Explore the vast range of titles available.

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.

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.

Positron emission tomography (PET) is a diagnostic nuclear imaging modality that relies on automated protocols to prepare agents labeled with a positron-emitting radionuclide (e.g., 18 F). In recent years, new reactions have appeared for the 18 F-labeling of agents that are difficult to access by applying traditional radiochemistry, for example those requiring 18 F incorporation into unactivated (hetero)arenes. However, automation of these new methods for translation to the clinic has progressed slowly because extensive modification of manual protocols is typically required when implementing novel 18 F-labeling methodologies within automated modules. Here, we describe the workflow that led to the automated radiosynthesis of the poly(ADP-ribose) polymerase (PARP) inhibitor [ 18 F]olaparib. First, we established a robust manual protocol to prepare [ 18 F]olaparib from the protected N -[2-(trimethylsilyl)ethoxy]methyl (SEM) arylboronate ester precursor in a 17% ± 5% ( n = 15; synthesis time, 135 min) non-decay-corrected (NDC) activity yield, with molar activity ( A m ) up to 34.6 GBq/µmol. Automation of the process, consisting of copper-mediated 18 F-fluorodeboronation followed by deprotection, was achieved on an Eckert & Ziegler Modular-Lab radiosynthesis platform, affording [ 18 F]olaparib in a 6% ± 5% ( n = 3; synthesis time, 120 min) NDC activity yield with A m up to 319 GBq/µmol. Automation of new methods for 18 F incorporation into unactivated (hetero)arenes for translation to the clinic has progressed slowly. This protocol describes a workflow leading to automated radiosynthesis of the PARP inhibitor [ 18 F]olaparib.

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 to γCF 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 -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 F-nuclear magnetic resonance of the γCF 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 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 F-based detection methods.

The neurofilament light chain protein (NfL) is a suggested general marker for neuronal loss. Its release from brain parenchyma into cerebral spinal fluid, and presumed detection in blood has seen it established as a first blood-based marker of disease activity and drug efficacy in multiple sclerosis (MS) and in the presymptomatic diagnosis and assessment of disease course for other neurodegenerative disorders. However, the lack of characterisation of its behaviour in circulation, largely due to its antibody-dependent measurement, have hampered the biological interpretation of these measurements, especially after acute injury such as in MS relapse or head trauma. Here, we describe a strategy for exploiting positron emission tomography (PET) imaging using isosteric protein mimics following the installation of a fluorine-18 label that is benign enough to provide sensitive, real-time information on the dynamics and trafficking of NfL protein. This circumvents the limits of current methods that integrate 18F into proteins through the bio-conjugation of bulky, unnatural groups, which we show perturb NfL's assembly and functional properties from those in the natural state. We use a visible-light-driven reaction to access radioactive isostere proteins that are unperturbed and so closely resemble their native form. In this way, generation of [18F]fluoroalkyl radicals that can be rapidly reacted at pre-defined sites on proteins creates mimics of proteinogenic side chains bearing near-zero-size labels to probe proteins in functionally 'true' form. These prosthetic-free, protein radiotracers can be generated in excellent radiochemical yield (up to 67%) via a semi-automated protocol in just 15 mins. High associated molar activities (precursor up to 102 GBq μmol-1) allowed high sensitivity dynamic observations in blood, brain and cerebrospinal fluid, enabling even the first unambiguous observations of spinal flow kinetics using proteins. These dynamics, including the high rate of spinal flow (on the order of mm per min) and drainage of NfL from CSF into sacral lymph nodes, now provides evidence that the slow fall rate of antibody-detected markers that is observed after acute neural insults is not due to a long half-life, but rather reflects sustained neuronal loss. This discovery will now help to better correlate clinical and radiological features of disease with NfL blood levels. Our methodology now demonstrates the broad potential of a near-zero-size labelling method for the functional study of proteins in whole organisms without interfering with their biological activity and native assembly.Competing Interest StatementA.W.J.P., B.J., V.G. and B.G.D. are listed on patents filed by Oxford University Innovations concerning protein editing.

Positron emission tomography (PET) is a diagnostic nuclear imaging modality that relies on automated protocols to prepare agents labeled with a positron-emitting radionuclide (e.g., 18F). In recent years, new reactions have appeared for the 18F-labeling of agents that are difficult to access by applying traditional radiochemistry, for example those requiring 18F incorporation into unactivated (hetero)arenes. However, automation of these new methods for translation to the clinic has progressed slowly because extensive modification of manual protocols is typically required when implementing novel 18F-labeling methodologies within automated modules. Here, we describe the workflow that led to the automated radiosynthesis of the poly(ADP-ribose) polymerase (PARP) inhibitor [18F]olaparib. First, we established a robust manual protocol to prepare [18F]olaparib from the protected N-[2-(trimethylsilyl)ethoxy]methyl (SEM) arylboronate ester precursor in a 17% ± 5% (n = 15; synthesis time, 135 min) non-decay-corrected (NDC) activity yield, with molar activity (Am) up to 34.6 GBq/µmol. Automation of the process, consisting of copper-mediated 18F-fluorodeboronation followed by deprotection, was achieved on an Eckert & Ziegler Modular-Lab radiosynthesis platform, affording [18F]olaparib in a 6% ± 5% (n = 3; synthesis time, 120 min) NDC activity yield with Am up to 319 GBq/µmol.Positron emission tomography (PET) is a diagnostic nuclear imaging modality that relies on automated protocols to prepare agents labeled with a positron-emitting radionuclide (e.g., 18F). In recent years, new reactions have appeared for the 18F-labeling of agents that are difficult to access by applying traditional radiochemistry, for example those requiring 18F incorporation into unactivated (hetero)arenes. However, automation of these new methods for translation to the clinic has progressed slowly because extensive modification of manual protocols is typically required when implementing novel 18F-labeling methodologies within automated modules. Here, we describe the workflow that led to the automated radiosynthesis of the poly(ADP-ribose) polymerase (PARP) inhibitor [18F]olaparib. First, we established a robust manual protocol to prepare [18F]olaparib from the protected N-[2-(trimethylsilyl)ethoxy]methyl (SEM) arylboronate ester precursor in a 17% ± 5% (n = 15; synthesis time, 135 min) non-decay-corrected (NDC) activity yield, with molar activity (Am) up to 34.6 GBq/µmol. Automation of the process, consisting of copper-mediated 18F-fluorodeboronation followed by deprotection, was achieved on an Eckert & Ziegler Modular-Lab radiosynthesis platform, affording [18F]olaparib in a 6% ± 5% (n = 3; synthesis time, 120 min) NDC activity yield with Am up to 319 GBq/µmol.

BackgroundDuring transitions of care, including hospital discharge, patients are at risk of drug-related problems (DRPs).AimTo investigate the impact of pharmacist-led services, specifically medication reconciliation at admission and/or interprofessional ward rounds on the number of DRPs at discharge.MethodIn this retrospective, single-center cohort study, we analyzed routinely collected data of patients discharged from internal medicine wards of a regional Swiss hospital that filled their discharge prescriptions in the hospital’s community pharmacy between June 2016 and May 2019. Patients receiving one of the two or both pharmacist-led services (Study groups: Best Care = both services; MedRec = medication reconciliation at admission; Ward Round = interprofessional ward round), were compared to patients receiving standard care (Standard Care group). Standard care included medication history taken by a physician and regular ward rounds (physicians and nurses). At discharge, pharmacists reviewed discharge prescriptions filled at the hospital’s community pharmacy and documented all DRPs. Multivariable Poisson regression analyzed the independent effects of medication reconciliation and interprofessional ward rounds as single or combined service on the frequency of DRPs.ResultsOverall, 4545 patients with 6072 hospital stays were included in the analysis (Best Care n = 72 hospital stays, MedRec n = 232, Ward Round n = 1262, and Standard Care n = 4506). In 1352 stays (22.3%) one or more DRPs were detected at hospital discharge. The combination of the two pharmacist-led services was associated with statistically significantly less DRPs compared to standard care (relative risk: 0.33; 95% confidence interval: 0.16, 0.65). Pharmacist-led medication reconciliation alone showed a trend towards fewer DRPs (relative risk: 0.75; 95% confidence interval: 0.54, 1.03).ConclusionOur results support the implementation of pharmacist-led medication reconciliation at admission in combination with interprofessional ward rounds to reduce the number of DRPs at hospital discharge.