An amino acid-based heterofunctional cross-linking reagent (original) (raw)
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This is a multidisciplinary research project in the field of bioconjugation chemistry. Bioconjugation chemistry, in its fundamental aspects, refers to the site specific covalent modification of bio-molecules adding or modulating desired characteristics. The recent advances in bioconjugation approaches have enabled the progressive construction of groundbreaking monoclonal antibodies (mAbs) biotherapies, such as antibody drug conjugates (ADCs) and bispecific antibodies. However, there is still a long list of limitations encountered by the currents approaches such as poor plasma stability, postmodification structural stability and heterogeneity of the produced conjugates, which are usually tackled by expensive and complex biological-based techniques. Therefore, in this study, we aimed to develop novel conjugation chemistry suitable for the construction of antibody-protein conjugates with promising anti-cancer activity. Production of protein conjugates is usually achieved through targeting thiolate groups of their cysteine residues. We aimed to develop an effective reduction method for activation of thiolates towards conjugation reactions. We developed and evaluated a novel one-pot method by using water soluble azide-derivatised ethylene glycols (PEGazides) to quench excess trialkylphosphines prior to thiol alkylation reactions. The rates of oxidation of trialkylphosphines with a series of PEG-azides were determined and the x 4.5.1 Determination of the reactivity of methyl 3,5-bis(2haloacetamido)benzoate (4.22 and 4.24) and methyl 3,4-bis(2haloacetamido)benzoate (4.23 and 4.25
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Journal of Medicinal Chemistry, 2011
The synthesis and biological evaluation of hydrophilic heterobifunctional cross-linkers for conjugation of antibodies with highly cytotoxic agents are described. These linkers contain either a negatively charged sulfonate group or a hydrophilic, noncharged PEG group in addition to an amine-reactive Nhydroxysuccinimide (NHS) ester and sulfhydryl reactive termini. These hydrophilic linkers enable conjugation of hydrophobic organic molecule drugs, such as a maytansinoid, at a higher drug/antibody ratio (DAR) than hydrophobic SPDB and SMCC linkers used earlier without triggering aggregation or loss of affinity of the resulting conjugate. AntibodyÀmaytansinoid conjugates (AMCs) bearing these sulfonate-or PEG-containing hydrophilic linkers were, depending on the nature of the targeted cells, equally to more cytotoxic to antigen-positive cells and equally to less cytotoxic to antigen-negative cells than conjugates made with SPDB or SMCC linkers and thus typically displayed a wider selectivity window, particularly against multidrug resistant (MDR) cancer cell lines in vitro and tumor xenograft models in vivo.
Catalytic Cleavage of Disulfide Bonds in Small Molecules and Linkers of Antibody–Drug Conjugates
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In cells, catalytic disulfide cleavage is an essential mechanism in protein folding and synthesis. However, detailed enzymatic catalytic mechanism relating cleavage of disulfide bonds in xenobiotics is not well understood. This study reports an enzymatic mechanism of cleavage of disulfide bonds in xenobiotic small molecules and antibody conjugate (ADC) linkers. The chemically stable disulfide bonds in substituted disulfide-containing pyrrolobenzodiazepine (PBD, pyrrolo[2,1-c][1,4]benzodiazepine) monomer prodrugs in presence of glutathione or cysteine were found to be unstable in incubations in whole blood of humans and rats. It was shown the enzymes involved were thioredoxin (TRX) and glutaredoxin (GRX). For a diverse set of drug-linker conjugates, we determined that TRX in the presence of TRX-reductase and NADPH generated the cleaved products that are consistent with catalytic disulfide cleavage and linker immolation. GRX was less rigorously studied; in the set of compounds studied, its role in the catalytic cleavage was also confirmed. Collectively, these in vitro experiments demonstrate that TRX as well as GRX can catalyze the cleavage of disulfide bonds in both small molecules and linkers of ADCs.
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Co-delivery systems of siRNA and chemotherapeutic drugs have been developed as an attractive strategy to optimize the efficacy of chemotherapy towards cancer cells with multidrug resistance. In these typical systems, siRNAs are usually associated to drugs within a carrier but without covalent interactions with the risk of a premature release and degradation of the drugs inside the cells. To address this issue, we propose a covalent approach to co-deliver a siRNA-drug conjugate with a redox-responsive self-immolative linker prone to intracellular glutathione-mediated disulfide cleavage. Herein, we report the use of two disulfide bonds connected by a pentane spacer or a p-xylene spacer as self-immolative linker between the primary amine of the anticancer drug doxorubicin (Dox) and the 2′-position of one or two ribonucleotides in RNA. Five Dox-RNA conjugates were successfully synthesized using two successive thiol-disulfide exchange reactions. The Dox-RNA conjugates were annealed with ...
Bioorganic & Medicinal Chemistry Letters, 2008
A flexible, trifunctional poly(ethylene glycol)-succinamide-Lysine-Lysine-maleimide (PEG-SU-Lys-Lys-mal) linker was employed to simultaneously allow biotin tagging and cell-surface targeting through an integrin α 4 β 1-binding peptidomimetic that was regiospecifically conjugated to an IgG1-derived Fc fragment with an engineered C-terminal selenocysteine residue. The resulting antibody derivative mediates Fc receptor binding by virtue of the Fc protein and selectively targets cancer cells expressing human integrin α 4 β 1. The PEG-SU-Lys-Lys-mal linker may have general utility as an organic tether for the construction of antibody-drug conjugates.
Synthesis of disulfide-rich heterodimeric peptides through an auxiliary N, N-crosslink
Communications Chemistry, 2018
Insulins, relaxins, and other insulin-like peptides present a longstanding synthetic challenge due to their unique cysteine-rich heterodimeric structure. While their three disulfide signature is conserved within the insulin superfamily, sequences of the constituent chains exhibit considerable diversity. As a result, methods which rely on sequence-specific strategies fail to provide universal access to these important molecules. Biomimetic methods utilizing native and chemical linkers to tether the A-chain N-terminus to the B-chain C-terminus, entail complicated installation, and require a unique proteolytic site, or a two-step chemical release. Here we present a strategy employing a linkage of the A- and B-chains N-termini offering unrestricted access to these targets. The approach utilizes a symmetrical linker which is released in a single chemical step. The simplicity, efficiency, and scope of the method are demonstrated in the synthesis of insulin, relaxin, a 4-disulfide insulin ...