Disulfide Cleavage Research Articles

Functionalizable, Side Chain-Immolative Poly(benzyl ether)s.

Herein, we report a poly(benzyl ether)-based self-immolative polymer (SIP) with pendant pyridine disulfide groups. Cleavage of the side-chain disulfides leads to the formation of phenolates, which initiate depolymerization from the side chain. Due to the higher density of the disulfide groups compared to that of the chain-end-capping group, which normally is responsible for initiating depolymerization of SIPs, the side chain-immolative polymer (ScIP) can be readily degraded in the solid state where the mobility of polymer chains is substantially limited. The ScIP was also further modified through the thiol-disulfide exchange reaction to prepare ScIP-g-poly(ethylene glycol) graft polymers and organogels, which were also able to undergo complete reductive self-immolative degradation.

Redox Dual-Responsive and O2‑Evolving Theranostic Nanosystem for Highly Selective Chemotherapy against Hypoxic Tumors.

Activatable theranostic agents, which combine fluorescent reporters with masked chemotherapeutic agents that are activated by tumor-associated stimuli, would be attractive candidates to improve the tumor selectivity of chemotherapy. This work reports a ROS/GSH dual-activatable and O2‑evolving theranostic nanosystem (RA-S-S-Cy@PLGA NPs) for highly selective therapy against hypoxic tumors and in situ fluorescence-tracking of cancer chemotherapy.Methods: In this system, the newly designed theranostic agent (RA-S-S-Cy) is composed of a disulfide bond as a cleavable linker, a near infrared (NIR) active fluorophore as a fluorescent tracker, and a natural cyclopeptide RA-V as the active anti-cancer agent. Upon reaction with the high level of intracellular glutathione (GSH), disulfide cleavage occurs, resulting in concomitant active drug RA-V release and significant NIR fluorescence increase. To further improve the tumor targeting of RA-S-S-Cy and achieve redox dual-responsiveness, RA-S-S-Cy was incorporated into the c(RGDfK)-targeted PLGA nanoparticles together with an O2-generating agent (catalase) to produce RA-S-S-Cy@PLGA NPs.Results: The cell-specific and redox dual-activatable release of RA-V lead to enhanced therapeutic outcomes in vivo and in vitro. More significantly, the RA-S-S-Cy@PLGA NPs were successfully applied for monitoring of drug release and chemotherapeutic efficacy in situ by “turn-on” NIR fluorescence.Conclusions: RA-S-S-Cy@PLGA NPs would be efficient theranostic nanosystems for more precise therapy against hypoxic tumors and provides a potential tool for deeper understanding of drug release mechanisms.

Construction of a Glutathione-Responsive and Silica-Based Nanocomposite for Controlled Release of Chelator Dimercaptosuccinic Acid.

Dimercaptosuccinic acid (DMSA) is an oral heavy metal chelator. Although DMSA is the most acceptable chelator in the urinary excretion of toxic elements from children and adults, its defects in plasma binding and the membrane permeability limit its interaction with intracellular elements and affect its efficacy in chelation therapy. Herein, a novel nanocomposite composed of mesoporous silica nanoparticles (MSNs), disulfide bond, and DMSA was synthesized and characterized with a scanning/transmission electron microscope, IR and Raman spectra, and TGA analysis. The in vitro interactions with glutathione (GSH) and cellular uptake assays showed that it was able to be stable in extracellular environments such as in blood, be internalized by cells, and release DMSA inside via GSH-triggered disulfide cleavage reaction. The in vitro adsorption assays showed that MSNs-SH as its intracellular metabolite had strong adsorbability for models of Hg2+ or Pb2+. The hemolysis and cell viability assays showed that it was compatible with blood and cells even at a concentration of 1000 μg·mL−1. All above could not only enable it to be a GSH-responsive drug delivery system (DDS) for DMSA delivery but also to be a solution for its defects and efficacy. Thus, introduction of intelligent DDS might open a new avenue for DMSA-based chelation therapy.

Characterization of Protein Disulfide Linkages by MS In-Source Dissociation Comparing to CID and ETD Tandem MS.

Direct characterization of disulfide linkages in proteins by mass spectrometry has been challenging. Here, we report analysis of disulfide linkages in insulin variant, endothelin 3, and relaxin 2 by in-source dissociation (ISD) during LC-MS. A duplet insulin peptide from Glu-C digestion that contains peptides p1 and p2 (from chains A and B, respectively) was selected as a model peptide. This duplet peptide has an inter-chain disulfide bond between p1 and p2, and an intra-chain disulfide bond in p1. To compare the gas-phase fragmentation, it was subjected to ISD MS and MS/MS methods, including collision-induced dissociation (CID) and electron transfer dissociation (ETD). The pattern and efficiency of peptide backbone and disulfide cleavage varied with these dissociation methods. ETD, CID, and ISD were able to generate single backbone, double backbone, and triple (double backbone and single disulfide bond) cleavages in this model peptide, respectively. Specifically, CID did not cleave disulfide bonds and ETD was able to only cleave the inter-chain disulfide bond at low efficiency, limiting their usage in this disulfide analysis. In contrast, ISD was able to cleave the intra-chain disulfide bond in addition to peptide backbone, creating multiple fragment ions that allow accurate assignment of both intra- and inter-chain disulfide linkages. ISD was also successfully applied to determine double disulfide linkages in endothelin 3 and relaxin 2 peptides. This study contributes to the fundamental understanding of disulfide bond cleavages in different gas-phase fragmentations and provides an efficient cleavage strategy for identification of disulfide bonds in proteins by ISD ESI-MS. Graphical Abstract.

Exploration of Pyrrolobenzodiazepine (PBD)-Dimers Containing Disulfide-Based Prodrugs as Payloads for Antibody-Drug Conjugates.

A number of cytotoxic pyrrolobenzodiazepine (PBD) monomers containing various disulfide-based prodrugs were evaluated for their ability to undergo activation (disulfide cleavage) in vitro in the presence of either glutathione (GSH) or cysteine (Cys). A good correlation was observed between in vitro GSH stability and in vitro cytotoxicity toward tumor cell lines. The prodrug-containing compounds were typically more potent against cells with relatively high intracellular GSH levels (e.g., KPL-4 cells). Several antibody-drug conjugates (ADCs) were subsequently constructed from PBD dimers that incorporated selected disulfide-based prodrugs. Such HER2 conjugates exhibited potent antiproliferation activity against KPL-4 cells in vitro in an antigen-dependent manner. However, the disulfide prodrugs contained in the majority of such entities were surprisingly unstable toward whole blood from various species. One HER2-targeting conjugate that contained a thiophenol-derived disulfide prodrug was an exception to this stability trend. It exhibited potent activity in a KPL-4 in vivo efficacy model that was approximately three-fold weaker than that displayed by the corresponding parent ADC. The same prodrug-containing conjugate demonstrated a three-fold improvement in mouse tolerability properties in vivo relative to the parent ADC, which did not contain the prodrug.

Mechano-redox control of integrin de-adhesion.

How proteins harness mechanical force to control function is a significant biological question. Here we describe a human cell surface receptor that couples ligand binding and force to trigger a chemical event which controls the adhesive properties of the receptor. Our studies of the secreted platelet oxidoreductase, ERp5, have revealed that it mediates release of fibrinogen from activated platelet αIIbβ3 integrin. Protein chemical studies show that ligand binding to extended αIIbβ3 integrin renders the βI-domain Cys177-Cys184 disulfide bond cleavable by ERp5. Fluid shear and force spectroscopy assays indicate that disulfide cleavage is enhanced by mechanical force. Cell adhesion assays and molecular dynamics simulations demonstrate that cleavage of the disulfide induces long-range allosteric effects within the βI-domain, mainly affecting the metal-binding sites, that results in release of fibrinogen. This coupling of ligand binding, force and redox events to control cell adhesion may be employed to regulate other protein-protein interactions.

Reduction-Triggered Transformation of Disulfide-Containing Micelles at Chemically Tunable Rates.

A dilemma exists between the circulation stability and cargo release/mass diffusion at desired sites when designing delivery nanocarriers and in vivo nanoreactors. Reported herein are disulfide-crosslinked (DCL) micelles exhibiting reduction-triggered switching of crosslinking modules and synchronized hydrophobic-to-hydrophilic transition. Tumor cell targeted DCL micelles undergo cytoplasmic milieu triggered disulfide cleavage and self-immolative decaging reactions at chemically adjustable rates, generating primary amine moieties. Extensive amidation reactions with neighboring ester moieties then occur because of the high local concentration and suppression of the apparent amine pKa value within the hydrophobic cores, thus leading to the transformation of crosslinking modules and formation of tracelessly crosslinked (TCL) micelles, with hydrophilic cores, inside live cells. We further integrate this design principle with theranostic nanocarriers for selective intracellular drug transport guided by enhanced magnetic resonance (MR) imaging performance.

A Trimethoprim Conjugate of Thiomaltose Has Enhanced Antibacterial Efficacy In Vivo.

Trimethoprim is one of the most widely used antibiotics in the world. However, its efficacy is frequently limited by its poor water solubility and dose limiting toxicity. Prodrug strategies based on conjugation of oligosaccharides to trimethoprim have great potential for increasing the solubility of trimethoprim and lowering its toxicity, but they have been challenging to develop due to the sensitivity of trimethoprim to chemical modifications, and the rapid degradation of oligosaccharides in serum. In this report, we present a trimethoprim conjugate of maltodextrin termed TM-TMP, which increased the water solubility of trimethoprim by over 100 times, was stable to serum enzymes, and was active against urinary tract infections in mice. TM-TMP is composed of thiomaltose conjugated to trimethoprim, via a self-immolative disulfide linkage, and releases 4'-OH-trimethoprim (TMP-OH) after disulfide cleavage, which is a known metabolic product of trimethoprim and is as potent as trimethoprim. TM-TMP also contains a new maltodextrin targeting ligand composed of thiomaltose, which is stable to hydrolysis by serum amylases and therefore has the metabolic stability needed for in vivo use. TM-TMP has the potential to significantly improve the treatment of a wide number of infections given its high water solubility and the widespread use of trimethoprim.

Anti-inflammatory polymersomes of redox-responsive polyprodrug amphiphiles with inflammation-triggered indomethacin release characteristics

Inflammation serves as a natural defense mechanism to protect living organisms from infectious diseases. Nonsteroidal anti-inflammatory drugs (NSAIDs) can help relieve inflammatory reactions and are clinically used to treat pain, fever, and inflammation, whereas long-term use of NSAIDs may lead to severe side effects including gastrointestinal damage and cardiovascular toxicity. Therefore, it is of increasing importance to configure new dosing strategies and alleviate the side effects of NSAIDs. Towards this goal, glutathione (GSH)-responsive disulfide bonds and hydrogen peroxide (H2O2)-reactive phenylboronic ester linkages were utilized as triggering moieties in this work to design redox-responsive prodrug monomers and polyprodrug amphiphiles based on indomethacin (IND) drug. Note that IND is a widely prescribed NSAID in the clinic. Starting from three types of redox-reactive IND prodrug monomers, redox-responsive polyprodrug amphiphiles were synthesized through reversible addition-fragmentation chain transfer (RAFT) polymerizations of prodrug monomers using poly(ethylene oxide) (PEO)-based macroRAFT agent. The resultant polyprodrug amphiphiles with high IND loading contents (>33 wt%) could self-assemble into polymersomes with PEO shielding coronas and redox-responsive bilayer membranes composed of IND prodrugs. Upon incubation with GSH or H2O2, controlled release of intact IND in the active form from polyprodrug polymersomes was actuated by GSH-mediated disulfide cleavage reaction and H2O2-mediated oxidation of phenylboronic ester moieties, respectively, followed by self-immolative degradation events. Furthermore, in vitro studies at the cellular level revealed that redox-responsive polymersomes could efficiently relieve inflammatory responses induced by lipopolysaccharide (LPS) in RAW264.7 macrophage cells.

Deducing disulfide patterns of cysteine-rich proteins using signature fragments produced by top-down mass spectrometry.

Direct mapping of protein disulfide patterns using top-down mass spectrometry (MS) is often hampered by inadequate fragmentation at the disulfide-enclosing region, and insufficient structural information provided by the fragments. Here we used electron-transfer/high energy collision dissociation (EThcD) to improve the fragmentation efficiency, and developed strategies that minimize the false positive identification of fragments and deconvolute the signals representing specific modifications made to the disulfide-cleavage-induced fragments. We observed clear correlations between unique modification (attachment or removal of H or SH) patterns and the number of disulfide bonds that enclose the corresponding region. Using the characteristic signature fragments, we in part localized the Cys-bridging sites in disulfide-scrambled lysozymes, and reduced the number of putative disulfide patterns from 104 to 6. The results demonstrated the feasibility of direct analysis of complex disulfide patterns using top-down MS.

Surface design and preparation of multi-functional magnetic nanoparticles for cancer cell targeting, therapy, and imaging.

Recently, theranostic candidates based on superparamagnetic iron oxide nanoparticles (SPIONs) providing the combination of therapy and diagnosis have become one of the most promising system in cancer research. However, poor stability, premature drug release, lack of specific tumor cell targeting, and complicated multi-step synthesis processes still hinder them for potential clinical applications. In this research, the multi-functional magnetic nanoparticles (MNPs-DOX) were prepared via a simple assembly process for targeted delivery of doxorubicin (DOX) and enhanced magnetic resonance (MR) imaging detection. Firstly, the multi-functional copolymer coating, polyamidoamine (PAMAM), was designed and synthesized by Michael addition reaction, where N,N-bis(acryloyl)cystamine served as backbone linker, and DOX, dopamine (DA), and polyethylene glycol (PEG) acted as comonomers. The PAMAM was then directly assembled to the surface of SPIONs by the ligand exchange reaction with SPIONs forming the MNPs-DOX. The hydrophilic PEG moieties provide the nanoparticles with colloidal stability and good-dispersity in aqueous solution. Comparing with the quick release of free DOX, the drug release behavior of MNPs-DOX exhibited a sustained drug release. Because the chemical cleavage of disulfide in the polymer backbone, a high cumulative drug release up to 60% in GSH within 48 h was observed, rather than only 26% in PBS (pH 7.4) without GSH. The MR imaging detection experiment showed that the MNPs-DOX had an enhanced T2 relaxivity of 126 mM−1 S−1 for MR imaging. The results of the cytotoxicity assays showed a remarkable killing effect of cancer cells by MNPs-DOX due to the FA tumor-targeting ligand, comparing with non-targeted drug molecules. All the results showed that the as prepared multi-functional magnetic nanoparticles may serve as a promising theranostic candidate for targeted anticancer drug delivery and efficient detection through MR imaging in medical application.

Sodium Hypochlorite Pentahydrate Crystals (NaOCl·5H2O): A Convenient and Environmentally Benign Oxidant for Organic Synthesis

The novel oxidant of sodium hypochlorite pentahydrate (NaOCl·5H2O) crystals is now available for industrial and laboratory use. It is superior to conventional aqueous sodium hypochlorite solutions (aq. NaOCl). The crystalline material is 44% NaOCl and contains minimal amounts of sodium hydroxide and sodium chloride, and the aqueous solution, which is prepared from NaOCl·5H2O and water, has a pH of 11–12. Examples of the selective organic synthesis using NaOCl·5H2O involve the oxidations of primary and secondary alcohols, selective oxidations to sulfoxide and sulfone, oxidative cleavage of disulfide to sulfonyl chloride and bromide, oxaziridine synthesis, and oxidative dearomatization of phenols.

C‐O and C‐S coupling reaction of 1,2‐di(pyrimidin‐2‐yl) disulfides with phenols/thiophenols promoted by copper(I) chloride

AbstractAn efficient protocol for C‐O and C‐S bonds formation by the cross‐coupling reaction of 1,2‐di(pyrimidin‐2‐yl) disulfides with phenols/thiophenols promoted by copper(I) chloride was established. It was discovered that variously substituted di(hetero)aryl disulfides and phenols were well tolerated. This strategy is the conversion of disulfides into hetero‐aryl ethers and thioethers by a copper‐promoted chemoselective C‐S bond cleavage of disulfides.

Methanogenic heterodisulfide reductase (HdrABC-MvhAGD) uses two noncubane [4Fe-4S] clusters for reduction.

In methanogenic archaea, the carbon dioxide (CO2) fixation and methane-forming steps are linked through the heterodisulfide reductase (HdrABC)-[NiFe]-hydrogenase (MvhAGD) complex that uses flavin-based electron bifurcation to reduce ferredoxin and the heterodisulfide of coenzymes M and B. Here, we present the structure of the native heterododecameric HdrABC-MvhAGD complex at 2.15-angstrom resolution. HdrB contains two noncubane [4Fe-4S] clusters composed of fused [3Fe-4S]-[2Fe-2S] units sharing 1 iron (Fe) and 1 sulfur (S), which were coordinated at the CCG motifs. Soaking experiments showed that the heterodisulfide is clamped between the two noncubane [4Fe-4S] clusters and homolytically cleaved, forming coenzyme M and B bound to each iron. Coenzymes are consecutively released upon one-by-one electron transfer. The HdrABC-MvhAGD atomic model serves as a structural template for numerous HdrABC homologs involved in diverse microbial metabolic pathways.

Methanogenic archaea metabolism

Structural Biology Most of the methane on Earth is produced by the metabolism of methanogenic archaea. The final step involves a reaction between methyl-coenzyme M and coenzyme B to give CoM-S-S-CoB and methane. Wagner et al. report a high-resolution structure of the methanogenic heterodisulfide reductase (HdtABC)-[NiFe]-hydrogenase, the enzyme that reduces the disulfide and couples this to the reduction of ferredoxin in an energy-conserving process known as flavin-based electron bifurcation (FBEB) (see the Perspective by Dobbek). The reduced ferredoxin, in turn, drives the first step of methanogenesis. The structure shows how two noncubane [4Fe-4S] clusters perform disulfide cleavage and gives insight into the mechanism of FBEB. Science , this issue p. [699][1]; see also p. [642][2] [1]: /lookup/doi/10.1126/science.aan0425 [2]: /lookup/doi/10.1126/science.aao2439

Efficient One-Pot Synthesis of 2,4-Disubstituted Thiazoles and Dimeric Thiazoles Directly from Acyl Chlorides and β-Azido Disulfides

A simple and effective one-pot cascade procedure to provide 2,4-disubstituted thiazoles and symmetrical dimeric thiazoles directly from commercially available acid chlorides and β-azido disulfides in moderate to high yields is described. This cascade transformation consists of disulfide cleavage, thiocarbonylation, phosphine-promoted intramolecular Staudinger/aza-Wittig reaction and dehydrogenation to form the corresponding thiazoles. The application of our methodology is demonstrated by the concise two-step synthesis of anticancer agent SMART and its O-linked dimer in 64% and 46% overall yields, respectively. This directed cascade reaction could be easily handled and scaled up under mild conditions, enabling the construction of focused thiazole derivatives for further pharmacological evaluation.

Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections.

Multiplexing, labeling for multiple immunostains in the very same cell or tissue section in situ, has raised considerable interest. The methods proposed include the use of labeled primary antibodies, spectral separation of fluorochromes, bleaching of the fluorophores or chromogens, blocking of previous antibody layers, all in various combinations. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, and scarcity of specialized skills or facilities. We have validated a method based on common primary and secondary antibodies and diffusely available fluorescent image scanners. It entails rounds of four-color indirect immunofluorescence, image acquisition, and removal (stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulfide cleavage and a detergent or by a chaotropic salt treatment, this latter followed by antigen refolding. More than 30 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software. Multiplexing on routine tissue sections is a high throughput tool for in situ characterization of neoplastic, reactive, inflammatory, and normal cells.

Synthesis of 2-arylbenzothiazoles via direct condensation between in situ generated 2-aminothiophenol from disulfide cleavage and carboxylic acids

Abstract In this work we describe a simple and efficient general methodology for 2-arylbenzothiazole preparation employing disulfides and carboxylic acids. The reaction is promoted by tributylphosphine that acts both in disulfide bond cleavage and as activating agent for coupling with carboxylic acids. The reaction scope was studied using bis(2-aminophenyl)disulfide and different carboxylic acids with donor/withdrawing substituents, which resulted in the desired 2-arylbenzothiazole with moderate to good yields. The method was tested with success in preparation of the amyloid probe 2-(4-aminophenyl)-6-methoxybenzothiazole that employed a substituted bis(2-aminophenyl)disulfide.

Assessment of disulfide and hinge modifications in monoclonal antibodies.

During the last years there was a substantial increase in the use of antibodies and related proteins as therapeutics. The emphasis of the pharmaceutical industry is on IgG1, IgG2, and IgG4 antibodies, which are therefore in the focus of this article. In order to ensure appropriate quality control of such biopharmaceuticals, deep understanding of their chemical degradation pathways and the resulting impact on potency, pharmacokinetics, and safety is required. Criticality of modifications may be specific for individual antibodies and has to be assessed for each molecule. However, some modifications of conserved structure elements occur in all or at least most IgGs. In these cases, criticality assessment may be applicable to related molecules or molecule formats. The relatively low dissociation energy of disulfide bonds and the high flexibility of the hinge region frequently lead to modifications and cleavages. Therefore, the hinge region and disulfide bonds require specific consideration during quality assessment of mAbs. In this review, available literature knowledge on underlying chemical reaction pathways of modifications, analytical methods for quantification and criticality are discussed. The hinge region is prone to cleavage and is involved in pathways that lead to thioether bond formation, cysteine racemization, and iso‐Asp (Asp, aspartic acid) formation. Disulfide or sulfhydryl groups were found to be prone to reductive cleavage, trisulfide formation, cysteinylation, glutathionylation, disulfide bridging to further light chains, and disulfide scrambling. With regard to potency, disulfide cleavage, hinge cleavage, disulfide bridging to further light chains, and cysteinylation were found to influence antigen binding and fragment crystallizable (Fc) effector functionalities. Renal clearance of small fragments may be faster, whereas clearance of larger fragments appears to depend on their neonatal Fc receptor (FcRn) functionality, which in turn may be impeded by disulfide bond cleavage. Certain modifications such as disulfide induced aggregation and heterodimers from different antibodies are generally regarded critical with respect to safety. However, the detection of some modifications in endogenous antibodies isolated from human blood and the possibility of in vivo repair mechanisms may reduce some safety concerns.

Synthesis and Characterization of Stimuli-Responsive Star-Like Polypept(o)ides: Introducing Biodegradable PeptoStars.

Star-like polymers are one of the smallest systems in the class of core crosslinked polymeric nanoparticles. This article reports on a versatile, straightforward synthesis of three-arm star-like polypept(o)ide (polysarcosine-block-polylysine) polymers, which are designed to be either stable or degradable at elevated levels of glutathione. Polypept(o)ides are a recently introduced class of polymers combining the stealth-like properties of the polypeptoid polysarcosine with the functionality of polypeptides, thus enabling the synthesis of materials completely based on endogenous amino acids. The star-like homo and block copolymers are synthesized by living nucleophilic ring opening polymerization of the corresponding N-carboxyanhydrides (NCAs) yielding polymeric stars with precise control over the degree of polymerization (Xn = 25, 50, 100), Poisson-like molecular weight distributions, and low dispersities (Đ = 1.06-1.15). Star-like polypept(o)ides display a hydrodynamic radius of 5 nm (μ2 < 0.05) as determined by dynamic light scattering (DLS). While star-like polysarcosines and polypept(o)ides based on disulfide containing initiators are stable in solution, degradation occurs at 100 × 10-3 m glutathione concentration. The disulfide cleavage yields the respective polymeric arms, which possess Poisson-like molecular weight distributions and low dispersities (Đ = 1.05-1.12). Initial cellular uptake and toxicity studies reveal that PeptoStars are well tolerated by HeLa, HEK 293, and DC 2.4 cells.