Disulfides, imines, and metal coordination within a single system: interplay between three dynamic equilibria

We report a system in which three distinct dynamic linkages, disulfide (S-S), imine (C=N), and coordinative (N-->metal) bonds were shown to be capable of simultaneous reversible exchange. The "disulfide layer" of the system under study consists of two homo-disulfides, bis(4-aminophenyl) disulfide 1 and bis(4-methoxyphenyl) disulfide 2 that equilibrate in the presence of catalytic amount of triethylamine to favor the formation of a hetero-disulfide product, 4-aminophenyl-4'-methoxyphenyl disulfide 3. The addition of 2-formylpyridine and a metal salt strongly perturbed this 1+2<-->3 equilibrium through the formation of metal complexes incorporating disulfide 1 as a subcomponent. CuI perturbed the equilibrium by a factor of 3.3, and FeII by a factor of 179, in both cases in favor of the homo-disulfides. The disulfide equilibrium could be further modified, following metal-complex formation, by coordinative (transmetallation: substitution of FeII for CuI) or covalent (imine exchange: the substitution of one amine residue for another) exchange. Thus, although the three kinds of dynamic linkages were demonstrated to be mutually compatible, changes at one kind of linkage could be used to predictably perturb an equilibrium involving another.     

Reversed-phase liquid chromatography in-line with negative ionization electrospray mass spectrometry for the characterization of the disulfide-linkages of an immunoglobulin gamma antibody

In this report, we present a new approach for the determination of the disulfide bond connectivity in proteins using negative ionization mass spectrometry of nonreduced enzymatic digests. The mass spectrometric analysis in negative ion mode was optimized to allow in-line analysis coupled directly to the HPLC system used for the separation of the peptides resulting from enzymatic digestion. We determined the disulfide structure of a human immunoglobulin gamma 2 (IgG2) antibody containing 18 unique cysteine residues linked via 11 unique disulfide bonds. The efficiency of the gas-phase dissociation of disulfide-linked peptides using negative electrospray ionization was evaluated for an ion trap mass spectrometer and an orthogonal acceleration time-of-flight mass spectrometer. Both mass spectrometry techniques provided efficient in-source fragmentation for the identification of the disulfide-linked peptides of the antibody. Both instruments were limited in the number of disulfide bonds that could be dissociated. Seven of the 11 unique disulfide linkages have been determined, including the linkage of the light chain to the heavy chain. Only the disulfide connectivity of the hinge peptide H6H7H8H9 (C6C7VEC8PPC9PAPPVAGPSVFLFPPKPK) could not be determined (numbering the cysteine residues sequentially from the N-terminus and labeling the heavy chain cysteines "H" and the light chain cysteines "L"). However, we identified the dimer of peptide C6C7VEC8PPC9PAPPVAGPSVFLFPPKPK linked via four disulfide bonds based on the unique molecular weight of this dipeptide. The established linkages were H1 to H2, H10 to H11, H12 to H13, L1 to L2, L3 to L4, and L5 to H3H4. The intrachain linkages of the light chain (L1 to L2, L3 to L4), and heavy chain (H10 to H11, H12 to H13) domains were identical to the linkages found in IgG1 antibodies.     

Rhodium-catalyzed arylthiolation reaction of nitroalkanes, diethyl malonate, and 1,2-diphenylethanone with diaryl disulfides: control of disfavored equilibrium reaction

In the presence of catalytic amounts of RhH(PPh₃)₄ and 1,2-bis(diphenylphosphino)ethane (dppe), 1-nitroalkanes reacted with a diaryl disulfide giving 1-arylthio-1-nitroalkanes in air. The equilibrium to form thermodynamically disfavored products was shifted by the rhodium-catalyzed oxidation of thiols to disulfides and water. The thiolation reaction of cyclic nitroalkanes proceeded in high yields provided that suitable diaryl disulfides were employed depending on the substrate: di(p-chlorophenyl) disulfide was used for the thiolation reaction of 1-nitroalkanes, 1-nitrocyclopentane and 1-nitrocycloheptane with acidic α-protons (pK_a 16 and 17); di(p-methoxyphenyl) disulfide for 1-nitrocyclobutane and 1-nitrocyclohexane with less acidic α-protons (pK_a ca. 18). Related reactivities were observed in the thiolation reactions of malonate and 1,2-diphenylethanone.     

Racemic and Quasi‐Racemic X‐ray Structures of Cyclic Disulfide‐Rich Peptide Drug Scaffolds

Cyclic disulfide‐rich peptides have exceptional stability and are promising frameworks for drug design. We were interested in obtaining X‐ray structures of these peptides to assist in drug design applications, but disulfide‐rich peptides can be notoriously difficult to crystallize. To overcome this limitation, we chemically synthesized the L ‐ and D ‐forms of three prototypic cyclic disulfide‐rich peptides: SFTI‐1 (14‐mer with one disulfide bond), cVc1.1 (22‐mer with two disulfide bonds), and kB1 (29‐mer with three disulfide bonds) for racemic crystallization studies. Facile crystal formation occurred from a racemic mixture of each peptide, giving structures solved at resolutions from 1.25 Å to 1.9 Å. Additionally, we obtained the quasi‐racemic structures of two mutants of kB1, [G6A]kB1, and [V25A]kB1, which were solved at a resolution of 1.25 Å and 2.3 Å, respectively. The racemic crystallography approach appears to have broad utility in the structural biology of cyclic peptides.     

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.     

Bioinformatics approaches for disulfide connectivity prediction

Protein structure prediction with computational methods has gained much attention in the research fields of protein engineering and protein folding studies. Due to the vastness of conformational space, one of the major tasks is to restrain the flexibility of protein structure and reduce the search space. Many studies have revealed that, with the information of disulfide connectivity available, the search in conformational space can be dramatically reduced and lead to significant improvements in the prediction accuracy. As a result, predicting disulfide connectivity using bioinformatics approaches is of great interest nowadays. In this mini-review, the prediction of disulfide connectivity in proteins will be discussed in four aspects: (1) how the problem formulated and the computational techniques used in the literatures; (2) the effects of the features adopted to encode the information and the biological meanings implied; (3) the problems encountered and limitations of disulfide connectivity prediction; and (4) the practical usages of predicted disulfide bond information in molecular simulation and the prospects in the future.     

Characterization of disulfide linkages and disulfide bond scrambling in recombinant human macrophage colony stimulating factor by fast-atom bombardment mass spectrometry of enzymatic digests

Fast-atom bombardment mass spectrometry was used to study disulfide bonding patterns in heat-denatured human recombinant macrophage colony stimulating factor (rhM-CSF). The heat-denaturated protein was studied by analysis of the pattern of peptides in the proteolytic digests. Native rhM-CSF is a homodimer with intramolecular disulfide linkages between Cys7–Cys90, Cys48–Cys139, and Cys102–Cys146 and intermolecular linkages between Cys31-Cys31, and the pairs Cys157 and Cys159. Brief heating for 1 min leads to partial disulfide bond scrambling. In addition to the native disulfide bonds between Cys7–Cys90, Cys48–Cys139, and Cys31-Cys31, nonnative disulfide bonds were detected between Cys48–Cys90 and Cys48–Cys102. When heated for 5 min the disulfide bonds of rhM-CSF are completely scrambled and lead to nonnative intramolecular disulfide bonds between Cys48–Cys102 and Cys90–Cys102 and one intermolecular disulfide bond between Cys102–Cys102.     

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.     

双(O,O-二烃基硫代磷酰基)二硫物与二氯化汞的反应

双(O,O-二乙基硫代磷酰基)二硫物(Ⅰ)及双(O,O-二正丁基硫代磷酸基)二硫物(Ⅰ)与二氯化汞于室温下在乙醇中反应,视反应物的摩尔比为1:1或1:2.     

Transferable potentials for phase equilibria. 8. United-atom description for thiols, sulfides, disulfides, and thiophene

An extension of the transferable potentials for phase equilibria united-atom (TraPPE-UA) force field to thiol, sulfide, and disulfide functionalities and thiophene is presented. In the TraPPE-UA force field, nonbonded interactions are governed by a Lennard-Jones plus fixed point charge functional form. Partial charges are determined through a CHELPG analysis of electrostatic potential energy surfaces derived from ab initio calculations at the HF/6-31g+(d,p) level. The Lennard-Jones well depth and size parameters for four new interaction sites, S (thiols), S(sulfides), S(disulfides), and S(thiophene), were determined by fitting simulation data to pure-component vapor-equilibrium data for methanethiol, dimethyl sulfide, dimethyl disulfide, and thiophene, respectively. Configurational-bias Monte Carlo simulations in the grand canonical ensemble combined with histogram-reweighting methods were used to calculate the vapor-liquid coexistence curves for methanethiol, ethanethiol, 2-methyl-1-propanethiol, 2-methyl-2-propanethiol, 2-butanethiol, pentanethiol, octanethiol, dimethyl sulfide, diethyl sulfide, ethylmethyl sulfide, dimethyl disulfide, diethyl disulfide, and thiophene. Excellent agreement with experiment is achieved, with unsigned errors of less than 1% for saturated liquid densities and less than 3% for critical temperatures. The normal boiling points were predicted to within 1% of experiment in most cases, although for certain molecules (pentanethiol) deviations as large as 5% were found. Additional calculations were performed to determine the pressure-composition behavior of ethanethiol+n-butane at 373.15 K and the temperature-composition behavior of 1-propanethiol+n-hexane at 1.01 bar. In each case, a good reproduction of experimental vapor-liquid equilibrium separation factors is achieved; both of the coexistence curves are somewhat shifted because of overprediction of the pure-component vapor pressures.     

Determination of the disulfide bond pattern of the endogenous and recombinant angiogenesis inhibitor endostatin by mass spectrometry.

Endostatin, a C-terminal fragment of collagen XVIII, is a promising protein drug which is in development for cancer therapy due to its anti-angiogenic activity. Although several endogenous molecular forms of human endostatin differing in their N-terminal length and their post-translational modifications (18.5-22 kDa) have been discovered, only one recombinant form of 20 kDa is used in clinical trials. This protein, recombinantly expressed in Pichia pastoris, contains four cysteines forming two disulfide bonds (Cys1-Cys4 and Cys2-Cys3). In contrast, there are conflicting data about the disulfide pattern of endogenous material. This report presents the disulfide analyses of both the endogenous circulating endostatins isolated from human hemofiltrate and the recombinant protein. The determination of the disulfide pattern was performed by Edman degradation, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization ion trap mass spectrometry (ESI-ITMS) performed in the off-line nanospray mode. All native and recombinant endostatins exhibited a Cys1-Cys4 (Cys(162)-Cys(302)) and Cys2-Cys3 (Cys(264)-Cys(294)) linkage. For a clear discussion of fragmented disulfide-bridged peptide chains obtained from MS(n) experiments, a modified general nomenclature is proposed.     

Identification of intermolecular disulfide linkages in underivatised peptides using negative ion electrospray mass spectrometry. A joint experimental and theoretical study

The [M--H](-) ion of a symmetrical peptide containing one intermolecular disulfide linkage cleaves through the disulfide link to produce up to four fragment anions. Two of these characteristic fragments are formed by a cleavage initiated from the Cys enolate anion on the peptide backbone. The other fragment anions are formed by a cleavage directed from an anion site on the disulfide side chain. In the case of an unsymmetrical peptide containing one intermolecular disulfide, the [M--H](-) anion may cleave through the disulfide unit to give a maximum of eight cleavage anions. These fragmentations are low-energy processes as determined by theoretical calculations carried out at the HF/6-31G(d)//AM1 level of theory. Collision-induced mass spectra of the fragment anions may provide the sequence of the peptide.     

Determination of disulfide bond patterns in laminin beta1 chain N-terminal domains by nano-high-performance liquid chromatography/matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry

The disulfide bonding patterns in the N-terminal (LN) domains of the basement membrane protein laminin beta1 have not been investigated so far. We report an in-depth mass spectrometric analysis using offline nano-high-performance liquid chromatography/matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (nano-HPLC/MALDI-TOF/TOF-MS) for determining the disulfide bond patterns in the LN-domain of recombinant mouse laminin beta1 chain for the first time. Mass spectra were recorded and the putatively disulfide-linked peptides were subjected to LIFT-TOF/TOF-MS to confirm the disulfide bond. Screening the fragment ion mass spectra of disulfide-linked peptides for characteristic 66-amu patterns (34 u +32 u), arising from symmetric and asymmetric cleavage of disulfide bonds, facilitated their identification. Using various enzymes for proteolytic digestion of a recombinant laminin beta1 chain N-terminal protein fragment, a linear bonding pattern of the eight cysteine residues in the LN-domain of the laminin beta1 chain was observed with a (1-2, 3-4, 5-6, 7-8) connectivity of cysteines. The identical disulfide-bonding pattern was found in E4, the N-terminal laminin beta1 chain fragment derived by elastase digestion of mouse tumor laminin-111, confirming that this pattern also occurs in native laminin.     

Phosphate diesters and DNA hydrolysis by dinuclear Zn(II) complexes featuring a disulfide bridge and H-bond donors

The dinuclear ligand 1 based on the bis-(2-amino-pyridinyl-6-methyl)amine (BAPA) metal binding unit and featuring a two-atom disulfide bridge was synthesized and studied as hydrolytic catalysts for phosphate diesters. The Zn(II) complexes of BAPA are known to elicit the cooperation between the metal ion and the hydrogen-bond donating amino groups to greatly increase the rate of cleavage of phosphate diesters. The reactivity of the dinuclear complex 1·Zn(II)₂ toward bis-p-nitrophenyl phosphate and plasmid DNA was investigated and compared with that of reference complexes devoid of the disulfide bridge or of the hydrogen-bond donating amino groups. The dimetallic Zn(II) complex produces remarkable accelerations of the rate of cleavage of both the substrates accompanied by significant differences. In the case of BNP, the presence of the disulfide bridge does not lead to the improvement of the cooperative action of the two metal ions expected as the result of better preorganization. On the other hand, in the case of DNA the complex 1·Zn(II)₂ is much more reactive that the corresponding reference devoid of the disulfide bridge. Hence, different requisites must be fulfilled by a good catalyst for the cleavage of the two substrates. Moreover, binding studies with DNA indicated that the presence of two metal ions in the complex or of the pyridine amino groups, but not of the disulfide bridge, results into an enhanced affinity of the complexes toward this substrate.     

Formation of a sandwich-structure assisted, relatively long-lived sulfur-centered three-electron bonded radical anion in the reduction of a bis(1-substituted-uracilyl) disulfide in aqueous solution

The one-electron reduction of bis[1-(2',3',5'-tri-O-acetylribosyl)uracil-4-yl] disulfide, initiated by hydrated electrons in a radiation chemical study, has been shown to yield 1-(2',3',5'-tri-O-acetylribosyl)-4-thiouracil as a stable molecular product. The reduction reaction leads, in the first instance, to a transient, albeit remarkably stable disulfide radical anion. This is characterized by a 2-center-3-electron bond with two bonding sigma-electrons and an antibonding sigma*-electron in the sulfur-sulfur bridge, (-S therefore S-)(-). It receives its stability from a sandwich-structure with the two uracilyl moieties facing each other (possibly further assisted by the 2',3',5'-tri-O-acetylribosyl substituents). A considerable lengthening of the original disulfide bridge from 2.02 to 2.73 A in the radical anion seems to facilitate the interaction of the heterocycles and leads to a gain in stabilization energy of 24 and 33 kcal/mol (100 and 140 kJ/mol) as evaluated by UMP2/cc-pVTZ and UMP2/cc-pVDZ calculations, respectively. The (-S therefore S-)(-) bonded radical anion shows a broad optical absorption band with lambdamax=450 nm, epsilonmax=6000 M(-1) cm(-1), and a half-width of 1.0 eV. It exists in equilibrium with the conjugated 1-(2',3',5'-tri- O-acetylribosyl)uracil-4-yl thiyl radical -S(*), and the corresponding thiolate, -S(-). The rate determining step for the disappearance of the disulfide radical anion appears to be protonation of both the radical anion and the free thiolate by reaction with H(+)aq. Absolute rate constants have been measured for these protonation processes, for the formation of the stable thiouridine product, and for the electron transfer from the disulfide radical anion to molecular oxygen. With the (-S therefore S-)(-) <--> -S(*) + -S(-) equilibrium lying very much on the left-hand side, the reduced disulfide system exhibits predominantly reducing properties whereas any oxidizing property of the conjugated thiyl radical has only little if any chance to materialize. Besides attaching directly to the disulfide bridge, the hydrated electrons react also, with about equal efficiency, with the uracil moiety of the investigated compound. This leads to a structurally totally different and electronically distinguishable species than that with the reduced disulfide bridge. In particular, there is no face-to-face interaction between the two heterocyclic moieties and no increased electron density in the S-S bond. The C-centered radicals resulting from the reduction of the uracil and possibly also generated from the ribosyl moieties initiate further cleavage of the S-S bond and thus contribute to the formation of thiouridine. The overall yield of the latter, as determined from steady-state gamma-radiolysis, indicates a small chain process (G=1.54 micromol/J). Possible mechanisms are discussed.     

Intramolecular disulfide bond between catalytic cysteines in an intein precursor

Protein splicing is a self-catalyzed and spontaneous post-translational process in which inteins excise themselves out of precursor proteins while the exteins are ligated together. We report the first discovery of an intramolecular disulfide bond between the two active-site cysteines, Cys1 and Cys+1, in an intein precursor composed of the hyperthermophilic Pyrococcus abyssi PolII intein and extein. The existence of this intramolecular disulfide bond is demonstrated by the effect of reducing agents on the precursor, mutagenesis, and liquid chromatography-mass spectrometry (LC-MS) with tandem MS (MS/MS) of the tryptic peptide containing the intramolecular disulfide bond. The disulfide bond inhibits protein splicing, and splicing can be induced by reducing agents such as tris(2-carboxyethyl)phosphine (TCEP). The stability of the intramolecular disulfide bond is enhanced by electrostatic interactions between the N- and C-exteins but is reduced by elevated temperature. The presence of this intramolecular disulfide bond may contribute to the redox control of splicing activity in hypoxia and at low temperature and point to the intriguing possibility that inteins may act as switches to control extein function.     

Palladium‐Mediated Direct Disulfide Bond Formation in Proteins Containing S‐Acetamidomethyl‐cysteine under Aqueous Conditions

One of the applied synthetic strategies for correct disulfide bond formation relies on the use of orthogonal Cys protecting groups. This approach requires purification before and after the deprotection steps, which prolongs the entire synthetic process and lowers the yield of the reaction. A major challenge in using this approach is to be able to apply one‐pot synthesis under mild conditions and aqueous media. In this study, we report the development of an approach for rapid disulfide bond formation by employing palladium chemistry and S‐acetamidomethyl‐cysteine [Cys(Acm)]. Oxidation of Cys(Acm) to the corresponding disulfide bond is achieved within minutes in a one‐pot operation by applying palladium and diethyldithiocarbamate. The utility of this reaction was demonstrated by the synthesis of the peptide oxytocin and the first total chemical synthesis of the protein thioredoxin‐1. Our investigation revealed a critical role of the Acm protecting group in the disulfide bond formation, apparently due to the generation of a disulfiram in the reaction pathway, which significantly assists the oxidation step.     

PDI-Regulated Disulfide Bond Formation in Protein Folding and Biomolecular Assembly

Disulfide bonds play a pivotal role in maintaining the natural structures of proteins to ensure their performance of normal biological functions. Moreover, biological molecular assembly, such as the gluten network, is also largely dependent on the intermolecular crosslinking via disulfide bonds. In eukaryotes, the formation and rearrangement of most intra- and intermolecular disulfide bonds in the endoplasmic reticulum (ER) are mediated by protein disulfide isomerases (PDIs), which consist of multiple thioredoxin-like domains. These domains assist correct folding of proteins, as well as effectively prevent the aggregation of misfolded ones. Protein misfolding often leads to the formation of pathological protein aggregations that cause many diseases. On the other hand, glutenin aggregation and subsequent crosslinking are required for the formation of a rheologically dominating gluten network. Herein, the mechanism of PDI-regulated disulfide bond formation is important for understanding not only protein folding and associated diseases, but also the formation of functional biomolecular assembly. This review systematically illustrated the process of human protein disulfide isomerase (hPDI) mediated disulfide bond formation and complemented this with the current mechanism of wheat protein disulfide isomerase (wPDI) catalyzed formation of gluten networks.     

A novel salt bridge mechanism highlights the need for nonmobile proton conditions to promote disulfide bond cleavage in protonated peptides under low-energy collisional activation

The gas-phase fragmentation mechanisms of small models for peptides containing intermolecular disulfide links have been studied using a combination of tandem mass spectrometry experiments, isotopic labeling, structural labeling, accurate mass measurements of product ions, and theoretical calculations (at the MP2/6-311 + G(2d,p)//B3LYP/3-21G(d) level of theory). Cystine and its C-terminal derivatives were observed to fragment via a range of pathways, including loss of neutral molecules, amide bond cleavage, and S-S and C-S bond cleavages. Various mechanisms were considered to rationalize S-S and C-S bond cleavage processes, including charge directed neighboring group processes and nonmobile proton salt bridge mechanism. Three low-energy fragmentation pathways were identified from theoretical calculations on cystine N-methyl amide: (1) S-S bond cleavage dominated by a neighboring group process involving the C-terminal amide N to form either a protonated cysteine derivative or protonated sulfenyl amide product ion (44.3 kcal mol(-1)); (2) C-S bond cleavage via a salt bridge mechanism, involving abstraction of the alpha-hydrogen by the N-terminal amino group to form a protonated thiocysteine derivative (35.0 kcal mol(-1)); and (3) C-S bond cleavage via a Grob-like fragmentation process in which the nucleophilic N-terminal amino group forms a protonated dithiazolidine (57.9 kcal mol(-1)). Interestingly, C-S bond cleavage by neighboring group processes have high activation barriers (63.1 kcal mol(-1)) and are thus not expected to be accessible during low-energy CID experiments. In comparison to the energetics of simple amide bond cleavage, these S-S and C-S bond cleavage reactions are higher in energy, which helps rationalize why bond cleavage processes involving the disulfide bond are rarely observed for low-energy CID of peptides with mobile proton(s) containing intermolecular disulfide bonds. On the other hand, the absence of a mobile proton appears to "switch on" disulfide bond cleavage reactions, which can be rationalized by the salt bridge mechanism. This potentially has important ramifications in explaining the prevalence of disulfide bond cleavage in singly protonated peptides under MALDI conditions.     

One-pot synthesis of unsymmetrical disulfides using 1-chlorobenzotriazole as oxidant: Interception of the sulfenyl chloride intermediate

A high-yielding and low temperature one-pot procedure is described for unsymmetrical disulfide synthesis from two different thiols using 1-chlorobenzotriazole (BtCl) as oxidant. The mechanism of the coupling involves in situ trapping of the sulfenyl chloride intermediate R¹SCl by nucleophilic benzotriazole (BtH) to form R¹SBt, which protects R¹SCl from forming the homodimer R¹SSR¹. The methodology is applicable to all types of thiol (aliphatic, aromatic, heteroaromatic), with a variation developed for aliphatic-aliphatic couplings. Differentially N-protected cysteines couple to afford the unsymmetrical cystine derivatives in high yield (90%), which serves as a model for the one-pot intermolecular coupling of cysteine-containing peptides to form peptide disulfide heterodimers. Minimal exchange in aromatic-aromatic disulfide synthesis is noted on account of the mild conditions.