radical-mediated C–C bonds grafted onto easy-to-access cysteine-derived dehydroalanine-containing proteins as starting materials. The heteroaryl–sulfonyl reagent required for generating the key carbon-centered C• radicals that install the sidechain can be synthesized in two to six steps from commercially available precursors. This workflow allows the nonexpert to create fluorinated proteins within 24 h, starting from a corresponding purified cysteine-containing protein precursor, without the need for bespoke biological systems. As an example, we readily introduce three γCF 2 -containing methionines in all three progressive oxidation states (sulfide, sulfoxide and sulfone) as d -/ l - forms into histone eH3.1 at site 4 (a relevant lysine to methionine oncomutation site), and each can be detected by 19 F-nuclear magnetic resonance of the γCF 2 group, as well as the two diastereomers of the sulfoxide, even when found in a complex protein mixture of all three. The site-directed editing of C–H→C–F enables the use of γCF 2 as a highly sensitive, ‘zero-size-zero-background’ label in protein sidechains, which may be used to probe biological phenomena, protein structures and/or protein–ligand interactions by 19 F-based detection methods. A robust, mild and fast approach for the posttranslational, site-directed fluorination of protein sidechains, detectable via 19 F-based magnetic resonance methods.