Disulfide Bonds In Peptides Research Articles

Aryl Radicals Generated from Aryl Pinacol Boronates Modify Peptides and Proteins

We report here a distinct reaction, which generates aryl radicals from aryl pinacol boronates under mild aqueous conditions and can be used for peptide and protein modifications. The strategy leverages the versatile reactivity of aryl pinacol boronates to form aryl radicals in presence of ammonium persulfate (APS). The formed aryl radicals insert readily into peptide disulfide bonds while tolerating other functionalities. On the protein level, the reactivity extends beyond the disulfide bonds. The methodology benefits from the accessibility of starting aryl pinacol boronates, as well as biocompatible conditions. In contrast to conventional methods used for aryl radical generation, the strategy is metal‐free, does not require photoinduction and can be readily performed under aqueous conditions. The mechanism of the reactions was investigated by radical‐trapping experiments, spectroscopic analysis and oxygen scavenging. The presented approach broadens the application of aryl pinacol boronate esters in radical reactions.

Aromatic diselenide catalytic reactions for the efficient formation of multiple disulfide bonds in peptides

Multiple disulfide bonds (MBDs) play a critical role in maintaining the three-dimensional structure of peptides and proteins affording their pharmaceutical and biological activities. The efficient synthesis of correct MBDs in peptides and proteins remains a challenging task in chemical synthesis these compounds. In this work, aromatic diselenides including diphenyl diselenide (DPDS), bis(2-carboxyphenyl)diselenide (BCPDS), and 2,2′-dipyridyl diselenide (DPDDS) played as catalysts and were successfully used for the synthesis of correct MBDs in peptides. Good yields were achieved by use of the catalytic reactions under mild conditions, and the reaction time decreased with the trend of DPDDS < BCPDS < DPDS. A reaction mechanism was proposed, in which the formation of a selenenylsulfide intermediate played a critical role in the formation of correct MBDs in peptides. On the other hand, phenylselenenyl chloride (PSC) can oxidize fully reduced peptides to their correct MDBs forms.

Identification of Peptides in Spider Venom Using Mass Spectrometry.

Spider venoms are composed of hundreds of proteins and peptides. Several of these venom toxins are cysteine-rich peptides in the mass range of 3-9kDa. Small peptides (<3kDa) can be fully characterized by mass spectrometry analysis, while proteins are generally identified by the bottom-up approach in which proteins are first digested with trypsin to generate shorter peptides for MS/MS characterization. In general, it is sufficient for protein identification to sequence two or more peptides, but for venom peptidomics it is desirable to completely elucidate peptide sequences and the number of disulfide bonds in the molecules. In this chapter, we describe a methodology to completely sequence and determine the number of disulfide bonds of spider venom peptides in the mass range of 3-9kDa by multiple enzyme digestion, mass spectrometry of native and digested peptides, de novo analysis, and sequence overlap alignment.

Thiuram Disulfides for the Fast and Efficient Synthesis of Peptide Disulfide Bonds Under Aqueous Conditions

AbstractDisulfide bonds play a critical role in the pharmaceutical and biological properties of peptides. In this work, disulfiram and thiram were investigated to synthesize disulfide bonds in peptides. Excellent purities of single disulfide bond‐containing peptides were achieved with a fast reaction rate. Furthermore, one‐pot regioselective synthesis of two disulfide bonds was successfully achieved by the combination of disulfiram or thiram with trans‐[PtBr2(CN)4]2− under aqueous conditions.

L-Methionine Selenoxide as an Oxidizing and Deprotection Reagent for the Synthesis of Multiple Disulfide Bonds in Peptides.

The regioselective synthesis of multiple disulfide bonds in peptides has been a significant challenge in synthetic peptide chemistry. In this work, two disulfide bonds in peptides were regioselectively synthesized via an approach of MetSeO oxidation and deprotection reaction (SeODR), in which the first disulfide bond was constructed through oxidation of dithiol by MetSeO in a neutral buffer, and the second disulfide bond was then directly constructed through the deprotection of two Acm groups or one Acm group and one Thz group by MetSeO in acidic media. Synthesis of two disulfide bonds by the SeODR approach was achieved through a one-pot manner. Moreover, the SeODR approach is compatible with the synthesis of peptides containing methionine residues. Both H+ and Br- drastically promoted the reaction rate of SeODR. The mechanistic picture for the SeODR approach was delineated, in which the formation of a stable Se-X-S bridge as the transition state plays a critical role. The SeODR approach was also utilized to construct the three disulfide bonds in linaclotide, conferring a reasonable yield.

N-Halosuccinimides mediated deprotection of cysteine-S protecting groups for one-pot regioselective synthesis of disulfide bonds in peptides under mild aqueous conditions

Cysteine (Cys) with side chain protected by groups acetamidomethyl (Acm), 1,3-thiazolidine-4-carbonyl (Thz) and t-butyl (tBu) has been commonly used for the synthesis of correct disulfide bonds in peptides. Deprotection of these protected groups can be achieved by use of metal salts under harshly removing conditions. In this work, N-chlorosuccinimide (NCS) and N-bromosuccinimide (NBS) are demonstrated to deprotect these protecting groups to directly form an intramolecular disulfide bond in peptide. In addition, all the deprotection reactions were complete within 10 min, conferring fast reaction rates. Moreover, formation of a hydrolytic stable halosulfonium cation was revealed to play a critical role in these deprotection reactions. When NCS and NBS were employed for deprotecting these groups, two disulfide bonds in α-conotoxin SI, α-conotoxin IMI and apamin were synthesized regioselectively by an one-pot approach, rendering satisfied yields.

Polymer Supported Methionine Selenoxide as an Excellent Immobilized Oxidant for the Formation of Disulfide Bonds in Peptides

AbstractTwo PEG‐based polymer supported methionine selenoxide (MeSeO) named TentaGel‐MetSeO and PEG‐MetSeO were synthesized and used as immobilized oxidants for the purpose of formation of disulfide bonds in peptides under aqueous conditions. Peptide disulfide bonds were successfully synthesized by use of the two immobilized oxidants. Moreover, the oxidation of the methionine residue to its sulfoxide derivate was not observed. By comparison, the yield of disulfide‐containing peptide via PEG‐MetSeO oxidation is higher than that of the disulfide‐containing peptide via TentaGel‐MetSeO oxidation. PEG‐MetSeO can be used at least 10 cycles without the significant yield decrease. The two immobilized oxidants can be separated from the reaction mixture using filtration, making the peptide synthesis more easily.

In vitro Inhibition of HIV-1 by Cyclotide-Enriched Extracts of Viola tricolor.

Since viral infectious diseases continue to be a global health threat, new antiviral drugs are urgently needed. A unique class of therapeutic compounds are antimicrobial peptides (AMPs). They can be found in humans, bacteria and plants. Plants express a wide variety of such defense peptides as part of their innate immune system to protect from invading pathogens. Cyclotides are non-classical AMPs that share a similar structure. Their unique topology consists of a circular peptide backbone and disulfide bonds. In previous studies they have been attributed to a wide range of biological activities. To identify novel cyclotides with antiviral activity, we established a library of plant extracts largely consisting of cyclotide-rich species and screened them as inhibitors of HIV-1 infection. Subsequent extraction and fractionation revealed four cyclotide-containing subfractions from Viola tricolor with antiviral activity. These subfractions inhibited HIV-1 infection with IC50 values between 0.6 and 11.2 μg/ml, and selectivity indices of up to 8.1. The identification and characterization of antiviral cyclotides and the determination of the antiviral mechanisms may allow to develop novel agents to combat viral infections. Therefore, cyclotides represent a natural source of bioactive molecules with prospects for development as therapeutics.

Carboxypeptidase Y Assisted Disulfide-Bond Identification with Linearized Database Search.

A disulfide bond is an important protein post-translational modification and plays a key role in regulating protein oxidation status, protein structure, and stability. Analysis of a disulfide bond using mass spectrometry is challenging because there lacks an efficient method to separate the disulfide-linked peptides from a complex protein digest, and the MS data requires sophisticated interpretation. Here, we developed a novel disulfide bond identification strategy, termed as "carboxypeptidase Y assisted disulfide-bond identification (CADI)". CADI is able to significantly reduce sample complexity by depleting ∼90% of the linear peptides while keeping the disulfide-bonded peptides. Furthermore, all CADI data can be directly analyzed by widely used protein database search engines, such as Mascot and MaxQuant. Our data show that CADI is able to sensitively identify disulfide bonds in peptides and proteins. However, CADI has not yet achieved a satisfied in-depth coverage on complex mammalian cell lysates due to the limited enzymatic activity of carboxypeptidase Y and low occurrences of disulfide bonds in a proteome. Altogether, CADI is a useful method that can get disulfide-linked peptides enriched and analyzed with regular search engines. CADI holds great potentials to deepen the analysis of disulfide bond and other types of cross-linked peptides on the proteome scale.

Insight on the Order of Regioselective Ultrafast Formation of Disulfide Bonds in (Antimicrobial) Peptides and Miniproteins.

Disulfide-rich peptides and proteins are among the most fascinating bioactive molecules. The difficulties associated with the preparation of these targets have prompted the development of various chemical strategies. Nevertheless, the production of these targets remains very challenging or elusive. Recently, we introduced a strategy for one-pot disulfide bond formation, tackling most of the previous limitations. However, the effect of the order of oxidation remained an underexplored issue. Herein we report on the complete synthetic flexibility of the approach with respect to the order of oxidation of three disulfide bonds in targets that lack the knot motif. In contrast, our study reveals an essential order of disulfide bond formation in the EETI-II knotted miniprotein. This synthetic strategy was applied for the synthesis of novel analogues of the plectasin antimicrobial peptide with enhanced activities against methicillin-resistant Staphylococcus aureus (MRSA), a notorious human pathogen.

Insight on the Order of Regioselective Ultrafast Formation of Disulfide Bonds in (Antimicrobial) Peptides and Miniproteins

AbstractDisulfide‐rich peptides and proteins are among the most fascinating bioactive molecules. The difficulties associated with the preparation of these targets have prompted the development of various chemical strategies. Nevertheless, the production of these targets remains very challenging or elusive. Recently, we introduced a strategy for one‐pot disulfide bond formation, tackling most of the previous limitations. However, the effect of the order of oxidation remained an underexplored issue. Herein we report on the complete synthetic flexibility of the approach with respect to the order of oxidation of three disulfide bonds in targets that lack the knot motif. In contrast, our study reveals an essential order of disulfide bond formation in the EETI‐II knotted miniprotein. This synthetic strategy was applied for the synthesis of novel analogues of the plectasin antimicrobial peptide with enhanced activities against methicillin‐resistant Staphylococcus aureus (MRSA), a notorious human pathogen.

The Redox-Active Conopeptide Derived from the Venom Duct Transcriptome of Conus lividus Assists in the Oxidative Folding of Conotoxin.

The tetrapeptides Li504 and Li520, differing in the modification of the 4-trans-hydroxylation of proline, are novel conopeptides derived from the venom duct transcriptome of the marine cone snail Conus lividus. These predicted mature peptides are homologous to the active site motif of oxidoreductases that catalyze the oxidation, reduction, and rearrangement of disulfide bonds in peptides and proteins. The estimated reduction potential of the disulfide of Li504 and Li520 is within the range of disulfide reduction potentials of oxidoreductases, indicating that they may catalyze the oxidative folding of conotoxins. Conformational features of Li504 and Li520 include the trans configuration of the Cys1-Pro2/Hyp2 peptide bond with a type 1 turn that is similar to the active site motif of glutaredoxin that regulates the oxidation of cysteine thiols to disulfides. Li504- and Li520-assisted oxidative folding of α-conotoxin ImI confirms that Li520 improves the yield of the natively folded peptide by concomitantly decreasing the yield of the non-native disulfide isomer and thus acts as a miniature disulfide isomerase. The geometry of the Cys1-Hyp2 peptide bond of Li520 shifts between the trans and cis configurations in the disulfide form and thiol/thiolate form, which regulates the deprotonation of the N-terminal cysteine residue. Hydrogen bonding of the hydroxyl group of 4-trans-hydroxyproline with the interpeptide chain unit in the mixed disulfide form may play a vital role in shifting the geometry of the Cys1-Hyp2 peptide bond from cis to trans configuration. The Li520 conopeptide together with similar peptides derived from other species may constitute a new family of "redox-active" conopeptides that are integral components of the oxidative folding machinery of conotoxins.

Expanding the Toolbox for Peptide Disulfide Bond Formation via l-Methionine Selenoxide Oxidation.

In this study, l-methionine selenoxide (MetSeO) was used as an oxidant for the construction of peptide disulfide bonds. Excellent yields for various disulfide-containing peptides were achieved via the MetSeO oxidation method in different solvents and on a resin. Most importantly, the construction of disulfide bonds can be performed in the trifluoroacetic acid cocktail used for the cleavage of peptides from the resin, which obviates the steps of peptide purification and lyophilization. This facilitates and simplifies the synthesis of disulfide-containing peptides. Kinetic and mechanistic studies of the reaction between MetSeO and dithiothreitol (DTT, a model compound of dicysteine-containing peptide) show that the reaction is first order in both [MetSeO] and [DTT], and a reaction mechanism is proposed that can help us gain insights into the reaction of the oxidative synthesis of disulfide bonds via MetSeO oxidation.

General synthetic strategy for regioselective ultrafast formation of disulfide bonds in peptides and proteins

Despite six decades of efforts to synthesize peptides and proteins bearing multiple disulfide bonds, this synthetic challenge remains an unsolved problem in most targets (e.g., knotted mini proteins). Here we show a de novo general synthetic strategy for the ultrafast, high-yielding formation of two and three disulfide bonds in peptides and proteins. We develop an approach based on the combination of a small molecule, ultraviolet-light, and palladium for chemo- and regio-selective activation of cysteine, which enables the one-pot formation of multiple disulfide bonds in various peptides and proteins. We prepare bioactive targets of high therapeutic potential, including conotoxin, RANTES, EETI-II, and plectasin peptides and the linaclotide drug. We anticipate that this strategy will be a game-changer in preparing millions of inaccessible targets for drug discovery.

General Synthetic Strategy for Regioselective Ultrafast Formation of Disulfide Bonds in Peptides and Proteins

General Synthetic Strategy for Regioselective Ultrafast Formation of Disulfide Bonds in Peptides and Proteins

Formation of peptide disulfide bonds through a trans-dibromido-Pt(IV) complex oxidation reaction: Kinetic and mechanistic analyses

A trans-dibromido-Pt(IV) complex [PtBr2(en)2]Br2 was synthesized and evaluated for the synthesis of peptide disulfide bonds in this work. The reactions between the Pt(IV) complex and dicysteine-containing peptides were carried out in various aqueous solutions. As a result, excellent yields with fast reaction rates were achieved. Methionine residue was intact when the Pt(IV) complex reacted with a dicysteine and methionine-containing peptide. 3,6-Dioxa-1,8-octanedithiol (DODT) was selected as a model compound of the dicysteine-containing peptide. Kinetic studies for the reaction between the Pt(IV) complex and DODT were performed in different pH solutions. It is first-order both in [Pt(IV)] and in [DODT]. A reaction mechanism was proposed accordingly. The kinetic and mechanistic results demonstrated that the reaction rate for the formation of peptide disulfide bond via the Pt(IV) oxidation is increased with the increase of pH of the reaction medium. The protolytic constants of the two thiol groups in peptide also play a critical role in reaction rate. On the other hand, kinetic studies demonstrated that the Pt(IV) complex trans-[PtBr2(en)2]2+ can be used in an acidic medium for the purpose of synthesis of disulfide bonds.

A study to develop platinum(iv) complex chemistry for peptide disulfide bond formation.

Platinum(iv) complexes with a heterocyclic ligand and an ancillary ligand have been investigated and applied for treating various tumour cell lines. Another application of the Pt(iv) complexes in forming peptide disulfide bonds was investigated in this work. For development of Pt(iv) complex chemistry for disulfide bond formation in peptides, two Pt(iv) complexes, [PtCl2(phen)(en)]Cl2 and [PtCl2(bpy)(en)]Cl2, were synthesized and characterized using elemental analysis, ESI-MS and NMR. Subsequently, they were investigated as oxidants for the formation of disulfide bonds in various peptides. Excellent purities and yields of disulfide-containing peptides were achieved when the reactions were carried out in aqueous solution. The reactions were completed rapidly in a wide range of pH values even in acidic medium at room temperature. An intramolecular disulfide bond was formed in each of the peptides in a solution containing two dithiol-containing peptides, making the Pt(iv) complexes useful for generating disulfide-containing peptide libraries. In addition, the two Pt(iv) complexes can be used as oxidants for the synthesis of disulfide bonds on a resin, which is a more convenient method to synthesize disulfide-containing peptides through automation.

2,2'-Dipyridyl diselenide: A chemoselective tool for cysteine deprotection and disulfide bond formation.

There are many examples of bioactive, disulfide-rich peptides and proteins whose biological activity relies on proper disulfide connectivity. Regioselective disulfide bond formation is a strategy for the synthesis of these bioactive peptides, but many of these methods suffer from a lack of orthogonality between pairs of protected cysteine (Cys) residues, efficiency, and high yields. Here, we show the utilization of 2,2'-dipyridyl diselenide (PySeSePy) as a chemical tool for the removal of Cys-protecting groups and regioselective formation of disulfide bonds in peptides. We found that peptides containing either Cys(Mob) or Cys(Acm) groups treated with PySeSePy in trifluoroacetic acid (TFA) (with or without triisopropylsilane (TIS) were converted to Cys-S-SePy adducts at 37 °C and various incubation times. This novel Cys-S-SePy adduct is able to be chemoselectively reduced by five-fold excess ascorbate at pH 4.5, a condition that should spare already installed peptide disulfide bonds from reduction. This chemoselective reduction by ascorbate will undoubtedly find utility in numerous biotechnological applications. We applied our new chemistry to the iodine-free synthesis of the human intestinal hormone guanylin, which contains two disulfide bonds. While we originally envisioned using ascorbate to chemoselectively reduce one of the formed Cys-S-SePy adducts to catalyze disulfide bond formation, we found that when pairs of Cys(Acm) residues were treated with PySeSePy in TFA, the second disulfide bond formed spontaneously. Spontaneous formation of the second disulfide is most likely driven by the formation of the thermodynamically favored diselenide (PySeSePy) from the two Cys-S-SePy adducts. Thus, we have developed a one-pot method for concomitant deprotection and disulfide bond formation of Cys(Acm) pairs in the presence of an existing disulfide bond.

Construction of disulfide bonds in peptides via immobilized platinum(IV) complex oxidation

A novel TentaGel resin supported platinum(IV) complex named TentaGel-Pt(IV) was synthesized and characterized by SEM, XPS, ICP-MS and UV–vis spectrophotometry. The formation of intramolecular disulfides in eleven dithiol-containing peptides of variable lengths using TentaGel-Pt(IV) was investigated at room temperature. The disulfide-containing peptides were formed in excellent oxidation yields and were easily separated by filtration upon completion of the reaction. Excellent reusability of TentaGel-Pt(IV) was also achieved. The mechanism for construction of the disulfide bond in peptides via TentaGel-Pt(IV) oxidation was proposed.

Hydrogen halide assisted formation of peptide disulfides by a platinum(IV) complex oxidation in aqueous medium, a mechanistic study

A new Pt(IV) complex trans-[Pt(OH)2(phen)(en)]2+ was synthesized and characterized by elemental analysis, MS and 1H NMR. It was used as an oxidant for construction of disulfide bond in peptide in various solutions. When the oxidation reaction was carried out in HBr solution, the highest oxidation rate and yield were achieved; no side reactions were occurred on the side chain of methionine, tyrosine and tryptophan residues of peptide. A novel reaction mechanism was proposed through the HPLC studies.