Influence of external force on properties and reactivity of disulfide bonds

The mechanochemistry of the disulfide bridge--that is, the influence of an externally applied force on the reactivity of the sulfur-sulfur bond--is investigated by unrestricted Kohn-Sham theory. Specifically, we apply the COGEF (constrained geometry simulates external force) approach to characterize the mechanochemistry of the disulfide bond in three different chemical environments: dimethyl disulfide, cystine, and a 102-atom model of the I27 domain in the titin protein. Furthermore, the mechanism of the thiol-disulfide reduction reaction under the effect of an external force is investigated by considering the COGEF potential for the adduct and transition-state clusters. With the unrestricted Becke-three-parameter-Lee-Yang-Parr (UB3LYP) exchange-correlation functional in the 6-311++G(3df,3pd) orbital basis, the rupture force of dimethyl disulfide is 3.8 nN at a disulfide bond elongation of 35 pm. The interaction with neighboring groups and the effect of conformational rigidity of the protein environment have little influence on the mechanochemical characteristics. Upon stretching, we make the following observations: the diradical character of the disulfide bridge increases; the energy difference between the singlet ground state and low-lying triplet state decreases; and the disulfide reduction is promoted by an external force in the range 0.1-0.4 nN. Our model of the interplay between force and reaction mechanism is in qualitative agreement with experimental observations.     

Single-molecule force spectroscopy measurements of bond elongation during a bimolecular reaction

It is experimentally challenging to directly obtain structural information of the transition state (TS), the high-energy bottleneck en route from reactants to products, for solution-phase reactions. Here, we use single-molecule experiments as well as high-level quantum chemical calculations to probe the TS of disulfide bond reduction, a bimolecular nucleophilic substitution (S N2) reaction. We use an atomic force microscope in force-clamp mode to apply mechanical forces to a protein disulfide bond and obtain force-dependent rate constants of the disulfide bond reduction initiated by a variety of nucleophiles. We measure distances to the TS or bond elongation (Delta x), along a 1-D reaction coordinate imposed by mechanical force, of 0.31 +/- 0.05 and 0.44 +/- 0.03 A for thiol-initiated and phosphine-initiated disulfide bond reductions, respectively. These results are in agreement with quantum chemical calculations, which show that the disulfide bond at the TS is longer in phosphine-initiated reduction than in thiol-initiated reduction. We also investigate the effect of solvent environment on the TS geometry by incorporating glycerol into the aqueous solution. In this case, the Delta x value for the phosphine-initiated reduction is decreased to 0.28 +/- 0.04 A whereas it remains unchanged for thiol-initiated reduction, providing a direct test of theoretical calculations of the role of solvent molecules in the reduction TS of an S N2 reaction. These results demonstrate that single-molecule force spectroscopy represents a novel experimental tool to study mechanochemistry and directly probe the sub-?ngstr?m changes in TS structure of solution-phase reactions. Furthermore, this single-molecule method opens new doors to gain molecular level understanding of chemical reactivity when combined with quantum chemical calculations.     

Atomistic evidence of how force dynamically regulates thiol/disulfide exchange

The intricate coupling of mechanical force and chemical reactivity has been increasingly revealed in recent years by force spectroscopy experiments on the thiol/disulfide exchange reaction. We here aimed at elucidating the underlying dynamic effects of force on the reaction center for the case of disulfide bond reduction by dithiothreitol at forces of 200-2000 pN, by combining transition path sampling and quantum/classical mechanical simulations. Reaction rates and their dependence on force as quantified by Δx(r), the distance between reactant and transition state, are in good agreement with experiments but indicate a shift of the transition state structure at high forces. Indeed, while an associate S(N)2 mechanism prevails, force causes a move of the transition state to a longer length of the cleaving bond and a shorter length of the forming disulfide bond. Our results highlight the distribution of force into various degrees of freedom, which implies that care must be taken when correlating Δx(r) with a single order parameter of the reaction.     

Dwell time analysis of a single-molecule mechanochemical reaction

Force-clamp spectroscopy is a novel technique for studying mechanochemistry at the single-bond level. Single disulfide bond reduction events are accurately detected as stepwise increases in the length of polyproteins that contain disulfide bonds and that are stretched at a constant force with the cantilever of an atomic force microscope (AFM). The kinetics of this reaction has been measured from single-exponential fits to ensemble averages of the reduction events. However, exponential fits are notoriously ambiguous to use in cases of kinetic data showing multiple reaction pathways. Here we introduce a dwell time analysis technique, of widespread use in the single ion channel field, that we apply to the examination of the kinetics of reduction of disulfide bonds measured from single-molecule force-clamp spectroscopy traces. In this technique, exponentially distributed dwell time data is plotted as a histogram with a logarithmic time scale and a square root ordinate. The advantage of logarithmic histograms is that exponentially distributed dwell times appear as well-defined peaks in the distribution, greatly enhancing our ability to detect multiple kinetic pathways. We apply this technique to examine the distribution of dwell times of 4488 single disulfide bond reduction events measured in the presence of two very different kinds of reducing agents: tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) and the enzyme thioredoxin (TRX). A different clamping force is used for each reducing agent to obtain distributions of dwell times on a similar time scale. In the case of TCEP, the logarithmic histogram of dwell times showed a single peak, corresponding to a single reaction mechanism. By contrast, similar experiments done with TRX showed two well-separated peaks, marking two distinct modes of chemical reduction operating simultaneously. These experiments demonstrate that dwell time analysis techniques are a powerful approach to studying chemical reactions at the single-molecule level.     

Molecular tensile machines: intrinsic acceleration of disulfide reduction by dithiothreitol

Significant tension on the order of 1 nN is self-generated along the backbone of bottlebrush macromolecules due to steric repulsion between densely grafted side chains. The intrinsic tension is amplified upon adsorption of bottlebrush molecules onto a substrate and increases with grafting density, side chain length, and strength of adhesion to the substrate. These molecules were employed as miniature tensile machines to study the effect of mechanical force on the kinetics of disulfide reduction by dithiothreitol (DTT). For this purpose, bottlebrush macromolecules containing a disulfide linker in the middle of the backbone were synthesized by atom transfer radical polymerization (ATRP). The scission reaction was monitored through molecular imaging by atomic force microscopy (AFM). The scission rate constant increases linearly with the concentration of DTT and exponentially with mechanical tension along the disulfide bond. Moreover, the rate constant at zero force is found to be significantly lower than the reduction rate constant in bulk solution, which suggests an acidic composition of the water surface with pH = 3.7. This work demonstrates the ability of branched macromolecules to accelerate chemical reactions at specific covalent bonds without applying an external force.     

Synthesis of a [2]rotaxane incorporating a "magic sulfur ring" by the thiol-ene click reaction

The mild and highly efficient thiol-ene click reaction has been used to construct a rotaxane incorporating dibenzo-24-crown-8 (DB24C8) and a dibenzylammonium-derived thread in high yield under the irradiation of UV light. A rotaxane containing a disulfide linkage in the macrocycle was also synthesized by the thiol-ene click reaction. It has been demonstrated that the formation of the [2]rotaxane with the disulfide bond in the macrocycle occurs by a mechanism that is different to the threading-followed-by-stoppering process. The successful construction of a rotaxane directly from its constituent components, the macrocycle containing a disulfide linkage and the dibenzylammonium hexafluorophosphate salt, suggests that the space within the macrocycle incorporating the disulfide linkage is smaller than the phenyl unit and a plausible reaction mechanism has been proposed as follows: A small amount of the initiator forms two radicals upon the absorption of UV irradiation; the radicals act as a "key" to "unlock" the disulfide bond in the macrocycle. The resulting crown ether like moiety in the macrocycle is clipped around the ammonium ion center in the dumb-bell-shaped compound. The [2]rotaxane is generated upon recombination of the disulfide linkage.     

Force‐Induced Reversal of β‐Eliminations: Stressed Disulfide Bonds in Alkaline Solution

Understanding the impact of tensile forces on disulfide bond cleavage is not only crucial to the breaking of cross‐linkers in vulcanized materials such as strained rubber, but also to the regulation of protein activity by disulfide switches. By using ab initio simulations in the condensed phase, we investigated the response of disulfide cleavage by β‐elimination to mechanical stress. We reveal that the rate‐determining first step of the thermal reaction, which is the abstraction of the β‐proton, is insensitive to external forces. However, forces larger than about 1 nN were found to reshape the free‐energy landscape of the reaction so dramatically that a second channel is created, where the order of the reaction steps is reversed, turning β‐deprotonation into a barrier‐free follow‐up process to C?S cleavage. This transforms a slow and force‐independent process with second‐order kinetics into a unimolecular reaction that is greatly accelerated by mechanical forces.     

Disulfide Bond Cleavage: A Redox Reaction Without Electron Transfer

By using Car–Parrinello molecular dynamics (CPMD) simulations we have simulated a mechanically induced redox reaction. Previous single‐molecule atomic force microscopy (AFM) experiments demonstrated that the reduction of disulfide bonds in proteins with the weak reducing agent dithiothreitol depends on a mechanical destabilization of the breaking bond. With reactive molecular dynamics simulations the single steps of the reaction mechanism can be elucidated and the motion of the electrons can be monitored. The simulations show that the redox reaction consists of the heterolytic cleavage of the S? S bond followed by a sequence of proton transfers.     

Contrasting the individual reactive pathways in protein unfolding and disulfide bond reduction observed within a single protein

Identifying the dynamics of individual molecules along their reactive pathways remains a major goal of modern chemistry. For simple chemical reactions, the transition state position is thought to be highly localized. Conversely, in the case of more complex reactions involving proteins, the potential energy surfaces become rougher, resulting in heterogeneous reaction pathways with multiple transition state structures. Force-clamp spectroscopy experimentally probes the individual reaction pathways sampled by a single protein under the effect of a constant stretching force. Herein, we examine the distribution of conformations that populate the transition state of two different reactions; the unfolding of a single protein and the reduction of a single disulfide bond, both occurring within the same single protein. By applying the recently developed static disorder theory, we quantify the variance of the barrier heights, σ(2), governing each distinct reaction. We demonstrate that the unfolding of the I27 protein follows a nonexponential kinetics, consistent with a high value of σ(2) ～ 18 (pN nm)(2). Interestingly, shortening of the protein upon introduction of a rigid disulfide bond significantly modulates the disorder degree, spanning from σ(2) ～ 8 to ～21 (pN nm)(2). These results are in sharp contrast with the exponential distribution of times measured for an S(N)2 chemical reaction, implying the absence of static disorder σ(2) ～ 0 (pN nm)(2). Our results demonstrate the high sensitivity of the force-clamp technique to capture the signatures of disorder in the individual pathways that define two distinct force-induced reactions, occurring within the core of a single protein.     

Mechanistic study of a place-exchange reaction of Au nanoparticles with spin-labeled disulfides

The mechanism of a place-exchange reaction of ligand-protected Au nanoparticles was investigated using diradical disulfide spin labels. Analysis of reaction mixtures using a combination of GPC and EPR allowed us to determine concentration profile and propose a kinetic model for the reaction. In this model, only one branch of the disulfide ligand is adsorbed on the Au surface during exchange; the other branch forms mixed disulfide with the outgoing ligand. The two branches of the disulfide ligand therefore do not adsorb in adjacent positions on the surface of Au nanoparticles; this was ultimately proven by the powder EPR spectra of frozen exchange reaction mixtures. Our data also suggest the presence of different binding sites with different reactivity in the exchange reaction. The most-active sites are likely to be nanoparticle surface defects.     

Conformational simulations of aqueous solvated alpha-conotoxin GI and its single disulfide analogues using a polarizable force field model

The solution conformation of alpha-conotoxin GI and its two single disulfide analogues are simulated using a polarizable force field in combination with the molecular fragmentation quantum chemical calculation. The polarizability is explicitly described by allowing the partial charges and fragment dipole moments to be variables, with values coming from the linear-scaling energy-based molecular fragmentation calculations at the B3LYP/6-31G(d) level. In comparison with the full quantum chemical calculations, the fragmentation approaches can yield precise ground-state energies, dipole moments, and static polarizabilities for peptides. The B3LYP/6-31G(d) charges and fragment-centered dipole moments are introduced in calculations of electrostatic terms in both AmberFF03 and OPLS force fields. Our test calculations on the gas-phase glucagon (PDB code: 1gcn) and solvated alpha-conotoxin GI (PDB code: 1not) demonstrate that the present polarization model is capable of describing the structural properties (such as the relative conformational energies, intramolecular hydrogen bonds, and disulfide bonds) with accuracy comparable to some other polarizable force fields (ABEEM/MM and OPLS-PFF) and the quantum mechanics/molecular mechanics (QM/MM) hybrid model. The employment of fragment-centered dipole moments in calculations of dipole-dipole interactions can save computational time in comparison with those polarization models using atom-centered dipole moments without much loss of accuracy. The molecular dynamics simulations using the polarizable force field demonstrate that two single disulfide GI analogues are more flexible and less structured than the native alpha-conotoxin GI, in agreement with NMR experiments. The polarization effect is important in simulations of the folding/unfolding process of solvated proteins.     

EPR study of a place-exchange reaction on Au nanoparticles: two branches of a disulfide molecule do not adsorb adjacent to each other

An EPR study of a place-exchange reaction of a diradical disulfide with butanethiol-protected Au nanoparticles showed that the two branches of the disulfide molecule do not adsorb adjacent to each other on the Au surface. The most likely mechanism includes adsorption of only one branch of the disulfide molecule in the exchange process. The exchange reaction was found to be zeroth-order with respect to the diradical, indicative of a dissociative "SN1"-type mechanism.     

Stabilization of the Cysteine‐Rich Conotoxin MrIA by Using a 1,2,3‐Triazole as a Disulfide Bond Mimetic

The design of disulfide bond mimetics is an important strategy for optimising cysteine‐rich peptides in drug development. Mimetics of the drug lead conotoxin MrIA, in which one disulfide bond is selectively replaced of by a 1,4‐disubstituted‐1,2,3‐triazole bridge, are described. Sequential copper‐catalyzed azide–alkyne cycloaddition (CuAAC; click reaction) followed by disulfide formation resulted in the regioselective syntheses of triazole–disulfide hybrid MrIA analogues. Mimetics with a triazole replacing the Cys4–Cys13 disulfide bond retained tertiary structure and full in vitro and in vivo activity as norepinephrine reuptake inhibitors. Importantly, these mimetics are resistant to reduction in the presence of glutathione, thus resulting in improved plasma stability and increased suitability for drug development.     

Rhodium-catalyzed disulfide exchange reaction

A system of RhH(PPh3)4, trifluoromethanesulfonic acid, and (p-tol)3P catalyzes the disulfide exchange reaction. Treatment of two symmetrical dialkyl disulfides with the catalyst provides an equilibrium mixture of three disulfides within 15 min in refluxing acetone. The catalyst is active after reaching the equilibrium, and addition of a disulfide to the mixture changes the ratio of the products. The use of 4 mol equiv excess of one of the disulfides provides the unsymmetrical disulfide in a yield exceeding 80%. Disulfide-containing peptides also undergo an exchange reaction. The reactions of diaryl disulfides and dialkyl disulfides are even faster, and reach equilibrium within 5 min at room temperature in the presence of the rhodium complex and 1,2-bis(diphenylphosphino)ethane (dppe). This exchange reaction is considerably affected by the substituents on the disulfides. Treatment of diphenyl disulfide, di(p-tolyl) disulfide, and bis(sec-butyl) disulfide yields phenyl p-tolyl disulfide at room temperature with unchanged bis(sec-butyl) disulfide; random disproportionation occurs at reflux. The rhodium catalysis can be used for the exchange reaction of disulfides and diselenides giving selenosulfides as well as disulfides and ditellurides giving tellurinosulfides.     

Direct MALDI‐MS analysis of the disulfide bonds in peptide using thiosalicylic acid as a reactive matrix

The ability of a thiol‐containing molecule, thiosalicylic acid (TSA), to function as a reactive matrix for matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry analysis of peptides has been investigated. Although TSA has reducing characteristics, the use of TSA did not cause a reduction‐induced MALDI in‐source decay, probably because of the weak interactions between the thiol group in TSA and the carboxyl oxygen in the peptide. In contrast, when peptides containing disulfide bonds were analyzed by MALDI with TSA as the matrix, the disulfide bond was partially cleaved owing to the reaction with TSA, producing TSA‐adducted peptides. The reaction between the disulfide bond and TSA was suggested to be occurred in solution. The comparison of the MALDI mass spectra obtained using conventional matrix and TSA allows us to count the number of disulfide bonds in the peptides. Copyright © 2017 John Wiley & Sons, Ltd.     

Disulfide bond cleavage induced by a platinum(II) methionine complex

The cleavage of a disulfide bond and the redox equilibrium of thiol/disulfide are strongly related to the levels of glutathione (GSH)/oxidized glutathione (GSSG) or mixed disulfides in vivo. In this work, the cleavage of a disulfide bond in GSSG induced by a platinum(II) complex [Pt(Met)Cl2] (where Met = methionine) was studied and the cleavage fragments or their platinated adducts were identified by means of electrospray mass spectrometry, high-performance liquid chromatography, and ultraviolet techniques. The second-order rate constant for the reaction between [Pt(Met)Cl2] and GSSG was determined to be 0.4 M(-1) s(-1) at 310 K and pH 7.4, which is 100- and 12-fold faster than those of cisplatin and its monoaqua species, respectively. Different complexes were formed in the reaction of [Pt(Met)Cl2] with GSSG, mainly mono- and dinuclear platinum complexes with the cleavage fragments of GSSG. This study demonstrated that [Pt(Met)Cl2] can promote the cleavage of disulfide bonds. The mechanistic insight obtained from this study may provide a deeper understanding on the potential involvement of platinum complexes in the intracellular GSH/GSSG systems.     

RhCl3-catalyzed disulfide exchange reaction using water solvent in homogeneous and heterogeneous systems

RhCl₃ catalyzed the alkylthio exchange reaction of hydrophilic disulfides in water under homogeneous conditions, and equilibrium was attained in several hours. The reaction was applied to the exchange of unprotected glutathione disulfide. The reaction of dimethyl disulfide and hydrophilic disulfides under heterogeneous conditions also proceeded effectively. The mechanism turned out to be dependent on the water solubility of the substrates: The reaction of bis(3-hydroxypropyl) disulfide took place in the dimethyl disulfide phase, whereas the reaction of bis(6-aminohexyl) disulfide dihydrochloride proceeded in the water phase.     

One-electron reductive template-directed ligation of oligodeoxynucleotides possessing a disulfide bond

Hypoxic X-radiolysis of diluted aqueous solutions was performed to generate hydrated electrons that induced one-electron reduction of oligodeoxynucleotides (ODNs) possessing a disulfide bond. Upon hypoxic irradiation of dinucleotides, two forms of dinucleotides were produced via intermolecular exchange of the disulfides and ligation that proceeded with a multiple turnover. In contrast to the efficient reaction induced by hypoxic irradiation, the reaction efficiency was dramatically decreased when irradiation was performed under aerobic conditions, presumably due to capturing reactive hydrated electrons by molecular oxygen. We subsequently applied these unique reaction characteristics to template-directed ligation. In the presence of a complementary template ODN, two ODNs possessing a disulfide bond produced a prescribed ODN with high regioselectivity via interstrand crossing upon hypoxic irradiation.     

Singlet oxygen reaction—II: Alkylthiosubstituted ethylene

The reaction of singlet oxygen with tetrakis(ethylthio)ethylene has been shown to afford diethylthiooxalate and diethyl disulfide. The expected diethylthiocarbonate was also obtained as a minor product. A similar reaction with bis(ethylthio)ethylene gave ethylthioglyoxalate together with diethyl disulfide. Formation of diethylthioacetaldehyde was also observed, and is suggested to proceed via the intermediary 1,2-dioxetane or perepoxide followed by preferential migration of the ethyltilio group. On the other hand, phenylthioethylene is oxidized with singlet oxygen to give a thiol ester together with disulfide. This suggests that the formation of disulfide probably occurs via a radical pathway. The photooxygenation of disulfide in alcohol was also studied.     

Novel synthesis of 2-arylbenzothiazoles mediated by ceric ammonium nitrate (CAN): a rebuttal

[reaction: see text] The reported synthesis of 2-arylbenzothiazoles mediated by ceric ammonium nitrate (CAN) is irreproducible; the only products formed in this reaction are bis-(p-tolyl) disulfide and p-tolyl p-toluenethiosulfonate.