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Cleavable linkers have become the subject of intense study in the field of chemical biology, particularly because of their applications in the construction of antibody-drug conjugates (ADC), where they facilitate lysosomal cleavage and liberation of drugs from their carrier protein. Due to lysosomes' acidic nature, acid-labile motifs have attracted much attention, leading to the development of hydrazone and carbonate linkers among several other entities. Continuing our efforts in designing new moieties, we present here a family of cyclic acetals that exhibit excellent plasma stability and acid lability, notably in lysosomes. Incorporated in ADC, they led to potent constructs with picomolar potency in vitro and similar in vivo efficacy as the commercially available ADC Kadcyla in mouse xenograft models.

Bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN alcohol) is the most prominent strained-alkyne scaffold in chemical biology. Described herein is the synthesis of an oxidized analogue – BCN acid – whose facile functionalization via amide bond formation yields more stable derivatives than the classically encountered carbamates.

International audience; Site-selective modification of proteins has been the object of intense studies over the past decades, especially in the therapeutic field. Prominent results have been obtained with recombinant proteins, for which site-specific conjugation is made possible by the incorporation of particular amino acid residues or peptide sequences. In parallel, methods for the site-selective and site-specific conjugation of native and natural proteins are starting to thrive, allowing the controlled functionalization of various types of amino acid residues. Pursuing the efforts in this field, we planned to develop a new type of site-selective method, aiming at the simultaneous conjugation of two amino acid residues. We reasoned that this should give higher chances of developing a site-selective strategy compared to the large majority of existing methods that solely target a single residue. We opted for the Ugi four-center three-component reaction to implement this idea, with the aim of conjugating the side-chain amine and carboxylate groups of two neighbouring lysine and aspartate/glutamate. Herein, we show that this strategy can give access to valuable antibody conjugates bearing several different payloads, and limits the potential conjugation sites to only six on the model antibody trastuzumab. Posttranslational modifications of proteins is Nature's way of generating a rich and diverse proteome from a more limited genetic coding capability. First occurrences of intentional, man-made-artificial-proteins modifications using a defined chemical-thus excluding the food-related Maillard reaction for example-could be dated back to the use of formaldehyde in the tanning industry or for the production of toxoids, 1,2 which evolved later on to immunization studies using chemically-modified bovine serum albumin in the 1900s and eventually led to Landsteiner's synthetic haptenes studies. 3,4 The field of protein modification has since largely benefited from the understanding of proteins' and amino acids' structures coupled to the parallel appearance of more efficient and precise analytical tools. This finally resulted in the development of bioconjugation reagents with excellent chemoselectivity towards various amino acids' side chains groups (i.e. residue-selectivity) that translated into major applications, notably in the pharmaceutical field with the generation of protein-fluorophore adducts for trafficking studies, or the polyethyleneglycol chains functionalization (PEGylation) of proteins to give less-immunogenic and more plasma-stable conjugates. 5,6 However, site selectivity quickly emerged as the main limitation of chemoselective strategies, due to the presence of multiple copies of each type of amino acid residue at the surface of proteins. Statistic conjugation of surface-accessible lysine residues with amine-selective reagents typically results in highly heterogeneous mixtures, containing up to millions of different adducts when large proteins such as antibodies are utilised. 7,8 Each of these adducts possessing distinct physicochemical properties, such chemoselective conjugation necessarily leads to mixtures with different in-vivo pharmacokinetic properties along with virtually no reproducibility in batch-to-batch production. 9,10 Regioselective (i.e., site-specific) methods were thus developed and are currently dominated by the use of recombinant proteins, incorporating exogenous amino acid residues-natural or unnatural-or peptide sequences that can be specifically targeted by a tailored reagent or strategy. 11-13 In parallel, site-selective chemical strategies for the conjugation of native and natural proteins have also flourished over the past few years, giving rise to methods targeting various types of amino acids-e.g. lysine, cysteine, tryptophan, tyrosine-that proved to be effective on proteins of all sorts of sizes, including antibodies. 14-28 With the aim of pursuing the efforts in this field, we could not help but notice that the vast majority of previously reported strategies for the site-selective conjugation of native proteins were focused on the modification of a unique residue. We hypothesized that targeting two different amino acid side chains simultaneously would lower the enormous subset of possibilities given by single-residue bioconjugation techniques, thus increasing our chances of developing a site-selective method by minimising the number

The bioconjugation of proteins—that is, the creation of a covalent link between a protein and any other molecule—has been studied for decades, partly because of the numerous applications of protein conjugates, but also due to the technical challenge it represents. Indeed, proteins possess inner physico-chemical properties—they are sensitive and polynucleophilic macromolecules—that make them complex substrates in conjugation reactions. This complexity arises from the mild conditions imposed by their sensitivity but also from selectivity issues, viz the precise control of the conjugation site on the protein. After decades of research, strategies and reagents have been developed to address two aspects of this selectivity: chemoselectivity—harnessing the reacting chemical functionality—and site-selectivity—controlling the reacting amino acid residue—most notably thanks to the participation of synthetic chemistry in this effort. This review offers an overview of these chemical bioconjugation strategies, insisting on those employing native proteins as substrates, and shows that the field is active and exciting, especially for synthetic chemists seeking new challenges.

Context Anonidium mannii (Annonaceae) has been traditionally used in Africa to treat stomach aches, schistosomiasis, and many other illnesses. However, few phytochemical study and no investigation on schistosomiasis have been conducted on this species. This neglected tropical disease, caused by a worm, comes second after malaria as the most devastating parasitical infection. Aim: The goal of this study was to evaluate the anti-Schistosoma mansoni activity of fractions and constituents from A. mannils stem bark and also to search efficient inhibitors of a recently discovered ectoenzyme of S. mansoni (S. mansoni nicotinamide adenine dinucleotide + catabolizing enzyme (SmNACED. Materials and Methods: The powdered stem bark of A. mannii was extracted with ethanol/distilled water (80:20). The extract was then subjected to a partial bioguided separation by chromatography means. The structures of compounds were elucidated using modern spectroscopic techniques. Furthermore, isolated and semisynthetic compounds were evaluated for their antischistosomal and cytotoxic activities. Results: Chemical investigation led to the isolation and identification of eight compounds, in the majority, obtained for the first time from this genus. In addition, acetylation reactions were carried out to afford a new semisynthetic derivative. Preliminary biological screening of the extracts and compounds showed very good activities from antiparasitic and enzymatic tests and also very good percentage of cell viability evaluation. Conclusion: Like praziquantel drug, gallic acid exhibited full anthelmintic activity at concentration of 100 mu M. On the other hand, piperolactam D showed important inhibition on SmNACE (IC50 10 mu M). Thus, standardization of bioactive fraction can help in improving traditional medicine. The optimization of those two compounds will enhance their selectivity/effectiveness and could be used as seed for the development of new remedies against schistosomiasis. Further, the study will be focus on other pathogens species of Schistosoma genus.

International audience; Small interfering RNAs (siRNAs) can down-regulate the expression of a target mRNA molecule in a sequence-specific manner, making them an attractive new class of drugs with broad potential for the treatment of diverse human diseases. Here, we report the synthesis of a series of cationic amphiphiles which were obtained by the coupling of amino acids and dipeptides onto a lipidic double chain. The new amphiphiles presenting a peptidic motif on a short hydrophilic spacer group were evaluated for selective gene silencing through RNA interference. Our results show that tryptophan residues boost siRNA delivery in an unexpected manner. The silencing experiments performed with very low concentrations of siRNA showed that the best formulations could induce significant death of tumor cells after silencing of polo-like kinase 1 which is implicated in cell cycle progression. In addition, these Trp containing peptide amphiphiles were highly efficient siRNA delivery vectors even in presence of competing serum proteins.

We report the synthesis and use of sydnone-based profluorophores as tools for imaging applications. These new probes display exquisite reactivity towards strain promoted cycloaddition reactions with cycloalkynes allowing fast, efficient and selective labeling in biological media. Styryl-pyridinium sydnone probes were found particularly interesting for click reactions to proceed selectively inside cells.

Antibody-Oligonucleotide Conjugates (AOCs) represent an emerging class of functionalized antibodies that have already been used in a wide variety of applications. While the impact of dye and drug conjugation on antibodies’ ability to bind their target has been extensively studied, little is known about the effect caused by the conjugation of hydrophilic and charged payloads such as oligonucleotides on the functions of an antibody. Previous observations of non-specific interactions of nucleic acids with untargeted cells prompted us to further investigate their impact on AOC binding abilities and cell selectivity. We synthesized a series of single- and double-stranded AOCs, as well as a human serum albumin-oligonucleotide conjugate, and studied their interactions with both targeted and non-targeted living cells using a time-resolved analysis of ligand binding assay. Our results indicate that conjugation of single strand oligonucleotides to proteins induce consistent non-specific interactions with cell surfaces while double strand oligonucleotides have little or no effect, depending on the preparation method.

International audience; A new alkaloid, manniindole 1, together with four known compounds: aristolactam AII 2, aristolactam BII 3, piperolactam D 4 and polycarpol 5 were isolated from the crude extract EtOHH2O (8:2) of the roots of Anonidium mannii by chromatographic separation. The structure elucidation was performed on the basis of a spectroscopic analysis (IR, HRESI MS, 1D and 2D NMR) as well as a comparison of their spectral data with those reported in the literature. For the first time, the crude extract and those isolated compounds were evaluated for their antischistosomal activity against Schistosoma mansoni and for cytotoxicity activity against Huh7 and A549 cells. Furthermore, they were also tested in vitro on the recent characterized Schistosoma mansoni NADþ catabolizing enzyme (SmNACE) for their impact on this enzyme which is localized on the outer surface of the adult parasite. Compound 2 displayed quite good worm killing capability, while 4 showed significant inhibition of SmNACE

A new alkaloid, manniindole

Controlled protein functionalization holds great promise for a wide variety of applications. However, despite intensive research, the stoichiometry of the functionalization reaction remains difficult to control due to the inherent stochasticity of the conjugation process. Classical approaches that exploit peculiar structural features of specific protein substrates, or introduce reactive handles via mutagenesis, are by essence limited in scope or require substantial protein reengineering. We herein present equimolar native chemical tagging (ENACT), which precisely controls the stoichiometry of inherently random conjugation reactions by combining iterative low-conversion chemical modification, process automation, and bioorthogonal trans-tagging. We discuss the broad applicability of this conjugation process to a variety of protein substrates and payloads., Controlled protein functionalization holds great promise for a wide variety of applications.

Here we present the synthesis and evaluation of antibody-drug conjugates (ADCs), for which antibody and drug are non-covalently connected using complementary DNA linkers. These ADCs are composed of trastuzumab, an antibody targeting HER2 receptors overexpressed on breast cancer cells, and monomethyl auristatin E (MMAE) as a drug payload. In this new ADC format, trastuzumab conjugated to a 37-mer oligonucleotide (ON) was prepared and hybridized with its complementary ON modified at 5-end with MMAE (cON-MMAE) in order to obtain trastuzumab-DNA-MMAE. As an advantage, the cON-MMAE was completely soluble in water, which decreases overall hydrophobicity of toxic payload, an important characteristic of ADCs. The stability in the human plasma of these non-engineered ON-based linkers was investigated and showed a satisfactory half-life of 5.8 days for the trastuzumab-DNA format. Finally, we investigated the in vitro cytotoxicity profile of both the DNA-linked ADC and the ON-drug conjugates and compared them with classical covalently linked ADC. Interestingly, we found increased cytotoxicity for MMAE compared to cON-MMAE and an EC50 in the nanomolar range for trastuzumab-DNA-MMAE on HER2-positive cells. Although this proved to be less potent than classically linked ADC with picomolar range EC50, the difference in cytotoxicity between naked payload and conjugated payload was significant when an ON linker was used. We also observed an interesting increase in cytotoxicity of trastuzumab-DNA-MMAE on HER2-negative cells. This was attributed to enhanced non-specific interaction triggered by the DNA strand as it could be confirmed using ligand tracer assay.

As multidrug resistant pathogenic microorganisms are a serious health menace, it is crucial to continuously develop novel medicines in order to overcome the emerging resistance. The methylerythritol phosphate pathway (MEP) is an ideal target for antimicrobial development as it is absent in humans but present in most bacteria and in the parasite Plasmodium falciparum. Here, we report the synthesis and the steady-state kinetics of a novel potent inhibitor (MEPN3) of Escherichia coli YgbP/IspD, the third enzyme of the MEP pathway. MEPN3 inhibits E. coli YgbP/IspD in mixed type mode regarding both substrates. Interestingly, MEPN3 shows the highest inhibitory activity when compared to known inhibitors of E. coli YgbP/IspD. The mechanism of this enzyme was also studied by steady-state kinetic analysis and it was found that the substrates add to the enzyme in sequential manner.

International audience; Dengue virus (DENV), a mosquito-borne flavivirus, causes severe and potentially fatal symptoms in millions of infected individuals each year. Although dengue fever represents a major global public health problem, the vaccines or antiviral drugs proposed so far have not shown sufficient efficacy and safety, calling for new antiviral developments. Here we have shown that a mannoside glycolipid conjugate (MGC) bearing a trimannose head with a saturated lipid chain inhibited DENV productive infection. It showed remarkable cell promiscuity, being active in human skin dendritic cells, hepatoma cell lines and Vero cells, and was active against all four DENV serotypes, with an IC 50 in the low micromolar range. Time-of-addition experiments and structure-activity analyses revealed the importance of the lipid chain to interfere with an early viral infection step. This, together with a correlation between antiviral activity and membrane polarization by the lipid moiety indicated that the in-hibitor functions by blocking viral envelope fusion with the endosome membrane. These finding establish MGCs as a novel class of antivirals against the DENV.

International audience; Here, we introduce 4-azidophenyl glyoxal (APG) as an efficient plug-and-play reagent for the selective functionalisation of arginine residues in native antibodies. The selective reaction between APG and arginines’ guanidine groups allowed a facile introduction of azide groups on the monoclonal antibody trastuzumab (plug stage). These pre-functionalised antibody–azide conjugates were then derivatised during the “play stage” via a biorthogonal cycloaddition reaction with different strained alkynes. This afforded antibody-fluorophore and antibody–oligonucleotide conjugates, all showing preserved antigen selectivity and high stability in human plasma. Due to a lower content of arginines compared to lysines in native antibodies, this approach is thus attractive for the preparation of more homogeneous conjugates. This method proved to be orthogonal to classical lysine-based conjugation and allowed straightforward generation of dual-payload antibody.

The reduction–rebridging strategy is a powerful method for the preparation of stable and homogeneous antibody–drug conjugates (ADCs). In this communication, we describe the development of the arylene-dipropiolonitrile (ADPN) functional group for the rebridging of reduced disulphide bonds and its application in the preparation of potent and selective ADCs.

Simultaneous access to several targets has become the subject of intense studies in immuno-oncology. In order to benefit from the synergies provided by the activation of different signaling pathways in immunology and the knockdown of proteins involved in cancer cell survival, we established a bispecific approach. The activation of the innate immune response by delivering agonists of pattern-recognition receptors (PRR) such as RIG-I (retinoic acid-inducible I) represents a promising strategy. RIG-I detects short double-stranded RNA molecules ended by a 5'-di/triphosphate moiety (5'ppp-dsRNA). RIG-I activation promotes type I IFN secretion and cancer-cell selective apoptosis. To obtain a bifunctional molecule, the 5'ppp-dsRNA sequence was designed to silence PLK1 (polo-like kinase 1). Suppressing PLK1 expression with small interfering RNAs (siRNA) leads to cell cycle arrest and retards cancer cell growth. This concept of bifunctional RNAs has been validated by using non-targeted systems.1 To enhance this synergy, we conjugated this 5'ppp-siPLK1 to an antibody for a specific delivery to cancerous cells that overexpress erythropoietin-producing hepatocellular receptor A2 (EphA2) at their surface. Upon binding to EphA2 receptor, the antibody is well internalized, thus making it a good vehicle to deliver the bispecific 5'ppp-siPLK1. After EphA2-positive cells treatment, we observed RIG-I specific activation as well as PLK1 depletion. Both effects were correlated with cellular apoptosis and the mode of action was further confirmed with mechanistic and kinetic studies. Finally, while non-modified unconjugated siRNA has a very short half-life in plasma, we observed an increase in stability for the antibody-5'ppp-siPLK1 conjugates. These data suggest that anti-EphA2 receptor antibody could be used to deliver a bispecific RNA molecule. References:1H. Poeck, R. Besch, C. Maihoefer, M. Renn, D. Tormo, S. S. Morskaya, S. Kirschnek, E. Gaffal, J. Landsberg, J. Hellmuth, A. Schmidt, D. Anz, M. Bscheider, T. Schwerd, C. Berking, C. Bourquin, U. Kalinke, E. Kremmer, H. Kato, S. Akira, R. Meyers, G. Häcker, M. Neuenhahn, D. Busch, J. Ruland, S. Rothenfusser, M. Prinz, V. Hornung, S. Endres, T. Tüting and G. Hartmann, 5′-triphosphate-siRNA: turning gene silencing and Rig-I activation against melanoma, Nat. Med., 2008, 14, 1256-1263. Citation Format: Tony Rady, Stéphane Erb, Safia Deddouche-Grass, Renaud Morales, Hervé Bouchard, Guilhem Chaubet, Sarah Cianférani, Dmitri Wiederschain, Nicolas Basse, Alain Wagner. Antibody conjugated to a bispecific RNA molecule targeting RIG-I and PLK1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 691.

We report the discovery of a new bioorthogonal click-and-release reaction involving iminosydnones and strained alkynes. This transformation leads to two products resulting from the ligation and fragmentation of iminosydnones under physiological conditions. Optimized iminosydnones were successfully used to design innovative cleavable linkers for protein modification, thus opening up new areas in the fields of drug release and target-fishing applications. This click-and-release technology offers the possibility of exchanging tags on proteins for functionalized cyclooctynes under mild and bioorthogonal conditions.

Drugs, usually long acting and metabolically stable molecules, might be the source of adverse effects triggered by complex drug interactions, anaphylaxis and drug-induced coagulopathy. To circumvent this growing drug safety issue, we herein investigate the opportunity offered by bio-orthogonal chemistry for in vivo drug neutralization. We design a small-molecule anticoagulant drug (Warfarin) containing an azide group that acts as a safety pin. It allows drug deactivation and restoration of physiological coagulation via in vivo click reaction with a suitable cyclooctyne-based neutralizing agent. In this strategy, the new molecule formed by reaction of the drug and the antidote is deprived of biological activity and prone to fast renal clearance. This ‘Click & Clear' approach lays ground for new strategies in designing drugs with switchable biophysical properties., The interaction of long circulating drugs with biological molecules might trigger harmful side effects. Here, the authors design an anticoagulant compound that can be deactivated in vivo using click chemistry, paving the way to the design of drugs with tunable in vivo properties.

The biochemical characteristics of hetero-bifunctional cross-linkers used in bioconjugates are of essential importance to the desired features of the final adduct (i.e. antibody-drug conjugates). These include stability in biological media, chemical and biological reactivities, cleavability under defined conditions, and solubility. In our previous work, we introduced a new amino-to-thiol linker, maleimidomethyl dioxane (MD), as an alternative to classical maleimide conjugation, with increased hydrophilicity and serum stability due to succinimidyl ring-opening. In this work, we investigate the generality of linkers containing a dioxo-ring with regard to their ability to self-hydrolyze and their surprising stability at a low pH. We synthesized four FRET probes which allowed us to address the stability of the dioxo-ring and to study the maleimide ring-opening and the thiol-exchange processes by means of detecting and measuring the generation of fluorescence. It was found that the ring expansion (from a 5- to a 6-membered ring) improved the stability of the probes in aqueous media, and the increase of the chain length between the dioxo-ring and the succinimide ring (from methylene to ethylene) decreased the rate of succinimidyl ring-opening.

Antibody-oligonucleotide conjugates (AOCs) are a novel class of synthetic chimeric biomolecules that has been continually gaining traction in different fields of modern biotechnology. This is mainly due to the unique combination of the properties of their two constituents, exceptional targeting abilities and antibody biodistribution profiles, in addition to an extensive scope of oligonucleotide functional and structural roles. Combining these two classes of biomolecules in one chimeric construct has therefore become an important milestone in the development of numerous biotechnological applications, including imaging (DNA-PAINT), detection (PLA, PEA), and therapeutics (targeted siRNA/antisense delivery). Numerous synthetic approaches have been developed to access AOCs ranging from stochastic chemical bioconjugation to site-specific conjugation with reactive handles, introduced into antibody sequences through protein engineering. This Review gives a general overview of the current status of AOC applications with a specific emphasis on the synthetic methods used for their preparation. The reported synthetic techniques are discussed in terms of their practical aspects and limitations. The importance of the development of novel methods for the facile generation of AOCs possessing a defined constitution is highlighted as a priority in AOC research to ensure the advance of their new applications.

Chemical Biology is the science of designing chemical tools to dissect and manipulate biology at different scales. It provides the fertile ground from which to address important problems of our society, such as human health and environment.

International audience; A novel generation of pH-responsive photopolymerized diacetylenic amphiphile (PDA) micelles with a diameter of 10 nm was designed and optimized for the intracellular delivery of siRNAs. Dialysis and photopolymerization of the micelles allowed a strong reduction of the cytotoxicity of the nanovector, while the hydrophilic histidine headgroup permitted enhancing the siRNA delivery potential by improving the endosomal escape via imidazole protonation. These PDA-micellar systems were fully characterized by DLS, TEM, and DOSY-NMR experiments. The resulting bioactive complexes of PDA-micelles with siRNA were shown to have an optimal size below 100 nm.

The third generation of aminobiphenyl palladacycle pre-catalyst "G3-Xantphos" enables functionalization of peptides containing cysteine in high yields. The conjugation (bioconjugation) occurs chemoselectively at room temperature under biocompatible conditions. Extension of the method to protein functionalization allows selective bioconjugation of the trastuzumab antibody.

In droplet-based microfluidic, fluorosurfactants are essential to ensure the stability of the emulsion. Beyond this primary role, fluorosurfactants can be engineered to provide droplet inner surface specific interaction characteristics with analyte present in droplet content. Despite the high potency of such capture system in terms of micro-compartmentalisation and surface/analyte ratio, only few studies have reported the use of the water/fluorinated oil interphase to immobilize target molecules. The difficult synthesis of the required functionalized fluorosurfactants needed for each application may account for this relative desertion. To make microdroplet capture approaches more straightforward, we have investigated a ready-to-use click chemistry-based approach that enables intra-droplet chemical modification. This strategy, which avoids tedious synthesis of complex fluorosurfactant, opens access to a wide variety of functional heads via copper-free click chemistry using a pre-functionalized fluorosurfactant which can be easily obtained in large scale. To demonstrate the efficiency of the click chemistry-based microdroplet surface functionalization, we have synthesized an azide fluorosurfactant capable of stabilizing microdroplets and performed a series of intra-droplet surface functionalizations by introducing fluorescent-labeled cycloalkyne derivatives in the aqueous phase. By doing so, we were able to demonstrate via polarization fluorescence that molecules from the aqueous phase could be efficiently captured at the inner droplet surface. Furthermore, we also showed that the density of azide functions at the inner surface could be adjusted by diluting the functionalized surfactant with a non-functionalized one. Fluorescence polarization analysis revealed that these dilutions result in the production of microdroplets with controlled azide surface density.

This review will discuss recent development in the bioconjugation of lysine residues on antibodies. As several chemoselective reagents have already been developed for modifying amine groups, recent strategies now tend to aim at being site-specific. Four general methods have been listed: kinetically controlled, template-directed or enzymatic strategies as well as the use of chemically programmed antibodies.

We designed a convergent synthesis pathway that provides access to trifunctional oligoethyleneglycol-amine (OEG-amine) linkers. By applying the reductive coupling of a primary azide to bifunctional OEG-azide precursors, the corresponding symmetrical dialkylamine bearing two homo-functional end chain groups and a central nitrogen was obtained. These building blocks bear minimal structural perturbation compared to the native OEG backbone which makes them attractive for biomedical applications. The NMR investigations of the mechanism process reveal the formation of nitrile and imine intermediates which can react with the reduced free amine form. Additionally, these trifunctional OEG-amine linkers were employed in a coupling reaction to afford branched multifunctional PEG dendrons which are molecularly defined. These discrete PEG-based dendrons (n = 16, 18 and 36) could be useful for numerous applications where multivalency is required.

International audience; Recently, it has been shown that the efficiency of antitumoral drugs can be enhanced when combined with therapeutic siRNAs. In the present study, an original platform based on polydiacetylenic micelles containing a cationic head group able to efficiently deliver a small interfering RNA (siRNA) targeting the PLK-1 gene while offering a hydrophobic environment for encapsulation of lipophilic drugs such as camptothecin is developed. We demonstrate that the co-delivery of these two agents with our micellar system results in a synergistic tumor cell killing of cervical and breast cancer cell lines in vitro. The combined drugs are active in a subcutaneous in vivo cancer model. Altogether, the results show that our nanometric micellar delivery system can be used for the development of new drug–siRNA combo-therapies.

Polydiacetylenic nanofibers (PDA-Nfs) obtained by photopolymerization of surfactant 1 were optimized for intracellular delivery of small interfering RNAs (siRNAs). PDA-Nfs/siRNA complexes efficiently silenced the oncogene Lim-1 in the renal cancer cells 786-O in vitro. Intraperitoneal injection of PDA-Nfs/siLim1 downregulated Lim-1 in subcutaneous tumor xenografts obtained with 786-O cells in nude mice. Thus, PDA-Nfs represent an innovative system for in vivo delivery of siRNAs.

Thiols are among the most frequently used functional groups in the field of bioconjugation. While there exists a variety of heterobifunctional reagents that allow for coupling thiols to other functions (e.g., amines, carboxylic acids), there is no specific reagent for creating heteroconjugates using two different thiols. In response to the ever-increasing demand for bioconjugation tools, we have developed p-(maleimide)-phenylpropionitrile (MAPN)-an efficient reagent for kinetically resolved thiol-to-thiol coupling. In a comparative study with its closest commercially available analogue, p-phenylenedimaleimide, MAPN has shown substantial advantages for the preparation of thiol-thiol heteroconjugates. Namely, an antibody-drug conjugate (ADC) with mertansine (DM1), conjugated to the cysteine residues of Trastuzumab, was prepared for the first time.

A series of polydiacetylene (PDA) - based micelles were prepared from diacetylenic surfactant bearing polyethylene glycol, by increasing UV-irradiation times. These polymeric lipid micelles were analyzed by physicochemical methods, electron microscopy and NMR analysis. Cellular delivery of fluorescent dye suggests that adjusting the polymerization state is vital to reach the full in vitro potential of PDA-based delivery systems.

Bioconjugation methodologies have proven to play a central enabling role in the recent development of biotherapeutics and chemical biology approaches. Recent endeavours in these fields shed light on unprecedented chemical challenges to attain bioselectivity, biocompatibility, and biostability required by modern applications. In this review the current developments in various techniques of selective bond forming reactions of proteins and peptides were highlighted. The utility of each endogenous amino acid-selective conjugation methodology in the fields of biology and protein science has been surveyed with emphasis on the most relevant among reported transformations; selectivity and practical use have been discussed.

The selective destruction of tumour cells while sparing healthy tissues is one of the main challenges in cancer therapy. Antibody-drug conjugates (ADCs) are arguably the most rapidly expanding class of targeted cancer therapies. Efficient drug conjugation and release technologies are essential for the development of these new therapeutic agents. In response to the ever-increasing demand for efficient drug release systems, we have developed a new class of β-galactosidase-cleavable linkers for ADCs. Within this framework, novel payloads comprising a galactoside linker, the monomethyl auristatin E (MMAE) and cysteine-reactive groups were synthesized, conjugated with trastuzumab and evaluated both in vitro and in vivo. The ADCs with galactoside linkers demonstrated superior therapeutic efficacy in mice compared to the marketed trastuzumab emtansine used for the treatment of breast cancer.

We report a plug-and-play strategy for the preparation of functionally enhanced antibodies with a defined average degree of conjugation (DoC). The first stage (plug) allows the controllable and efficient installation of azide groups on lysine residues of a native antibody using 4-azidobenzoyl fluoride. The second step (play) allows for versatile antibody functionalization with a single payload or combination of payloads, such as a toxin, a fluorophore, or an oligonucleotide, via copper-free strain-promoted azide–alkyne cycloaddition (SPAAC). It is notable that in comparison to a classical N-hydroxysuccinimide ester (NHS) strategy, benzoyl fluorides show faster and more efficient acylation of lysine residues in a PBS buffer. This translates into better control of the DoC and enables the efficient and fast functionalization of delicate biomolecules at low temperature.

The pathogenesis of autoimmune diseases has not been completely elucidated yet, and only a few specific treatments have been developed so far. In autoimmune diseases mediated by pathogenic autoantibodies, such as systemic lupus erythematosus, the specific detection and analysis of autoreactive B cells is crucial for a better understanding of the physiopathology. Biological characterization of these cells may help to define new therapeutic targets. Very few techniques allowing the precise detection of autoreactive B cells have been described so far. Herein we propose a new flow cytometry technique for specific detection of anti-nucleosome B cells, which secrete autoantibodies in systemic lupus erythematosus, using labeled nucleosomes. We produced different fluorochrome-labeled nucleosomes, characterized them, and finally tested them in flow cytometry. Nucleosomes labeled via the cysteines present in H3 histone specifically bind to autoreactive B cells in the anti-DNA transgenic B6.56R mice model. The present work validates the use of fluorochrome-labeled nucleosomes via cysteines to identify anti-nucleosome B cells and offers new opportunities for the description of autoreactive B cell phenotype.

The concept of chelation-assisted copper catalysis was employed for the development of new azides that display unprecedented reactivity in the copper(I)-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) reaction. Azides that bear strong copper-chelating moieties were synthesized; these functional groups allow the formation of azide copper complexes that react almost instantaneously with alkynes under diluted conditions. Efficient ligation occurred at low concentration and in complex media with only one equivalent of copper, which improves the biocompatibility of the CuAAC reaction. Furthermore, such a click reaction allowed the localization of a bioactive compound inside living cells by fluorescence measurements.

Exquisite chemoselectivity for cysteine has been found for a novel class of remarkably hydrolytically stable reagents, 3-arylpropiolonitriles (APN). The efficacy of the APN-mediated tagging was benchmarked against other cysteine-selective methodologies in a model study on a series of traceable amino acid derivatives. The selectivity of the methodology was further explored on peptide mixtures obtained by trypsin digestion of lysozyme. Additionally, the superior stability of APN-cysteine conjugates in aqueous media, human plasma, and living cells makes this new thiol-click reaction a promising methodology for applications in bioconjugation.

The synthesis of two molecules, B1 and B2, based on elements of norbadione A, the natural Cs + chelator in mushrooms, associated, in the case of B2, with an 18-crown-6 ether is reported. Thermodynamic and kinetic analyses performed in water, ethanol and ethanol/water 9/1 v/v (M1) show in M1 and ethanol that B1 and B2 form stable complexes with Na + , K + and Cs + . Affinity constants, measured spectrophotometrically in ethanol and M1, by the use of the S pecfit program, are in the 10 5 and 10 6 range for B1 and B2, respectively. The second-order rate constants are in the 10 6 –10 7 M −1 s −1 range and the first-order rate constants about unity. The ratios of the second-order/first-order rate constants confirm the thermodynamic results in EtOH. The kinetic processes become much too fast to allow runs in M1. Molecular simulations in EtOH imply the existence of two isomers for each of the Cs + /B1 and Cs + /B2 complexes. With B1, the more stable one is that in which the two enolates are parallel and mimic the alkali-metal inclusion cavity already envisaged for norbadione A. With B2, two similar structures are extracted, in both of which Cs + is included in the crown ether and capped by the enolate. The affinity of B1 for Cs + is comparable to that of norbadione A, whereas that of B2 is higher. These results are encouraging as they introduce a new series of alkali chelators which can lead to molecules capable of complexing 137 Cs + for radioactive decontamination.

We report the synthesis and reactivity of 4-fluorosydnones, a unique class of mesoionic dipoles displaying exquisite reactivity towards both copper-catalyzed and strain-promoted cycloaddition reactions with alkynes. Synthetic access to these new mesoionic compounds was granted by electrophilic fluorination of σ-sydnone Pd(II) precursors in the presence of Selectfluor. Their reactions with terminal and cyclic alkynes were found to proceed very rapidly and selectively, affording 5-fluoro-1,4-pyrazoles with bimolecular rate constants up to 10(4) m(-1) s(-1) , surpassing those documented in the literature with cycloalkynes. Kinetic studies were carried out to unravel the mechanism of the reaction, and the value of 4-fluorosydnones was further highlighted by successful radiolabeling with [(18) F]Selectfluor.

The vast majority of antibody-drug conjugates (ADC) are prepared through amine-to-thiol conjugation. To date, N-Succinimidyl-4-(maleimidomethyl) cyclohexanecarboxylate (SMCC) has been one of the most frequently applied reagents for the preparation of ADC and other functional conjugates. However, SMCC-based conjugates suffer from limited stability in blood circulation and from a hydrophobic character of the linker, which may give rise to major pharmacokinetic implications. To address this issue, we have developed a heterobifunctional analogue of a SMCC reagent, i.e., sodium 4-(maleimidomethyl)-1,3-dioxane-5-carbonyl)oxy)-2,3,5,6- tetrafluorobenzenesulfonate (MDTF) for amine-to-thiol conjugation. By replacing the cyclohexyl ring in the SMCC structure with the 1,3-dioxane, we increased the hydrophilicity of the linker. A FRET probe based on MD linker was prepared and showed superior stability compared to the MCC linker in human plasma, as well as in a variety of aqueous buffers. A detailed investigation demonstrated an accelerated succinimide ring opening for MD linker, resulting in stabilized conjugates. Finally, the MDTF reagent was applied for the preparation of serum stable antibody-dye conjugate.

A multi-disciplinary approach was used to identify the first pharmacophore model for KCC2 blockers: several physico-chemical studies such as XRD and NMR were combined to molecular modelling techniques, SAR analysis and synthesis of constrained analogues in order to determine a minimal conformational space regrouping few potential bioactive conformations. These conformations were further compared to the conformational space of a different series of KCC2 blockers in order to identify the common pharmacophoric features. The synthesis of more potent analogues in this second series confirmed the usefulness of this KCC2 blocker pharmacophore model.

A screening strategy based on hyphenated capillary electrophoresis and inductively coupled plasma mass spectrometry (CE–ICP-MS) was developed to classify phosphorylated ligands according to their europium(III) binding affinity in a hydro-organic medium (sodium formate, pH 3.7, H2O/MeOH 90:10, v/v). Taking advantage of the high sensibility of ICP-MS for detecting phosphorus, this method enabled to assess the affinity of a variety of phosphorylated compounds, including phosphine oxides, thiophosphines, phosphonates, and phosphinates, in less than 1 h and using less than 5 ng of substance. By varying the total europium concentration, complexation constants could be determined according to a sequential multiple run strategy, which proved to be in excellent agreement with the values obtained by UV–Vis absorption spectrophotometric titrations.

Interest in cleavable linkers is growing due to the rapid development and expansion of chemical biology. The chemical constrains imposed by the biological conditions cause significant challenges for organic chemists. In this review we will present an overview of the cleavable linkers used in chemical biology classified according to their cleavage conditions by enzymes, nucleophilic/basic reagents, reducing agents, photo-irradiation, electrophilic/acidic reagents, organometallic and metal reagents, oxidizing reagents.

Antioxidative activity expressed as protection of thymidine has been investigated for a set of 30 pulvinic acid derivatives. A combination of in vitro testing and in silico modeling was used for synthesis of new potential antioxidants. Experimental data obtained from a primary screening test based on oxidation under Fenton conditions and by an UV exposure followed by back-titration of the amount of thymidine remaining intact have been used to develop a computer model for prediction of antioxidant activity. Structural descriptors of 30 compounds tested for their thymidine protection activity were calculated in order to define the structure-property relationship and to construct predictive models. Due to the potential nonlinearity, the counter-propagation artificial neural networks were assessed for modeling of the antioxidant activity of these compounds. The optimized model was challenged with 80 new molecules not present in the initial training set. The compounds with the highest predicted antioxidant activity were considered for synthesis. Among the predicted structures, some coumarine derivatives appeared to be especially interesting. One of them was synthesized and tested on in vitro assays and showed some antioxidant and radioprotective activities, which turned out as a promising lead toward more potent antioxidants.

Several phosphinoylimines have been synthesized in five steps by starting from the appropriate phosphane oxide and were then treated with methylmagnesium bromide to give both diastereoisomers in high yields and with promising diastereomeric ratios. Then N-[(tert-butyl)(phenyl)phosphinoyl]benzaldimine, which displayed the best results, was subjected to the 1,2-addition of various Grignard reagents to evaluate the best chiral induction due to the stereogenic phosphorus atom. The corresponding adducts were obtained in excellent yields and with moderate to excellent diastereoisomeric ratios.

An unprecedented and highly diastereoselective 6-endo-trig cyclization of 2-alkenyl-1,3-dithiolanes has been developed yielding trans-decalins, an important scaffold present in numerous di- and triterpenes. The novelty of this 6-endo-trig cyclization stands in the stepwise mechanism involving 2-alkenyl-1,3-dithiolane, acting as a novel latent initiator. It is suggested that the thioketal opens temporarily under the influence of TMSOTf, triggering the cationic 6-endo-trig cyclization, and closes after C-C bond formation and diastereoselective protonation to terminate the process. DFT calculations confirm this mechanistic proposal and provide a rationale for the observed diastereoselectivity. The reaction tolerates a wide range of functionalities and nucleophilic partners within the substrate. We have also shown that the one-pot 6-endo-trig cyclization followed by in situ 1,3-dithiolane deprotection afford directly the corresponding ketone. This improvement allowed the achievement of the shortest total synthesis of triptophenolide and the shortest formal synthesis of triptolide.