Synthesis of two closely spaced cysteine barbiturates containing peptides by copper-catalyzed oxidation of contryphan disulfide

In this report, we are documenting the synthesis of peptide barbiturate through copper-catalyzed oxidation of peptide disulfide. Single disulfide-containing contryphans are used as models to access possibility of anchoring of barbituric acid (BRB) onto the peptide disulfide. Current method permits anchoring of two molecules of BRB onto the polypeptide and yield of peptide barbiturates varies from 59 to 84%. Formation of cysteine sulfenic acid (Cys-SOH) during oxidation of disulfide was confirmed using chemical probe of Cys-SOH dimedone. Mass spectrometric studies have confirmed the presence of cysteine barbiturate in anchored peptides. Based on the nature of reactive oxygen species involved in oxidation of peptide disulfide, possible mechanisms were proposed for anchoring of BRB onto the peptide disulfide through Cys-SOH. This is the first report on anchoring of two molecules of BRB onto the closely spaced cysteine residues of single polypeptide.     

Proteomic analysis of peptides tagged with dimedone and related probes

Owing to its labile nature, a new role for cysteine sulfenic acid (–SOH) modification has emerged. This oxidative modification modulates protein function by acting as a redox switch during cellular signaling. The identification of proteins that undergo this modification represents a methodological challenge, and its resolution remains a matter of current interest. The development of strategies to chemically modify cysteinyl‐containing peptides for liquid chromatography–tandem mass spectrometry (LC‐MS/MS) analysis has increased significantly within the past decade. The method of choice to selectively label sulfenic acid is based on the use of dimedone or its derivatives. For these chemical probes to be effective on a proteome‐wide level, their reactivity toward –SOH must be high to ensure reaction completion. In addition, the presence of an adduct should not interfere with electrospray ionization, the efficiency of induced dissociation in MS/MS experiments or with the identification of Cys‐modified peptides by automated database searching algorithms. Herein, we employ a targeted proteomics approach to study the electrospray ionization and fragmentation effects of different –SOH specific probes and compared them to commonly used alkylating agents. We then extend our study to a whole proteome extract using shotgun proteomic approaches. These experiments enable us to demonstrate that dimedone adducts do not interfere with electrospray by suppressing the ionization nor impede product ion assignment by automated search engines, which detect a + 138 Da increase from unmodified peptides. Collectively, these results suggest that dimedone can be a powerful tool to identify sulfenic acid modifications by high‐throughput shotgun proteomics of a whole proteome. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterization by tandem mass spectrometry of stable cysteine sulfenic acid in a cysteine switch peptide of matrix metalloproteinases

Cysteine sulfenic acid (Cys-SOH) is an elusive intermediate in reactive oxygen species-induced oxidation reactions of many proteins such as peroxiredoxins and tyrosine phosphatases. Cys-SOH is proposed to play a vital role in catalytic and signaling functions. The formation of cysteine sulfinic acid (Cys-SO(2)H) and cysteine sulfonic acid (Cys-SO(3)H) has been implicated in the activation of matrix metalloproteinase-7 (MMP-7) and oxidation of thiol to cysteine sulfinic acid has been associated with the autolytic cleavage of MMP-7. We have examined the formation of cysteine sulfenic acid in a synthetic peptide PRCGVPDVA, which is a cysteine switch domain of MMP-7 and other matrix metalloproteases. We have prepared the cysteine sulfenic acid containing peptide, PRC(SOH)GVPDVA, by reaction with hydroxyl radicals generated by the Fenton reaction (Fe(+2)/H(2)O(2)). We characterized this modified peptide by tandem mass spectrometry and accurate mass measurement experiments. In addition, we used 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl) reagent to form an adduct with PRC(SOH)GVPDVA to provide additional evidence for the viability of PRC(SOH)GVPDVA in solution. We also characterized an intramolecular cysteine sulfinamide cross-link product PRC[S(O)N]GVPDVA based on tandem mass spectrometry and accurate mass measurement experiments. These results contribute to the understanding of a proteolytic cleavage mechanism that is traditionally associated with MMP activation.     

Peptide Cyclization Mediated by Metal-Free S-Arylation: S-Protected Cysteine Sulfoxide as an Umpolung of the Cysteine Nucleophile

Covalent linking of side chains provides a method to produce cyclic or stapled peptides that are important in developing peptide-based drugs. A variety of crosslinking formats contribute to fixing the active conformer and prolonging its biological activity under physiological conditions. One format uses the cysteine thiol to participate in crosslinking through nucleophilic thiolate anions or thiyl radicals to form thioether and disulfide bonds. Removal of the S-protection from an S-protected Cys derivative generates the thiol, which functions as a nucleophile. S-Oxidation of a protected Cys allows the formation of a sulfoxide that operates as an umpolung electrophile. Herein, the applicability of S-p-methoxybenzyl Cys sulfoxide (Cys(MBzl)(O)) to the formation of a thioether linkage between tryptophan and Cys has been investigated. The reaction of peptides containing Cys(MBzl)(O) and Trp with trifluoromethanesulfonic acid (TFMSA) or methanesulfonic acid (MSA) in TFA in the presence of guanidine hydrochloride (Gn ⋅ HCl) proceeded to give cyclic or stapled peptides possessing the Cys-Trp thioether linkage. In this reaction, strong acids such as TFMSA or MSA are necessary to activate the sulfoxide. Additionally, Gn ⋅ HCl plays a critical role in producing an electrophilic Cys derivative that combines with the indole by aromatic electrophilic substitution. The findings led us to conclude that the less-electrophilic Cys(MBzl)(O) serves as an acid-activated umpolung of a Cys nucleophile and is useful for S-arylation-mediated peptide cyclization.     

Fragmentation of protonated ions of peptides containing cysteine,cysteine sulfinic acid,and cysteine sulfonic acid

The oxidation of the sulfhydryl group in cysteine to sulfenic acid, sulfinic acid, and sulfonic acid in proteins is important in a number of enzymatic processes. In this study we examined the fragmentation of four peptides containing cysteine, cysteine sulfinic acid (Cys-SO(2)H), and cysteine sulfonic acid (Cys-SO(3)H) in an ion-trap mass spectrometer. Our results show that the presence of a Cys-SO(2)H in a peptide leads to preferential cleavage of the amide bond at the C-terminal side of the oxidized cysteine residue. The results are important for the determination of the site of the cysteine oxidation and might be useful for the sequencing of cysteine-containing peptides.     

A Type of Auxiliary for Native Chemical Peptide Ligation beyond Cysteine and Glycine Junctions

Native chemical ligation enables the chemical synthesis of proteins. Previously, thiol‐containing auxiliary groups have been used to extend the reaction scope beyond N‐terminal cysteine residues. However, the N‐benzyl‐type auxiliaries used so far result in rather low reaction rates. Herein, a new N^α‐auxiliary is presented. Consideration of a radical fragmentation for cleavage led to the design of a new auxiliary group which is selectively removed under mildly basic conditions (pH 8.5) in the presence of TCEP and morpholine. Most importantly and in contrast to previously described auxiliaries, the 2‐mercapto‐2‐phenethyl auxiliary is not limited to Gly‐containing sites and ligations succeed at sterically demanding junctions. The auxiliary is introduced in high yield by on‐resin reductive amination with commercially available amino acid building blocks. The synthetic utility of the method is demonstrated by the synthesis of two antimicrobial proteins, DCD‐1L and opistoporin‐2.     

Rethinking Cysteine Protective Groups: S‐Alkylsulfonyl‐l‐Cysteines for Chemoselective Disulfide Formation

The ability to reversibly cross‐link proteins and peptides grants the amino acid cysteine its unique role in nature as well as in peptide chemistry. We report a novel class of S‐alkylsulfonyl‐l ‐cysteines and N‐carboxy anhydrides (NCA) thereof for peptide synthesis. The S‐alkylsulfonyl group is stable against amines and thus enables its use under Fmoc chemistry conditions and the controlled polymerization of the corresponding NCAs yielding well‐defined homo‐ as well as block co‐polymers. Yet, thiols react immediately with the S‐alkylsulfonyl group forming asymmetric disulfides. Therefore, we introduce the first reactive cysteine derivative for efficient and chemoselective disulfide formation in synthetic polypeptides, thus bypassing additional protective group cleavage steps.     

Synthetic Methods of Phosphonopeptides

Phosphonopeptides are phosphorus analogues of peptides and have been widely applied as enzyme inhibitors and antigens to induce catalytic antibodies. Phosphonopeptides generally contain one aminoalkylphosphonic acid residue and include phosphonopeptides with C-terminal aminoalkylphosphonic acids and phosphonopeptides with a phosphonamidate bond. The phosphonamidate bond in the phosphonopeptides is generally formed via phosphonylation with phosphonochloridates, condensation with coupling reagents and enzymes, and phosphinylation followed by oxidation. Pseudo four-component condensation reaction of amides, aldehydes, alkyl dichlorophosphites, and amino/peptide esters is an alternative, convergent, and efficient strategy for synthesis of phosphonopeptides through simultaneous construction of aminoalkylphosphonic acids and formation of the phosphonamidate bond. This review focuses on the synthetic methods of phosphonopeptides containing a phosphonamidate bond.     

Peptide Cysteine Thiols Act as Photostabilizer of Avobenzone through Stabilizing the Transition State of Keto-Enol Tautomerization

Photostabilizers have been used to impart stability to an FDA-approved chemical UV-A filter avobenzone against the UV-A radiations and sunlight. The thiol group of glutathione plays a critical role in imparting the photostabilization activity of glutathione on avobenzone. The current report aims to evaluate the photostabilization activity of multiple thiols containing cysteine peptides on avobenzone. Cysteine-tripeptide and cysteine-pentapeptide were chemically synthesized and characterized using mass spectrometry. Synthetic peptides were assessed for their photostabilization activity on the enolic-form of the avobenzone under natural sunlight using UV spectroscopy in both protic and aprotic solvents. Unlike glutathione, which has pronounced activity in protic solvents, cysteine-pentapeptide exhibits similar photoprotection activity in both protic and aprotic solvents. Computational calculations using DFT suggest that peptide cysteine thiols may assist in the reversal of the photoketonization process of avobenzone thereby exhibiting the photoprotection activity to the enolic-form of avobenzone. Peptide cysteine thiols lower the activation energy barrier of keto-to-enol tautomerization of avobenzone by 30 kcal mol⁻¹ by assisting the proton shuttle through a six-membered transition state. The current report emphasizes the applications of peptide thiols in cosmetics and may help in the development of peptides as aesthetic medicines.     

Profiling the reactivity of cyclic C-nucleophiles towards electrophilic sulfur in cysteine sulfenic acid

Oxidation of a protein cysteine thiol to sulfenic acid, termed S-sulfenylation, is a reversible post-translational modification that plays a crucial role in regulating protein function and is correlated with disease states. The majority of reaction-based small molecule and immunochemical probes used for detecting sulfenic acids are based on the 5,5-dimethyl-1,3-cyclohexanedione (dimedone) scaffold, which is selective, but suffers from low reactivity. In addition, mechanistic details and features that diminish or enhance nucleophile reactivity remain largely unknown. A significant hurdle to resolving the aforementioned issues has been the chemically unstable nature of small-molecule sulfenic acid models. Herein, we report a facile mass spectrometry-based assay and repurposed dipeptide-based model to screen a library of cyclic C-nucleophiles for reactivity with sulfenic acid under aqueous conditions. Observed rate constants for ∼100 cyclic C-nucleophiles were obtained and, from this collection, we have identified novel compounds with more than 200-fold enhanced reactivity, as compared to dimedone. The increase in reactivity and retention of selectivity of these C-nucleophiles were validated in secondary assays, including a protein model for sulfenic acid. Together, this work represents a significant step toward developing new chemical reporters for detecting protein S-sulfenylation with superior kinetic resolution. The enhanced rates and varied composition of the C-nucleophiles should enable more comprehensive analyses of the sulfenome and serve as the foundation for reversible or irreversible nucleophilic covalent inhibitors that target oxidized cysteine residues in therapeutically important proteins.     

Chemical Synthesis of Human Insulin‐Like Peptide‐6

Human insulin‐like peptide‐6 (INSL‐6) belongs to the insulin superfamily and shares the distinctive disulfide bond configuration of human insulin. In this report we present the first chemical synthesis of INSL‐6 utilizing fluorenylmethyloxycarbonyl‐based (Fmoc) solid‐phase peptide chemistry and regioselective disulfide bond construction protocols. Due to the presence of an oxidation‐sensitive tryptophan residue, two new orthogonal synthetic methodologies were developed. The first method involved the identification of an additive to suppress the oxidation of tryptophan during iodine‐mediated S‐acetamidomethyl (Acm) deprotection and the second utilized iodine‐free, sulfoxide‐directed disulfide bond formation. The methodologies presented here offer an efficient synthetic route to INSL‐6 and will further improve synthetic access to other multiple‐disulfide‐containing peptides with oxidation‐sensitive residues.     

基于苯并吡喃腈的激活型半胱氨酸荧光探针

设计并合成了基于苯并吡喃腈为母体单元的近红外激活型荧光探针(E)-2-(苯并吡喃腈基)乙烯基-5-(二乙氨基)丙烯酸苯酯(DCM-AC),其结构中的丙烯酰酯键作为氨基酸激活反应的响应基团。 研究结果表明,探针分子DCM-AC对半胱氨酸具有高灵敏、选择性光谱响应,不仅能观察到明显的颜色变化,而且探针在710 nm处的荧光发射强度显著增强,相应的荧光增强比值与半胱氨酸的浓度(1.0~8.0 μmol/L)呈现良好的线性关系。 探针DCM-AC对半胱氨酸的检出限为2.8×10^-7 mol/L,能选择性检测半胱氨酸区别于结构类似的高半胱氨酸和谷胱甘肽,且不受其它氨基酸物质干扰。 通过质谱、核磁和紫外吸收光谱研究了DCM-AC检测半胱氨酸的反应激活机理:半胱氨酸先通过巯基与DCM-AC上的丙烯酰酯双键发生亲核加成,然后环化脱除内酰胺环状化合物。     

Post-translational modification of Rhodococcus R312 and Comamonas NI1 nitrile hydratases

Nitrile hydratases (NHases) are industrially significant iron- and cobalt-containing enzymes used in the large-scale synthesis of acrylamide. Previous reports have shown that the active site peptides of NHases are post-translationally modified by oxidation of cysteine residues, and that these modifications are essential for catalysis. We report mass spectrometric evidence of the oxidation states of the active site cysteines in the iron coordination spheres of two iron-containing nitrile hydratases, namely R312 NHase from Rhodococcus rhodochrous strain R312 and NI1 NHase from Comamonas testosteroni. At least one of these cysteines is oxidised to a sulfinic acid (SO(2)H) and there is also evidence suggesting an additional oxidation to a sulfenic acid (SOH). This is the first evidence for the presence of these oxidation states for full-length NHases and for Fe-NHases from different microorganisms. The presence of these covalent modifications was confirmed by performing mass spectrometry on the active site peptide of R312 NHase, under native, reduced and carboxymethylated conditions. We also show the nitrosylation of the iron by mass spectrometry, as well as the release of NO by photoirradiation.     

Fmoc-based solid-phase synthesis of adenylylated peptides using diester-type adenylylated amino acid derivatives

Phosphodiester-type adenylylated (AMPylated) Ser, Thr, and Tyr derivatives were developed for Fmoc solid phase peptide synthesis of AMPylated peptides. One-pot/sequential reaction consisting of condensation of an N-nonprotected adenosine derivative and Fmoc-Ser/Thr/Tyr-OAllyl using allyl-N,N-diisopropylchlorophosphoramidite and subsequent oxidation with m-chloroperbenzoic acid gave phosphotriester-type AMPylated Ser/Thr/Tyr derivatives. After Pd(0)-mediated deprotection of allyl groups, the resulting phosphodiester-type AMPylated Ser/Thr/Tyr derivatives were successfully incorporated into peptides by standard Fmoc solid phase peptide synthesis without significant side reactions including dehydroalanine formation.     

Cyclocondensation of 2-Aminobenzimidazole with Dimedone and Its Arylidene Derivatives

The reactions of 2-aminobenzimidazole with substituted benzaldehydes and dimedone, 2-arylidene derivatives of dimedone, 9-arylhexahydro-1H-xanthene-1,8(2H)-diones and also with dimedone and DMF have been studied. The direction of formation of the pyrimidine ring has been established and discussed. An X-ray structural investigation of 2,2-dimethyl-2,3-dihydrobenzimidazo[1,2-a]quinazolin-4(1H)-one has been carried out.     

N-Alkyl cysteine-assisted thioesterification of peptides

A new method for the preparation of peptide thioester by the post-solid phase peptide synthesis (SPPS) approach was developed. A series of N-alkyl cysteine derivatives were prepared and used as the C-terminus residue of the peptides prepared by the Fmoc SPPS. The synthetic peptides released from resin by TFA were readily converted to the peptide thioester in aqueous 3-mercaptopropionic acid (MPA) without significant side reactions.     

Reactive sulfur species: kinetics and mechanisms of the oxidation of cysteine by hypohalous acid to give cysteine sulfenic acid

Cysteine sulfenic acid has been generated in alkaline aqueous solution by oxidation of cysteine with hypohalous acid (HOX, X = Cl or Br). The kinetics and mechanisms of the oxidation reaction and the subsequent reactions of cysteine sulfenic acid have been studied by stopped-flow spectrophotometry between pH 10 and 14. Two reaction pathways were observed: (1) below pH 12, the condensation of two sulfenic acids to give cysteine thiosulfinate ester followed by the nucleophilic attack of cysteinate on cysteine thiosulfinate ester and (2) above pH 10, a pH-dependent fast equilibrium protonation of cysteine sulfenate that is followed by rate-limiting comproportionation of cysteine sulfenic acid with cysteinate to give cystine. The observation of the first reaction suggests that the condensation of cysteine sulfenic acid to give cysteine thiosulfinate ester can be competitive with the reaction of cysteine sulfenic acid with cysteine.     

Efficient Chemical Protein Synthesis using Fmoc-Masked N-Terminal Cysteine in Peptide Thioester Segments

We report an operationally simple method to facilitate chemical protein synthesis by fully convergent and one-pot native chemical ligations utilizing the fluorenylmethyloxycarbonyl (Fmoc) moiety as an N-masking group of the N-terminal cysteine of the middle peptide thioester segment(s). The Fmoc group is stable to the harsh oxidative conditions frequently used to generate peptide thioesters from peptide hydrazide or o-aminoanilide. The ready availability of Fmoc-Cys(Trt)-OH, which is routinely used in Fmoc solid-phase peptide synthesis, where the Fmoc group is pre-installed on cysteine residue, minimizes additional steps required for the temporary protection of the N-terminal cysteinyl peptides. The Fmoc group is readily removed after ligation by short exposure (<7 min) to 20 % piperidine at pH 11 in aqueous conditions at room temperature. Subsequent native chemical ligation reactions can be performed in presence of piperidine in the same solution at pH 7.     

Cysteine Derivatives Protect Against UV-lnduced Reactive Intermediates in Human Keratinocytes: The Role of Glutathione Synthesis

Abstract— Several recent studies have shown cysteine derivatives can protect against negative effects of UV exposure. In this study, an attempt was made to correlate cellular bioavailability and metabolism of cysteine derivatives with protection against UV-induced reactive intermediates. Human keratinocytes were treated with cysteine, N -ace-tylcysteine(NAC), cysteine-ethylester(CYSET) and N- acetylcysteine-ethylester. The uptake of the compounds and their metabolism to cysteine and eventually to glutathione(GSH) was measured. Large differences in uptake were observed, with CYSET resulting in the highest and NAC in the lowest intracellular thiol levels. The increase in intracellular GSH was similar for all derivatives with a maximum of 23-54% over the control level. Protective efficacy of the derivatives was measured as the inhibition of binding of UV-induced reactive intermediates from 8-methoxypsoralen. There was only a small difference between the compounds, with maximum protection of 25-31%. No relation was found between total intracellular thiol and protection. However, for NAC, there was a linear relation between GSH level and protective efficacy (r = 0.94). Even though this was not clear for the other derivatives(r = 0.55 for CYS; r = 0.60 for CYSET; r = 0.70 for NACET), it indicates that GSH synthesis is an important factor.
This was confirmed by experiments using cells with irreversibly inhibited GSH synthesis. Even though the total intracellular thiol level was comparable to uninhibited cells, protection was decreased. We conclude that the intracellular GSH increase is the most important factor in photoprotection by cysteine derivatives.     

含羟基和咪唑基团新手性肽核酸的设计与合成

以含丝氨酸和组氨酸残基的手性肽核酸单体替换经典肽核酸单体, 采用固相合成方法设计合成了五个新序列含羟基和咪唑基团的10聚体手性肽核酸, 经ESI-MS或MALDI-TOF-MS证实目标物结构正确.