Switching the Switch: Ligand Induced Disulfide Formation in HDAC8

Human histone deacetylase 8 is a well-recognized target for T-cell lymphoma and particularly childhood neuroblastoma. PD-404,182 was shown to be a selective covalent inhibitor of HDAC8 that forms mixed disulfides with several cysteine residues and is also able to transform thiol groups to thiocyanates. Moreover, HDAC8 was shown to be regulated by a redox switch based on the reversible formation of a disulfide bond between cysteines Cys₁₀₂ and Cys₁₅₃. This study on the distinct effects of PD-404,182 on HDAC8 reveals that this compound induces the dose-dependent formation of intramolecular disulfide bridges. Therefore, the inhibition mechanism of HDAC8 by PD-404,182 involves both, covalent modification of thiols as well as ligand mediated disulfide formation. Moreover, this study provides a deep molecular insight into the regulation mechanism of HDAC8 involving several cysteines with graduated capability to form reversible disulfide bridges.     

Defining the plant disulfide proteome

There is considerable interest in redox regulation and new targets for thioredoxin and glutaredoxin are now being identified. It would be of great benefit to the field to have a list of all possible candidates for redox regulation--that is all disulfide proteins in plant. We developed a simple and very powerful method for identifying proteins with disulfide bonds in vivo. In this method, free thiols in proteins are fully blocked by alkylation, following which disulfide cysteines are converted to sulfhydryl groups by reduction. Finally, proteins with sulfhydryls are isolated by thiol affinity chromatography. Our method is unique in that membrane proteins as well as water-soluble proteins are examined for their disulfide nature. By applying this method to Arabidopsis thaliana we identified 65 putative disulfide proteins, including 20 that had not previously been demonstrated to be regulated by redox state. The newly identified, possibly redox-regulated proteins include: violaxanthin de-epoxidase, two oxygen-evolving enhancer proteins, carbonic anhydrase, photosystem I reaction center subunit N, photosystem I subunit III, S-adenosyl-L-methionine carboxyl methyltransferase, guanylate kinase, and bacterial mutT homolog. Possible functions of disulfide bonding in these proteins are discussed.     

Redox-dependent formation of disulfide bonds in human replication protein A

Human replication protein A (RPA) is a single-stranded DNA (ssDNA)-binding protein with three subunits. The largest subunit, p70, contains a conserved (cysteine)(4)-type zinc-finger motif that has been implicated in the regulation of DNA replication and repair. Previous studies indicated that the ssDNA-binding activity of RPA could be redox-regulated via reversible oxidation of cysteines in the zinc-finger motif. We exposed recombinant human RPA to hydrogen peroxide and characterized the oxidized protein by liquid chromatography/tandem mass spectrometric (LC/MS/MS) analyses. Our results demonstrated that, upon H(2)O(2) treatment, four cysteines, which reside at the zinc-finger motif of the p70 subunit, could result in the formation of two pairs of intramolecular disulfides, Cys481-Cys486 and Cys500-Cys503; no cysteine sulfinic acid or cysteine sulfonic acid could be found. Moreover, the other 11 cysteines in this protein remained intact. The results demonstrated that the formation of disulfide bonds at the zinc-finger site was responsible for the redox regulation of the DNA-binding activity of RPA.     

Peptide cysteine thiyl radicals abstract hydrogen atoms from surrounding amino acids: the photolysis of a cystine containing model peptide

Peptide cysteine thiyl radicals were generated through UV-photolysis of disulfide precursors, in order to follow intramolecular reactions of those radicals with neighboring amino acids. When reactions were carried out in D(2)O, there was a significant incorporation of deuterium specifically into the C(alpha)-H bonds of glycine residues in positions i+1 and i-1 to the Cys residue, indicating a fast reversible H-atom transfer. This H-atom transfer occurred prior to the formation of final, nonradical products including free thiol, thioaldehyde, and aldehyde. Such fast H-atom transfer is relevant to biologic conditions of oxidative stress and to the stabilization of proteins against oxidation, where the formation of carbon-centered radicals in proteins may lead to fragmentation, intramolecular cross-linking, aggregation and/or epimerization.     

Theoretical and experimental studies of the spin trapping of inorganic radicals by 5,5-dimethyl-1-pyrroline N-oxide (DMPO). 2. Carbonate radical anion

Previous studies have shown that the enzyme-mediated generation of carbonate radical anion (CO(3)(.-)) may play an important role in the initiation of oxidative damage in cells. This study explored the thermodynamics of CO(3)(.-) addition to 5,5-dimethyl-1-pyrroline N-oxide (DMPO) using density functional theory at the B3LYP/6-31+G(**)//B3LYP/6-31G* and B3LYP/6-311+G* levels with the polarizable continuum model to simulate the effect of the bulk dielectric effect of water on the calculated energetics. Theoretical data reveal that the addition of CO(3)(.-) to DMPO yields an O-centered radical adduct (DMPO-OCO2) as governed by the spin (density) population on the CO(3)(.-). Electron paramagnetic resonance spin trapping with the commonly used spin trap, DMPO, has been employed in the detection of CO(3)(.-). UV photolysis of H(2)O(2) and DMPO in the presence of sodium carbonate (Na(2)CO(3)) or sodium bicarbonate (NaHCO(3)) gave two species (i.e., DMPO-OCO(2) and DMPO-OH) in which the former has hyperfine splitting constant values of a(N) = 14.32 G, a(beta)-Eta = 10.68 G, and a(gamma-H) = 1.37 G and with a shorter half-life compared to DMPO-OH. The origin of the DMPO-OH formed was experimentally confirmed using isotopically enriched H(2)(17)O(2) that indicates direct addition of HO(.) to DMPO. Theoretical studies on other possible pathways for the formation of DMPO-OH from DMPO-OCO(2) in aqueous solution and in the absence of free HO(.) such as in the case of enzymatically generated CO(3)(.-), show that the preferred pathway is via nucleophilc substitution of the carbonate moiety by H(2)O or HO(-). Nitrite formation has been observed as the end product of CO(3)(.-) trapping by DMPO and is partly dependent on the basicity of solution. The thermodynamic behavior of CO(3)(.-) in the aqueous phase is predicted to be similar to that of the hydroperoxyl (HO(2)(.)) radical.     

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.     

Screening for disulfide-rich peptides in biological sources by carboxyamidomethylation in combination with differential matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Peptides with biological functions often contain disulfide bridges connecting two cysteine residues. In an attempt to screen biological fluids for peptides containing cysteine residues, we have developed a sensitive and specific method to label cysteines selectively and detect the resulting molecular mass shift by differential mass spectrometry. First, reduction of disulfide bridges and carboxyamidomethylation of free thiols is adjusted to quantitatively achieve cysteine alkylation for complex peptide extracts. In a second step, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) before and after chemical derivatization is performed, followed by differential analysis to determine shifted peaks; shifted peaks belong to cysteine-containing peptides, other peaks remain unchanged. The number of cysteines can then be determined by the resulting molecular mass shift. Free, reduced cysteines are shifted by 57 u, two oxidized cysteines involved in disulfide bridges (cystine) result in a shift to higher mass per disulfide bridge of 116 u. Disulfide bridges connecting different amino acid chains like insulin break up during reduction. In this case, two peaks with lower molecular masses result from a single one in the unmodified sample. With this technique, we were able to identify cysteine-containing peptides and short fragments of proteins present in human blood filtrate.     

Balancing conformational and oxidative kinetic traps during the folding of bovine pancreatic trypsin inhibitor (BPTI) with glutathione and glutathione disulfide

The oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) has served as a paradigm for the folding of disulfide-containing proteins from their reduced form, as well as for protein folding in general. Many extracellular proteins and most pharmaceutically important proteins contain disulfide bonds. Under traditional conditions, 0.125 mM glutathione disulfide (GSSG) and no glutathione (GSH), the folding pathway of BPTI proceeds through a nonproductive route via N* (a two disulfide intermediate), or a productive route via N' (and other two disulfide intermediates which are in rapid equilibrium with N'). Both routes have the rearrangement of disulfide bonds as their rate-determining steps. However, the effects of the composition of the redox buffer, GSSG and GSH, on folding has not been extensively investigated. Interestingly, BPTI folds more efficiently in the presence of 5 mM GSSG and 5 mM GSH than it does under traditional conditions. These conditions, which are similar to those found in vivo, result in a doubly mixed disulfide between N' and glutathione, which acts as an oxidative kinetic trap as it has no free thiols. However, with 5 mM GSSG and 5 mM GSH the formation of the double mixed disulfide is compensated for by N* being less kinetically stable and the more rapid conversion of the singly mixed disulfides between N' and glutathione to native protein (N). Thus a major rate-determining step becomes the direct conversion of a singly mixed disulfide to N, a growth-type pathway. Balancing the formation of N* and its stability versus the formation of the doubly mixed disulfide and its stability results in more efficient folding. Such balancing acts may prove to be general for other disulfide-containing proteins.     

Synthesis of 2-thio-substituted benzothiazoles via a domino condensation/S-arylation/heterocyclization process

Condensation of carbon disulfide with thiols in the presence of K(2)CO(3) generates carbonotrithioate salts in situ, which undergo coupling with 2-iodoanilines and subsequent intramolecular condensation and elimination under assistance of CuBr to afford 2-thio-substituted benzothiazoles. Both aliphatic and aromatic thiols are compatible with this process, delivering the corresponding heterocycles with good diversity.     

Piatzwechselvorgänge der Liganden in festen Komplexsalzen. I. Intramolekulare Isomerisierung an Gemischten Hexahalogenokomplexen von Osmium(IV)

Ligand Exchange in Solid Complex Salts. I. Intramolecular Isomerization of Mixed Hexahalo-complexes of Osmium(IV) The isomerization of pure solid complex salts of the type cis- or trans-K₂[OsCl_nY_6?n], Y = Br, I; n = 2, 3, 4, is measured spectrophotometrically at 115, 125 and 135°C. By intramolecular rearrangement of the ligands statistical relations of the isomers are adjusted. For the solid-state exchange reactions a dissociative bond-breaking-and a twist-mechanism are discussed.     

Oxidative decarboxylation of levulinic acid by silver(I)/persulfate

The oxidative decarboxylation of levulinic acid (LA) by silver(I)/persulfate [Ag(I)/S?O?2?] has been investigated in this paper. The effects of buffer solution, initial pH value, time and temperature and dosages of Ag(I)/S?O?2? on the decarboxylation of LA were examined in batch experiments and a reaction scheme was proposed on basis of the reaction process. The experimental results showed that a solution of NaOH-KH?PO? was comparatively suitable for the LA decarboxylation reaction by silver(I)/persulfate. Under optimum conditions (temperature 160 °C, pH 5.0, and time 0.5 h), the rate of LA conversion in NaOH-KH?PO? solutions with an initial concentration of 0.01 mol LA reached 70.2%, 2-butanone (methyl ethyl ketone) was the single product in the gas phase and the resulted molar yield reached 44.2%.     

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.     

Synthesis and Crystal Structure of 1-（Trifluoromethyl）-1-（propioloyl）-（E）-3-oxo-（2-thienyl）-（2-naphthylamine）

1INTRODUCTIONTheSchiffbasesderivedfromb-diketonesandaliphaticamineshavebeenshowntoexistastheketo-amines.However,ifsubstituentseitherattheketooraminogrouparearomatic,itmaybeexpectedtheenoliminewillbethefavoredtautomericform[1].Recently,someSchiffbasesderivedfromTTA(4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione)andPMBPhavebeenstudiedbyWang[2]andYu[3]etal.Inordertostudytherelationshipbetweenthestructuresandperformancesofthesecompounds,thetitlecom-poundwillbereportedherein.2EXPERIMENT…     

Oxidative Coupling of Thiols to Disulfides in Solution with Tripropylammonium Halochromates, (C3H7)3NH[CrO3X], (X = F,Cl) Adsorbed on Alumina

A mild and efficient method for the oxidative coupling of thiols by tripropylammonium fluorochromate and tripropylammonium chlorochromate absorbed on alumina in solution is reported. Alumina absorbed tripropylammonium fluorochromate and tripropylammonium chlorochromate are efficient and new reagents, which are easily prepared and oxidize thiols to the corresponding disulfides quickly. The reactions are clean and readily controlled to stop at the disulfide stage without the formation of common over-oxidized side products. Thus the advantages of the easy procedure and work up, short reaction times, and excellent yields make this a viable alternative method.     

Hydrolytic Activity of Vanadate toward Serine-Containing Peptides Studied by Kinetic Experiments and DFT Theory

Hydrolysis of dipeptides glycylserine (Gly-Ser), leucylserine (Leu-Ser), histidylserine (His-Ser), glycylalanine (Gly-Ala), and serylglycine (Ser-Gly) was examined in vanadate solutions by means of (1)H, (13)C, and (51)V NMR spectroscopy. In the presence of a mixture of oxovanadates, the hydrolysis of the peptide bond in Gly-Ser proceeds under the physiological pH and temperature (37 °C, pD 7.4) with a rate constant of 8.9 × 10(-8) s(-1). NMR and EPR spectra did not show evidence for the formation of paramagnetic species, excluding the possibility of V(V) reduction to V(IV) and indicating that the cleavage of the peptide bond is purely hydrolytic. The pD dependence of k(obs) exhibits a bell-shaped profile, with the fastest hydrolysis observed at pD 7.4. Combined (1)H, (13)C, and (51)V NMR experiments revealed formation of three complexes between Gly-Ser and vanadate, of which only one complex, designated Complex 2, formed via coordination of amide oxygen and amino nitrogen to vanadate, is proposed to be hydrolytically active. Kinetic experiments at pD 7.4 performed by using a fixed amount of Gly-Ser and increasing amounts of Na(3)VO(4) allowed calculation of the formation constant for the Gly-Ser/VO(4)(3-) complex (K(f) = 16.1 M(-1)). The structure of the hydrolytically active Complex 2 is suggested also on the basis of DFT calculations. The energy difference between Complex 2 and the major complex detected in the reaction mixture, Complex 1, is calculated to be 7.1 kcal/mol in favor of the latter. The analysis of the molecular properties of Gly-Ser and their change upon different modes of coordination to the vanadate pointed out that only in Complex 2 the amide carbon is suitable for attack by the hydroxyl group in the Ser side chain, which acts as an effective nucleophile. The origin of the hydrolytic activity of vanadate is most likely a combination of the polarization of amide oxygen in Gly-Ser due to the binding to vanadate, followed by the intramolecular attack of the Ser hydroxyl group.     

On-Resin Preparation of Allenamidyl Peptides: A Versatile Chemoselective Conjugation and Intramolecular Cyclisation Tool

The ability to modify peptides and proteins chemoselectively is of continued interest in medicinal chemistry, with peptide conjugation, lipidation, stapling, and disulfide engineering at the forefront of modern peptide chemistry. Herein we report a robust method for the on-resin preparation of allenamide-modified peptides, an unexplored functionality for peptides that provides a versatile chemical tool for chemoselective inter- or intramolecular bridging reactions with thiols. The bridging reaction is biocompatible, occurring spontaneously at pH 7.4 in catalyst-free aqueous media. By this “click” approach, a model peptide was successfully modified with a diverse range of alkyl and aryl thiols. Furthermore, this technique was demonstrated as a valuable tool to induce spontaneous intramolecular cyclisation by preparation of an oxytocin analogue, in which the native disulfide bridge was replaced with a vinyl sulfide moiety formed by thia-Michael addition of a cysteine thiol to the allenamide handle.     

Copper-catalyzed direct thiolation of benzoxazole with diaryl disulfides and aryl thiols

The cross-coupling reaction of benzoxazole with aryl thiols using the CuI/2,2′-bipyridine complex as a catalyst in DMF at 80 °C under oxygen produced the corresponding aryl thioethers in moderate to good yields. The coupling reaction with diaryl disulfide also occurred under similar oxidative conditions.     

A fluorescence turn-on probe for cysteine and homocysteine based on thiol-triggered benzothiazolidine ring formation

We synthesized a new coumarin-based probe TP, containing a disulfide moiety, to detect biothiols in cells. A fluorescence turn-on response is induced by the thiol–disulfide exchange of the probe, with subsequent intramolecular benzothiazolidine ring formation giving rise to a fluorescent product. The probe exhibits an excellent selectivity for cysteine (Cys) and homocysteine (Hcy) over glutathione (GSH) and other amino acids. The fluorescent probe also exhibits a highly sensitive fluorescence turn-on response to Cys and Hcy with detection limits of 0.8 μM for Cys and 0.5 μM for Hcy. In addition, confocal fluorescence microscopy imaging using RAW264.7 macrophages demonstrates that the probe TP could be an efficient fluorescent detector for thiols in living cells.     

Complete Mapping of a Cystine Knot and Nested Disulfides of Recombinant Human Arylsulfatase A by Multi-Enzyme Digestion and LC-MS Analysis Using CID and ETD

Cystine knots or nested disulfides are structurally difficult to characterize, despite current technological advances in peptide mapping with high-resolution liquid chromatography coupled with mass spectrometry (LC-MS). In the case of recombinant human arylsulfatase A (rhASA), there is one cystine knot at the C-terminal, a pair of nested disulfides at the middle, and two out of three unpaired cysteines in the N-terminal region. The statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination. We used a unique approach to determine the status and linkage of each cysteine in rhASA, which was comprised of multi-enzyme digestion strategies (from Lys-C, trypsin, Asp-N, pepsin, and PNGase F) and multi-fragmentation methods in mass spectrometry using electron transfer dissociation (ETD), collision induced dissociation (CID), and CID with MS³ (after ETD). In addition to generating desired lengths of enzymatic peptides for effective fragmentation, the digestion pH was optimized to minimize the disulfide scrambling. The disulfide linkages, including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the post-translational modification of a cysteine to formylglycine, were all determined. In the assignment, the disulfide linkages were Cys138–Cys154, Cys143–Cys150, Cys282–Cys396, Cys470–Cys482, Cys471–Cys484, and Cys475–Cys481. For the unpaired cysteines, Cys20 and Cys276 were free cysteines, and Cys51 was largely converted to formylglycine (>70 %). A successful methodology has been developed, which can be routinely used to determine these difficult-to-resolve disulfide linkages, ensuring drug function and stability.      

I2/TBHP-mediated domino process: a convenient route to 1,3-oxazole derivatives

A practical and simple synthesis of 2,5-disubstituted oxazoles was developed via a TBHP (tert-butyl hydroperoxide)/I₂-mediated domino strategy. The reaction proceeds in series, giving rise to the formation of an intermolecular C–N bond and an intramolecular C–O bond, which yield oxazole derivatives simultaneously. The reaction gave the desired products from readily available substrates under mild conditions.