Chemoselective cysteine or disulfide modification via single atom substitution in chloromethyl acryl reagents† † Electronic supplementary information (ESI) available. See DOI: 10.1039/d1sc03250j
ABSTRACT: The development of bioconjugation chemistry has enabled the combination of various synthetic functionalities to proteins, giving rise to new classes of protein conjugates with functions well beyond what Nature can provide. Despite the progress in bioconjugation chemistry, there are no reagents developed to date where the reactivity can be tuned in a user-defined fashion to address different amino acid residues in proteins. Here, we report that 2-chloromethyl acryl reagents can serve as a simple yet versatile platform for selective protein modification at cysteine or disulfide sites by tuning their inherent electronic properties through the amide or ester linkage. Specifically, the 2-chloromethyl derivatives (acrylamide or acrylate) can be obtained via a simple and easily implemented one-pot reaction based on the coupling reaction between commercially available starting materials with different end-group functionalities (amino group or hydroxyl group). 2-Chloromethyl acrylamide reagents with an amide linkage favor selective modification at the cysteine site with fast reaction kinetics and near quantitative conversations. In contrast, 2-chloromethyl acrylate reagents bearing an ester linkage can undergo two successive Michael reactions, allowing the selective modification of disulfides bonds with high labeling efficiency and good conjugate stability. 2-Chloromethyl acryl derivatives (acrylamides and acrylates) can serve as simple and versatile bioconjugation reagents to achieve site-selective cysteine and disulfide modification on demand and with high efficiency.
Project description:Site-selective chemical conjugation of synthetic molecules to proteins expands their functional and therapeutic capacity. Current protein modification methods, based on synthetic and biochemical technologies, can achieve site selectivity, but these techniques often require extensive sequence engineering or are restricted to the N- or C-terminus. Here we show the computer-assisted design of sulfonyl acrylate reagents for the modification of a single lysine residue on native protein sequences. This feature of the designed sulfonyl acrylates, together with the innate and subtle reactivity differences conferred by the unique local microenvironment surrounding each lysine, contribute to the observed regioselectivity of the reaction. Moreover, this site selectivity was predicted computationally, where the lysine with the lowest p Ka was the kinetically favored residue at slightly basic pH. Chemoselectivity was also observed as the reagent reacted preferentially at lysine, even in those cases when other nucleophilic residues such as cysteine were present. The reaction is fast and proceeds using a single molar equivalent of the sulfonyl acrylate reagent under biocompatible conditions (37 °C, pH 8.0). This technology was demonstrated by the quantitative and irreversible modification of five different proteins including the clinically used therapeutic antibody Trastuzumab without prior sequence engineering. Importantly, their native secondary structure and functionality is retained after the modification. This regioselective lysine modification method allows for further bioconjugation through aza-Michael addition to the acrylate electrophile that is generated by spontaneous elimination of methanesulfinic acid upon lysine labeling. We showed that a protein-antibody conjugate bearing a site-specifically installed fluorophore at lysine could be used for selective imaging of apoptotic cells and detection of Her2+ cells, respectively. This simple, robust method does not require genetic engineering and may be generally used for accessing diverse, well-defined protein conjugates for basic biology and therapeutic studies.
Project description:An efficient method for chemoselective cysteine arylation of unprotected peptides and proteins using Au(III) organometallic complexes is reported. The bioconjugation reactions proceed rapidly (<5 min) at ambient temperature in various buffers and within a wide pH range (0.5-14). This approach provides access to a diverse array of S-aryl bioconjugates including fluorescent dye, complex drug molecule, affinity label, poly(ethylene glycol) tags, and a stapled peptide. A library of Au(III) arylation reagents can be prepared as air-stable, crystalline solids in one step from commercial reagents. The selective and efficient arylation procedures presented in this work broaden the synthetic scope of cysteine bioconjugation and serve as promising routes for the modification of complex biomolecules.
Project description:Many reagents have been developed for cysteine-specific protein modification. However, few of them allow for multi-functionalization of a single Cys residue and disulfide bridging bioconjugation. Herein, we report 3-bromo-5-methylene pyrrolones (3Br-5MPs) as a simple, robust, and versatile class of reagents for cysteine-specific protein modification. These compounds can be facilely synthesized via a one-pot mild reaction and they show comparable tagging efficiency but higher cysteine specificity than the maleimide counterparts. The addition of cysteine to 3Br-5MPs generates conjugates that are amenable to secondary addition by another thiol or cysteine, making 3Br-5MPs valuable for multi-functionalization of a single cysteine and disulfide bridging bioconjugation. The labeling reaction and subsequent treatments are mild enough to produce stable and active protein conjugates for biological applications.
Project description:Site-selective bioconjugation to native protein residues is a powerful tool for protein functionalization, with cysteine and lysine side chains being the most common points for attachment owing to their high nucleophilicity. We now report a strategy for histidine modification using thiophosphorodichloridate reagents that mimic post-translational histidine phosphorylation, enabling fast and selective labeling of protein histidines under mild conditions where various payloads can be introduced via copper-assisted alkyne-azide cycloaddition (CuAAC) chemistry. We establish that these reagents are particularly effective at covalent modification of His-tags, which are common motifs to facilitate protein purification, as illustrated by selective attachment of polyarginine cargoes to enhance the uptake of proteins into living cells. This work provides a starting point for probing and enhancing protein function using histidine-directed chemistry.
Project description:Reactions based on transition metals have found wide use in organic synthesis, in particular for the functionalization of small molecules. However, there are very few reports of using transition-metal-based reactions to modify complex biomolecules, which is due to the need for stringent reaction conditions (for example, aqueous media, low temperature and mild pH) and the existence of multiple reactive functional groups found in biomolecules. Here we report that palladium(II) complexes can be used for efficient and highly selective cysteine conjugation (bioconjugation) reactions that are rapid and robust under a range of bio-compatible reaction conditions. The straightforward synthesis of the palladium reagents from diverse and easily accessible aryl halide and trifluoromethanesulfonate precursors makes the method highly practical, providing access to a large structural space for protein modification. The resulting aryl bioconjugates are stable towards acids, bases, oxidants and external thiol nucleophiles. The broad utility of the bioconjugation platform was further corroborated by the synthesis of new classes of stapled peptides and antibody-drug conjugates. These palladium complexes show potential as benchtop reagents for diverse bioconjugation applications.
Project description:Rapid, site-selective modification of cysteine residues with chloromethyl-triazole derivatives generates pseudo-acyl sLys motifs, mimicking important post-translational modifications. Near-native biotinylation of peptide and protein substrates is shown to be site-selective and modified histone H4 retains functional activity.
Project description:We have investigated 4-halopyridines as selective, tunable, and switchable covalent protein modifiers for use in the development of chemical probes. Nonenzymatic reactivity of 4-chloropyridine with amino acids and thiols was ranked with respect to common covalent protein-modifying reagents and found to have reactivity similar to that of acrylamide, but could be switched to a reactivity similar to that of iodoacetamide upon stabilization of the positively charged pyridinium. Diverse, fragment-sized 4-halopyridines inactivated human dimethylarginine dimethylaminohydrolase-1 (DDAH1) through covalent modification of the active site cysteine, acting as quiescent affinity labels that required off-pathway catalysis through stabilization of the protonated pyridinium by a neighboring aspartate residue. A series of 2-fluoromethyl-substituted 4-chloropyridines demonstrated that the pKa and kinact /KI values could be predictably varied over several orders of magnitude. Covalent labeling of proteins in an Escherichia coli lysate was shown to require folded proteins, indicating that alternative proteins can be targeted, and modification is likely to be catalysisdependent. 4-Halopyridines, and quiescent affinity labels in general, represent an attractive strategy to develop reagents with switchable electrophilicity as selective covalent protein modifiers.
Project description:The maleimide motif is widely used for the selective chemical modification of cysteine residues in proteins. Despite widespread utilization, there are some potential limitations, including the irreversible nature of the reaction and, hence, the modification and the number of attachment positions. We conceived of a new class of maleimide which would address some of these limitations and provide new opportunities for protein modification. We report herein the use of mono- and dibromomaleimides for reversible cysteine modification and illustrate this on the SH2 domain of the Grb2 adaptor protein (L111C). After initial modification of a protein with a bromo- or dibromomaleimide, it is possible to add an equivalent of a second thiol to give further bioconjugation, demonstrating that bromomaleimides offer opportunities for up to three points of attachment. The resultant protein-maleimide products can be cleaved to regenerate the unmodified protein by addition of a phosphine or a large excess of a thiol. Furthermore, dibromomaleimide can insert into a disulfide bond, forming a maleimide bridge, and this is illustrated on the peptide hormone somatostatin. Fluorescein-labeled dibromomaleimide is synthesized and inserted into the disulfide to construct a fluorescent somatostatin analogue. These results highlight the significant potential for this new class of reagents in protein modification.
Project description:Site-selective protein modification is of significant interest in chemical biology research, with lysine residues representing a particularly challenging target. Whilst lysines are popular for bioconjugation, due to their nucleophilicity, solvent accessibility and the stability of the resultant conjugates, their high abundance means site-selectivity is very difficult to achieve. Antibody-drug conjugates (ADCs) present a powerful therapeutic application of protein modification, and have often relied extensively upon lysine bioconjugation for their synthesis. Here we discuss advances in methodologies for achieving site-selective lysine modification, particularly within the context of antibody conjugate construction, including the cysteine-to-lysine transfer (CLT) protocol which we have recently reported.
Project description:Efficient and site-specific chemical modification of proteins under physiological conditions remains a challenge. Here we report that 1,4-dinitroimidazoles are highly efficient bifunctional bioconjugation reagents for protein functionalization and peptide macrocyclization. Under acidic to neutral aqueous conditions, 1,4-dinitroimidazoles react specifically with cysteines via a cine-substitution mechanism, providing rapid, stable and chemoselective protein bioconjugation. On the other hand, although unreactive towards amine groups under neutral aqueous conditions, 1,4-dinitroimidazoles react with lysines in organic solvents in the presence of base through a ring-opening & ring-close mechanism. The resulting cysteine- and lysine-(4-nitroimidazole) linkages exhibit stability superior to that of commonly employed maleimide-thiol conjugates. We demonstrate that 1,4-dinitroimidazoles can be applied in site-specific protein bioconjugation with functionalities such as fluorophores and bioactive peptides. Furthermore, a bisfunctional 1,4-dinitroimidazole derivative provides facile access to peptide macrocycles by crosslinking a pair of cysteine or lysine residues, including bicyclic peptides of complex architectures through highly controlled consecutive peptide macrocyclization.