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The trifluoromethyl group (CF 3 ) is a key functionality in pharmaceutical and agrochemical development, greatly enhancing the efficacy and properties of resulting compounds. However, attaching the CF 3 group to heteroatoms such as sulfur, oxygen, and nitrogen poses challenges because of the lack of general synthetic methods and reliance on bespoke reagents. Here, we present a modular flow platform that streamlines the synthesis of heteroatom-CF 3 motifs. Our method uses readily available organic precursors in combination with cesium fluoride as the primary fluorine source, facilitating the rapid generation of N -trifluoromethyl(R) [NCF 3 (R)], SCF 3 (trifluoromethylthio), and OCF 3 (trifluoromethoxy) anions on demand without reliance on perfluoroalkyl precursor reagents. This strategy offers a more environmentally friendly synthesis of trifluoromethyl(heteroatom)-containing molecules, with the potential for scalability in manufacturing processes facilitated by flow technology.

Herein, we report an approach for generating thionyl fluoride (SOF 2 ) from the commodity chemicals thionyl chloride (SOCl 2 ) and potassium fluoride (KF). The methodology relies on a microfluidic device that can efficiently produce and dose this toxic gaseous reagent under extremely mild and safe conditions. Subsequently, the in situ-generated thionyl fluoride is reacted with an array of structurally and electronically differing carboxylic acids, leading to the direct and efficient synthesis of highly sought-after acyl fluorides. Importantly, our investigation also highlights the inherent modularity of this flow-based platform. We demonstrate the adaptability of this approach by not only synthesizing acyl fluorides but also directly converting carboxylic acids into a diverse array of valuable compounds such as esters, thioesters, amides, and ketones. This versatility showcases the potential of this approach for a wide range of synthetic applications, underscoring its significance in the realm of chemical synthesis., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

The introduction of fluoroalkylthioether groups has attracted the attention of the drug-discovery community given the special physicochemical and pharmacokinetic features they confer to bioactive compounds, yet these are often limited to standard SCF 3 and SCF 2 H moieties. Herein, two saccharin-based electrophilic reagents have been disclosed for the incorporation of uncommon SCF 2 CF 2 H and SCF 2 CF 3 motifs. Their reactivity performance, multigram-scale preparation, and divergent derivatization have been thoroughly investigated with a variety of nucleophiles, including natural products and pharmaceuticals.

Methods that site-selectively attach multivalent carbohydrate moieties to proteins can be used to generate homogeneous glycodendriproteins as synthetic functional mimics of glycoproteins. Here, we study aspects of the scope and limitations of some common bioconjugation techniques that can give access to well-defined glycodendriproteins. A diverse reactive platform was designed via use of thiol-Michael-type additions, thiol-ene reactions, and Cu(I)-mediated azide-alkyne cycloadditions from recombinant proteins containing the non-canonical amino acids dehydroalanine, homoallylglycine, homopropargylglycine, and azidohomoalanine., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Pentafluoroethylation of unactivated C(sp 2 )-X bonds (X = I, Br) using a storable, "ligandless" CuC 2 F 5 reagent prepared by controlled self-condensation of ready available TMSCF 3 -derived CuCF 3 has been developed. A thorough analysis by 19 F NMR and ESI-MS revealed the nature of this reagent in solution. The operational simplicity and robustness of this system enables the efficient, late-stage incorporation of C 2 F 5 units into a variety of (hetero)aryl and complex alkenyl halides such as glycals, nucleosides, and nucleobases.

GalNAc-glycopeptides derived from mucin MUC1 are an important class of tumor-associated antigens. α- O-glycosylation forces the peptide to adopt an extended conformation in solution, which is far from the structure observed in complexes with a model anti-MUC1 antibody. Herein, we propose a new strategy for designing potent antigen mimics based on modulating peptide/carbohydrate interactions by means of O → S/Se replacement at the glycosidic linkage. These minimal chemical modifications bring about two key structural changes to the glycopeptide. They increase the carbohydrate-peptide distance and change the orientation and dynamics of the glycosidic linkage. As a result, the peptide acquires a preorganized and optimal structure suited for antibody binding. Accordingly, these new glycopeptides display improved binding toward a representative anti-MUC1 antibody relative to the native antigens. To prove the potential of these glycopeptides as tumor-associated MUC1 antigen mimics, the derivative bearing the S-glycosidic linkage was conjugated to gold nanoparticles and tested as an immunogenic formulation in mice without any adjuvant, which resulted in a significant humoral immune response. Importantly, the mice antisera recognize cancer cells in biopsies of breast cancer patients with high selectivity. This finding demonstrates that the antibodies elicited against the mimetic antigen indeed recognize the naturally occurring antigen in its physiological context. Clinically, the exploitation of tumor-associated antigen mimics may contribute to the development of cancer vaccines and to the improvement of cancer diagnosis based on anti-MUC1 antibodies. The methodology presented here is of general interest for applications because it may be extended to modulate the affinity of biologically relevant glycopeptides toward their receptors.

Monoclonal antibodies have emerged as an important class of therapeutics in oncological and autoimmune diseases due to their several attractive properties, such as high binding affinity and specificity. However, it has recently become clear that antibodies recovered from serum show a significantly decreased potency owing to various reasons, including deamidation, oxidation, fragment antigen binding (Fab) exchange, and disulfide shuffling. Fab exchange and disulfide shuffling result because of the instability of disulfides in serum. Herein, we reported a 'one-pot' stapling strategy using isobutylene motifs to stabilise the interchain disulfides of antibodies. This general method was applied to a Fab fragment of the anti-HER2 antibody. The stapled Fab was completely stable in the presence of biological thiols. The approach was further applied to two different full-length IgGs, trastuzumab and rituximab, under mild and biocompatible conditions. The binding affinity of the antibody was enhanced, relative to its native form, after being stapled. The stapled structure maintained its effector functions and behaved similarly to its native form in vivo. This work provides a straightforward and scalable method for the stabilisation of antibodies in various formats.

The preparation of well-defined d- xylo and d- ribo glycosides represents a synthetic challenge due to the limited configurational availability of starting materials and the laborious synthesis of homogeneous 2-deoxy-β-glycosidic linkages, in particular that of the sugar-steroid motif, which represents the "stereoselective determining step" of the overall synthesis. Herein we describe the use of 2-deoxy-2-iodo-glycopyranosyl sulfoxides accessible from widely available d-xylose and d-ribose monosaccharides as privileged glycosyl donors that permit activation at very low temperature. This ensures a precise kinetic control for a complete 1,2- trans stereoselective glycosylation of particularly challenging steroidal aglycones., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

We herein present a flexible approach for the incorporation of CF 3 units into a predefined site of electron-rich alkenes that exploits the regiocontrolled introduction of an iodine handle and subsequent trifluoromethylation of the C(sp 2 )-I bond using fluoroform-derived "ligandless" CuCF 3 . The broad substrate scope and functional group tolerance together with the scalability and purity of the resulting products enabled the controlled, late-stage synthesis of single regioisomers of complex CF 3 -scaffolds, such as sugars, nucleosides (antivirals), and heterocycles (indoles and chromones), with potential for academic and industrial applications.

We present a new peptide-macrocyclization strategy with an isobutylene graft. The reaction is mild and proceeds rapidly and efficiently both for linear and cyclic peptides. The resulting isobutylene-grafted peptides possess improved passive membrane permeability due to the shielding of the polar backbone of the amides, as demonstrated by NMR spectroscopy and molecular dynamics simulations. The isobutylene-stapled structures are fully stable in human plasma and in the presence of glutathione. This strategy can be applied to bioactive cyclic peptides such as somatostatin. Importantly, we found that structural preorganization forced by the isobutylene graft leads to a significant improvement in binding. The combined advantages of directness, selectivity, and smallness could allow application to peptide macrocyclization based on this attachment of the isobutylene graft., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Chemical modification of proteins is essential for a variety of important diagnostic and therapeutic applications. Many strategies developed to date lack chemo- and regioselectivity as well as result in non-native linkages that may suffer from instability in vivo and adversely affect the protein's structure and function. We describe here the reaction of N-nucleophiles with the amino acid dehydroalanine (Dha) in a protein context. When Dha is chemically installed in proteins, the addition of a wide-range N-nucleophiles enables the rapid formation of amine linkages (secondary and tertiary) in a chemoselective manner under mild, biocompatible conditions. These new linkages are stable at a wide range of pH values (pH 2.8 to 12.8), under reducing conditions (biological thiols such as glutathione) and in human plasma. This method is demonstrated for three proteins and is shown to be fully compatible with disulfide bridges, as evidenced by the selective modification of recombinant albumin that displays 17 structurally relevant disulfides. The practicability and utility of our approach is further demonstrated by the construction of a chemically modified C2A domain of Synaptotagmin-I protein that retains its ability to preferentially bind to apoptotic cells at a level comparable to the native protein. Importantly, the method was useful for building a homogeneous antibody-drug conjugate with a precise drug-to-antibody ratio of 2. The kinase inhibitor crizotinib was directly conjugated to Dha through its piperidine motif, and its antibody-mediated intracellular delivery results in 10-fold improvement of its cancer cell-killing efficacy. The simplicity and exquisite site-selectivity of the aza-Michael ligation described herein allows the construction of stable secondary and tertiary amine-linked protein conjugates without affecting the structure and function of biologically relevant proteins.

A four-membered oxygen ring (oxetane) can be readily grafted into native peptides and proteins through site-selective bis-alkylation of cysteine residues present as disulfides under mild and biocompatible conditions. The selective installation of the oxetane graft enhances stability and activity, as demonstrated for a range of biologically relevant cyclic peptides, including somatostatin, proteins, and antibodies, such as a Fab arm of the antibody Herceptin and a designed antibody DesAb-Aβ against the human Amyloid-β peptide. Oxetane grafting of the genetically detoxified diphtheria toxin CRM 197 improves significantly the immunogenicity of this protein in mice, which illustrates the general utility of this strategy to modulate the stability and biological activity of therapeutic proteins containing disulfides in their structures., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Central scaffold topology and carbohydrate density are important features in determining the binding mechanism and potency of synthetic multivalent of poly- versus monodisperse carbohydrate systems against a model plant toxin (Ricinus communis agglutinin (RCA 120 )). Lower densities of protein receptors favour the use of heterogeneous, polydisperse glycoconjugate presentations, as determined by surface plasmon resonance and dynamic light scattering., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The emergence of multidrug-resistant Mycobacterium tuberculosis ( Mtb ) strains highlights the need to develop more efficacious and potent drugs. However, this goal is dependent on a comprehensive understanding of Mtb virulence protein effectors at the molecular level. Here, we used a post-expression cysteine (Cys)-to-dehydrolanine (Dha) chemical editing strategy to identify a water-mediated motif that modulates accessibility of the protein tyrosine phosphatase A (PtpA) catalytic pocket. Importantly, this water-mediated Cys-Cys non-covalent motif is also present in the phosphatase SptpA from Staphylococcus aureus , which suggests a potentially preserved structural feature among bacterial tyrosine phosphatases. The identification of this structural water provides insight into the known resistance of Mtb PtpA to the oxidative conditions that prevail within an infected host macrophage. This strategy could be applied to extend the understanding of the dynamics and function(s) of proteins in their native state and ultimately aid in the design of small-molecule modulators.

A method for the selective activation of thioglycosides that uses the N + -thiophilic reagent O-mesitylenesulfonylhydroxylamine (MSH) as a promoter is presented. The reaction proceeds via anomeric mesitylensulfonate intermediates, which could be isolated and fully characterized by placing a fluorine atom at the C2 position. In the presence of a soft Lewis acid, glycosylation reaction proceeds at ambient temperature with good yields. It is further demonstrated that it is possible to orthogonally activate S-ethyl in the presence of S-phenyl donors, enabling the design of sequential glycosylation strategies.

Oxetanes are four-membered ring oxygen heterocycles that are advantageously used in medicinal chemistry as modulators of physicochemical properties of small molecules. Herein, we present a simple method for the incorporation of oxetanes into proteins through chemoselective alkylation of cysteine. We demonstrate a broad substrate scope by reacting proteins used as apoptotic markers and in drug formulation, and a therapeutic antibody with a series of 3-oxetane bromides, enabling the identification of novel handles (S-to-S/N rigid, non-aromatic, and soluble linker) and reactivity modes (temporary cysteine protecting group), while maintaining their intrinsic activity. The possibility to conjugate oxetane motifs into full-length proteins has potential to identify novel drug candidates as the next-generation of peptide/protein therapeutics with improved physicochemical and biological properties., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Transition metal catalysis has emerged as a powerful strategy to expand synthetic flexibility of protein modification. Herein, we report a cationic Ru(ii) system that enables the first example of alkyne hydrosilylation between dimethylarylsilanes and O -propargyl-functionalized proteins using a substoichiometric amount or low-loading of Ru(ii) catalyst to achieve the first C-Si bond formation on full-length substrates. The reaction proceeds under physiological conditions at a rate comparable to other widely used bioorthogonal reactions. Moreover, the resultant gem -disubstituted vinylsilane linkage can be further elaborated through thiol-ene coupling or fluoride-induced protodesilylation, demonstrating its utility in further rounds of targeted modifications.

Herein we present a chemical approach for the ready preparation of d-sarmentosyl donors enabling the first total synthesis and structure validation of cardenolide N-1, a challenging 2,6-dideoxy-3-O-methyl-β-d-xylo-hexopyranoside extracted from Nerium oleander twigs that displays anti-inflammatory properties and cell growth inhibitory activity against tumor cells. The strategy highlights the synthetic value of the sequential methodology developed in our group for the synthesis of 2-deoxyglycosides. Key steps include Wittig-Horner olefination of a d-xylofuranose precursor, [I + ]-induced 6-endo cyclization, and 1,2-trans stereoselective glycosylation.

The cleavage of a protecting group from a protein or drug under bioorthogonal conditions enables accurate spatiotemporal control over protein or drug activity. Disclosed herein is that vinyl ethers serve as protecting groups for alcohol-containing molecules and as reagents for bioorthogonal bond-cleavage reactions. A vinyl ether moiety was installed in a range of molecules, including amino acids, a monosaccharide, a fluorophore, and an analogue of the cytotoxic drug duocarmycin. Tetrazine-mediated decaging proceeded under biocompatible conditions with good yields and reasonable kinetics. Importantly, the nontoxic, vinyl ether duocarmycin double prodrug was successfully decaged in live cells to reinstate cytotoxicity. This bioorthogonal reaction presents broad applicability and may be suitable for in vivo applications., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The unstrained S-allyl cysteine amino acid was site-specifically installed on apoptosis protein biomarkers and was further used as a chemical handle and ligation partner for 1,2,4,5-tetrazines by means of an inverse-electron-demand Diels-Alder reaction. We demonstrate the utility of this minimal handle for the efficient labeling of apoptotic cells using a fluorogenic tetrazine dye in a pre-targeting approach. The small size, easy chemical installation, and selective reactivity of the S-allyl handle towards tetrazines should be readily extendable to other proteins and biomolecules, which could facilitate their labeling within live cells., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nature has produced intricate machinery to covalently diversify the structure of proteins after their synthesis in the ribosome. In an attempt to mimic nature, chemists have developed a large set of reactions that enable post-expression modification of proteins at pre-determined sites. These reactions are now used to selectively install particular modifications on proteins for many biological and therapeutic applications. For example, they provide an opportunity to install post-translational modifications on proteins to determine their exact biological roles. Labelling of proteins in live cells with fluorescent dyes allows protein uptake and intracellular trafficking to be tracked and also enables physiological parameters to be measured optically. Through the conjugation of potent cytotoxicants to antibodies, novel anti-cancer drugs with improved efficacy and reduced side effects may be obtained. In this Perspective, we highlight the most exciting current and future applications of chemical site-selective protein modification and consider which hurdles still need to be overcome for more widespread use.

The ability of glycosyldiselenides to act as lectin ligands and their selective detection in plasma by (77)Se NMR is reported.

2-Deoxy-2-fluoroglycosyl iodides are privileged glycosyl donors for the stereoselective preparation of 1-Nu-β-fluorosugars, which are useful reagents for chemical site-selective protein glycosylation. Ready access to such β-fluorosugars enables the mild and efficient construction of well-defined fluoroglycoproteins.

We report the design and synthesis of an aquacarbonyl Ru(II) dication cis-[Ru(CO)2(H2O)4](2+) reagent for histidine (His)-selective metallation of interleukin (IL)-8 at site 33. The artificial, non-toxic interleukin (IL)-8-Ru(II)(CO)2 metalloprotein retained IL-8-dependent neutrophil chemotactic activity and was shown to spontaneously release CO in live cells.

The preparation of challenging 2-deoxy-2-iodo-β-D-allo precursors of 2-deoxy-β-D-ribo-hexopyranosyl units and other analogues is reported using a robust olefination-cyclization-glycosylation sequence. Here, we particularly focus on tuning the stereoelectronic properties of the alkenyl sulfides intermediates in order to improve the diastereoselectivity of the cyclization step and, hence, the efficiency of the overall transformation. Phosphine oxides with the general formula Ph2P(O)CH2SR (R = t-Bu, Cy, p-MeOPh, 2,6-di-ClPh, and 2,6-di-MePh) were easily synthesized and subsequently used in the olefination reaction with 2,3,5-tri-O-benzyl-D-ribose and -D-arabinose. The corresponding sugar-derived alkenyl sulfides were submitted to a 6-endo [I(+)]-induced cyclization, and the resulting 2-deoxy-2-iodohexopyranosyl-1-thioglycosides were used as glycosyl donors for the stereoselective synthesis of 2-deoxy-2-iodohexopyranosyl glycosides. Among the different S-groups studied, t-Bu derivative was the best performer for the synthesis of cholesteryl 2-deoxy-2-iodomannopyranosides, whereas for the synthesis of 2-deoxy-2-iodoallopyranosides none of the derivatives here studied proved superior to the phenyl analogue previously described. Glycosylation of cholesterol with different d-allo and d-manno derivatives produced 2-deoxy-2-iodoglycosides with stereoselectivities in the same order in each case, reinforcing the involvement of an oxocarbenium ion as the common intermediate of this crucial glycosylation step.