P−B Desulfurization: An Enabling Method for Protein Chemical Synthesis and Site‐Specific Deuteration

Cysteine‐mediated native chemical ligation is a powerful method for protein chemical synthesis. Herein, we report an unprecedentedly mild system (TCEP/NaBH₄ or TCEP/LiBEt₃H; TCEP=tris(2‐carboxyethyl)phosphine) for chemoselective peptide desulfurization to achieve effective protein synthesis via the native chemical ligation–desulfurization approach. This method, termed P−B desulfurization, features usage of common reagents, simplicity of operation, robustness, high yields, clean conversion, and versatile functionality compatibility with complex peptides/proteins. In addition, this method can be used for incorporating deuterium into the peptides after cysteine desulfurization by running the reaction in D₂O buffer. Moreover, this method enables the clean desulfurization of peptides carrying post‐translational modifications, such as phosphorylation and crotonylation. The effectiveness of this method has been demonstrated by the synthesis of the cyclic peptides dichotomin C and E and synthetic proteins, including ubiquitin, γ‐synuclein, and histone H2A.     

Controlling Peptide Self‐Assembly through a Native Chemical Ligation/Desulfurization Strategy

Self‐assembled peptides were synthesized by using a native chemical ligation (NCL)/desulfurization strategy that maintained the chemical diversity of the self‐assembled peptides. Herein, we employed oxo‐ester‐mediated NCL reactions to incorporate cysteine, a cysteine‐based dipeptide, and a sterically hindered unnatural amino acid (penicillamine) into peptides. Self‐assembly of the peptides resulted in the formation of self‐supporting gels. Microscopy analysis indicated the formation of helical nanofibers, which were responsible for the formation of gel matrices. The self‐assembly of the ligated peptides was governed by covalent and non‐covalent interactions, as confirmed by FTIR, CD, fluorescence spectroscopy, and MS (ESI) analyses. Peptide disassembly was induced by desulfurization reactions with tris(2‐carboxyethyl)phosphine (TCEP) and glutathione at 80 °C. Desulfurization reactions of the ligated peptides converted the Cys and penicillamine functionalities into Ala and Val moieties, respectively. The self‐supporting gels showed significant shear‐thinning and thixotropic properties.     

Desulfurization of cysteine‐containing peptides resulting from sample preparation for protein characterization by mass spectrometry

In this study, we have examined two cysteine modifications resulting from sample preparation for protein characterization by mass spectrometry (MS): (1) a previously observed conversion of cysteine into dehydroalanine, now found in the case of disulfide mapping and (2) a novel modification corresponding to conversion of cysteine into alanine. Using model peptides, the conversion of cysteine into dehydroalanine via β‐elimination of a disulfide bond was seen to result from the conditions of typical tryptic digestion (37°C, pH 7.0–9.0) without disulfide reduction and alkylation. Furthermore, the surprising conversion of cysteine into alanine was shown to occur by heating cysteine‐containing peptides in the presence of a phosphine (tris(2‐carboxyethyl)phosphine hydrochloride (TCEP)). The formation of alanine from cysteine, investigated by performing experiments in H₂O or D₂O, suggested a radical‐based desulfurization mechanism unrelated to β‐elimination. Importantly, an understanding of the mechanism and conditions favorable for cysteine desulfurization provides insight for the establishment of improved sample preparation procedures of protein analysis. Copyright © 2010 John Wiley & Sons, Ltd.     

Cysteine/Penicillamine Ligation Independent of Terminal Steric Demands for Chemical Protein Synthesis

The chemical ligation of two unprotected peptides to generate a natural peptidic linkage specifically at the C‐ and N‐termini is a desirable goal in chemical protein synthesis but is challenging because it demands high reactivity and selectivity (chemo‐, regio‐, and stereoselectivity). We report an operationally simple and highly effective chemical peptide ligation involving the ligation of peptides with C‐terminal salicylaldehyde esters to peptides with N‐terminal cysteine/penicillamine. The notable features of this method include its tolerance of steric hinderance from the side groups on either ligating terminus, thereby allowing flexible disconnection at sites that are otherwise difficult to functionalize. In addition, this method can be expanded to selective desulfurization and one‐pot ligation‐desulfurization reactions. The effectiveness of this method was demonstrated by the synthesis of VISTA (216‐311), PD‐1 (192‐288) and Eglin C.     

A tris (2-carboxyethyl) phosphine (TCEP) related cleavage on cysteine-containing proteins

Introduced in the late 1980s as a reducing reagent, Tris (2-carboxyethyl) phosphine (TCEP) has now become one of the most widely used protein reductants. To date, only a few studies on its side reactions have been published. We report the observation of a side reaction that cleaves protein backbones under mild conditions by fracturing the cysteine residues, thus generating heterogeneous peptides containing different moieties from the fractured cysteine. The peptide products were analyzed by high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptides with a primary amine and a carboxylic acid as termini were observed, and others were found to contain amidated or formamidated carboxy termini, or formylated or glyoxylic amino termini. Formamidation of the carboxy terminus and the formation of glyoxylic amino terminus were unexpected reactions since both involve breaking of carbon—carbon bonds in cysteine.     

Desulfurization Mechanism of Cysteine in Synthesis of Polypeptides

The free-radical-based selective desulfurization of cysteine residue is an e cient protocol to achieve ligations at alanine sites in the synthesis of polypeptide and proteins. In this work, the mechanism of desulfurization process has been studied using the density functional theory methods. According to the calculation results, the desulfurization of the thiol group occurs via a three-steps mechanism: the abstraction of hydrogen atom on the thiol group with the radical initiator VA-044 (2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride), the removal of S atom under the reductant TCEP (tris(2-carboxyethyl)phosphine), and theformation of RH molecule (with the regeneration of RS radical). The second step (desulfurization step) is the rate-determining step, and the adduct t-BuSH facilitates the desulfurization of cysteine via bene ting the formation of the precursor of the desulfurization step.     

Evidence for formation of diphenylphosphinic peroxy radical intermediate: Spin trapping and chemical reactivity of the new phosphorus radical generated from diphenylphosphinic chloride and superoxide

Diphenylphosphinic chloride reacts with the superoxide anion radical in acetonitrile under mild conditions to form a peroxyphosphorus radical intermediate which shows strong oxidizing abilities for the epoxidation of olefins, oxidation of sulfides to sulfoxides, desulfurization of thioamides to amides, and oxidation of triarylphosphines to phosphine oxides. The radical can be trapped using a spin trapping method.     

A novel ring opening reaction of peptide N-terminal thiazolidine with 2,2′-dipyridyl disulfide (DPDS) efficient for protein chemical synthesis

In the protein chemical synthesis via native chemical ligation (NCL) method with three peptide segments, the N-terminal cysteine residue of middle segment is generally protected by thiazolidine ring. In this paper, we show the novel method for thiazolidine ring opening using 2,2′-dipyridyl disulfide (DPDS). The N-terminal thiazolidine was converted into S-pyridylsulfenylated cysteine residue with DPDS under acidic conditions, and this N-terminally Cys peptide protected with disulfide was applicable to NCL reaction without purification and deprotection steps. DPDS treatment did not remove other Cys protecting groups generally used for regioselective disulfide bond formation reactions. These results indicate that this thiazolidine ring opening reaction is quite useful for the protein chemical synthesis with three-segment NCL strategy.     

Synthesis of peptides and proteins without cysteine residues by native chemical ligation combined with desulfurization.

The highly chemoselective reaction between unprotected peptides bearing an N-terminal Cys residue and a C-terminal thioester enables the total and semi-synthesis of complex polypeptides. Here we extend the utility of this native chemical ligation approach to non-cysteine containing peptides. Since alanine is a common amino acid in proteins, ligation at this residue would be of great utility. To achieve this goal, a specific alanine residue in the parent protein is replaced with cysteine to facilitate synthesis by native chemical ligation. Following ligation, selective desulfurization of the resulting unprotected polypeptide product with H(2)/metal reagents converts the cysteine residue to alanine. This approach, which provides a general method to prepare alanyl proteins from their cysteinyl forms, can be used to chemically synthesize a variety of polypeptides, as demonstrated by the total chemical syntheses of the cyclic antibiotic microcin J25, the 56-amino acid streptococcal protein G B1 domain, and a variant of the 110-amino acid ribonuclease, barnase.     

Tuning Catalysts to Tune the Products: Phosphine‐Catalyzed Aza‐Michael Addition Reaction of Hydrazones with Allenoates

A new tunable phosphine‐catalyzed aza‐Michael β‐addition reaction between allenoates and various hydrazones has been developed. These reactions are most‐efficiently promoted by a catalytic amount of phosphine catalysts. These atom‐economical reactions are operationally simple and their corresponding adducts can been achieved in high yields and high selectivity under mild reaction conditions. Further studies revealed that different phosphine catalyst can produce different adducts from the same starting materials.     

Regio- and chemoselective covalent immobilization of proteins through unnatural amino acids

A general approach was developed for the regio- and chemoselective covalent immobilization of soluble proteins on glass surfaces through an unnatural amino acid created by post-translationally modifying the cysteine residue in a CaaX recognition motif with functional groups suitable for "click" chemistry or a Staudinger ligation. Farnesyl diphosphate analogues bearing omega-azide or omega-alkyne moieties were attached to the cysteine residue in Cys-Val-Ile-Ala motifs at the C-termini of engineered versions of green fluorescent protein (GFP) and glutathione S-transferase (GST) by protein farnesyltransferase. The derivatized proteins were attached to glass slides bearing linkers containing azide ("click" chemistry) or phosphine (Staudinger ligation) groups. "Click"-immobilized proteins were detected by fluorescently labeled antibodies and remained attached to the slide through two cycles of stripping under stringent conditions at 80 degrees C. GFP immobilized by a Staudinger ligation was detected by directly imagining the GFP fluorophore over a period of 6 days. These methods for covalent immobilization of proteins should be generally applicable. CaaX recognition motifs can easily be appended to the C-terminus of a cloned protein by a simple modification of the corresponding gene, and virtually any soluble protein or peptide bearing a CaaX motif is a substrate for protein farnesyltransferase.     

Mechanism of Phosphine‐Catalyzed Allene Coupling Reactions: Advances in Theoretical Investigations

Organocatalysis has emerged as an effective strategy for chemical synthesis. Within this area, phosphine‐catalyzed coupling reactions have attracted considerable attention because of their versatility and wide range of applications in the construction of new C?C bonds. Recently, various experimental studies on the phosphine‐catalyzed coupling reaction of allenes have been reported, and mechanistic and computational studies have also progressed considerably. As a nucleophile, phosphine can react with an allene to form a zwitterionic phosphoniopropenide intermediate. After stepwise cycloaddition and proton transfer, the phosphine catalyst can be regenerated by C?P bond cleavage. Alternatively, the zwitterionic phosphoniopropenide intermediate could also be protonated by a Brønsted acid to generate a phosphonium intermediate, which can be used to construct new C?C bonds by electrophilic addition. In this review, we have summarized details of mechanistic studies of phosphine‐catalyzed allene coupling reactions that follow these two reaction modes. In addition to detailing the reaction pathway, the regioselectivity and diastereoselectivity of the phosphine‐catalyzed allene coupling reaction are also discussed in this review.     

Unexpected hydrodeiodo Sonogashira-Heck-Casser coupling reaction of 2,2'-diiodobiphenyls with acetylenes

2,2'-Diiodobiphenyl-4,4'-dicarboxylic acid dimethyl ester (3) undergoes either a ring-closure reaction with phenylacetylene to give 4 or hydrodeiodo phenylethynylation to give 5 under the catalytic conditions of Pd(OAc)(2)/CuI/phosphine in amines. In these reactions, the amine and the phosphine ligands play important roles in controlling the reactivity. Among the ligands we used, tris(o-tolyl)phosphine is the best ligand for hydrodeiodo phenylethynylation, while the bidentate phosphine ligand retards both of the reactions. On the basis of our results, we propose that 5 is formed through a fast hydrodeiodination, followed by a Sonogashira phenylethynylation. The results of the deuterium labeling experiments show that proton exchange between the acetylenic proton and the alkyl protons of amine occurs effectively under the reaction conditions. In addition, the hydrogen that replaces the iodide in the hydrodeiodination process arises mainly from the acetylenic proton.     

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.     

Fast Cysteine Bioconjugation Chemistry

Cysteine bioconjugation serves as a powerful tool in biological research and has been widely used for chemical modification of proteins, constructing antibody-drug conjugates, and enabling cell imaging studies. Cysteine conjugation reactions with fast kinetics and exquisite selectivity have been under heavy pursuit as they would allow clean protein modification with just stoichiometric amounts of reagents, which minimizes side reactions, simplifies purification and broadens functional group tolerance. In this concept, we summarize the recent advances in fast cysteine bioconjugation, and discuss the mechanism and chemical principles that underlie the high efficiencies of the newly developed cysteine reactive reagents.     

Sulfur and selenium: the role of oxidation state in protein structure and function

Sulfur and selenium occur in proteins as constituents of the amino acids cysteine, methionine, selenocysteine, and selenomethionine. Recent research underscores that these amino acids are truly exceptional. Their redox activity under physiological conditions allows an amazing variety of posttranslational protein modifications, metal free redox pathways, and unusual chalcogen redox states that increasingly attract the attention of biological chemists. Unlike any other amino acid, the "redox chameleon" cysteine can participate in several distinct redox pathways, including exchange and radical reactions, as well as atom-, electron-, and hydride-transfer reactions. It occurs in various oxidation states in the human body, each of which exhibits distinctive chemical properties (e.g. redox activity, metal binding) and biological activity. The position of selenium in the periodic table between the metals and the nonmetals makes selenoproteins ideal catalysts for many biological redox transformations. It is therefore apparent that the chalcogen amino acids cysteine, methionine, selenocysteine, and selenomethionine exhibit a unique biological chemistry that is the source of exciting research opportunities.     

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.     

Thermal and photocontrol of the equilibrium between a 2-phosphinoazobenzene and an inner phosphonium salt

The reversible conversion between a phosphine and a phosphonium salt has been achieved by external stimuli of light and heat. Two 2-phosphinoazobenzenes were successfully synthesized by desulfurization of the corresponding phosphine sulfides. One of the phosphines bearing an azo group was in equilibrium with an inner phosphonium salt and showed thermochromism, which is derived from the change of the equilibrium constant depending on the temperature. While the 2-phosphinoazobenzene reacted as a usual triarylphosphine, its reaction with water gave phosphine oxide bearing a hydrazine moiety via a mechanism similar to the Mitsunobu reaction. The 2-phosphinoazobenzene bearing a methyl group at the 4'-position of azobenzene was isomerized to the Z-isomer by irradiation. The Z-isomer was neither in equilibrium with an inner phosphonium salt nor hydrolyzed, in contrast to the E-isomer, because its geometry is difficult for an intramolecular nucleophilic attack. Photoisomerization caused the switching of the unique reactivity toward water. Such phosphines in equilibrium with the inner phosphonium salts are expected to be useful to control organic reactions by taking advantage of the photoisomerization of the azobenzene moiety.     

Characterization of active sites, determination of mechanisms of H(2)S, COS and CS(2) sorption and regeneration of ZnO low-temperature sorbents: past, current and perspectives

The intellectually and technically challenging pursuit of the emerging global environmentally "green" and energy-efficient infrastructure of the 21st century requires the development of a worldwide network of low- to medium-power fuel cell (FC) based portable electric power-generating devices and high-power biomass/clean coal "electric+chemical plants" with zero carbon footprint utilizing integrated coal gasification combined cycle with geologic carbon sequestration (IGCC-GCS) under energy-efficient low-temperature conditions. These emerging technologies require the deep and ultradeep desulfurization of gaseous feeds, since sulfur compounds, especially hydrogen sulfide H(2)S are highly corrosive and poisonous to both technological processes and the environment. Therefore, it is of crucial importance for both academic and industrial research communities to have a solid understanding of the atomic-level structures of active sites and molecular-level mechanisms of surface chemical reactions of the novel deep and ultradeep desulfurization materials, especially desulfurization sorbents. This review critically analyzes the recent literature (last ～20 years) on the experimental determination of molecular and atomic-level nature of adsorption sites, effects of desulfurization promoters, mechanisms of chemical reactions of H(2)S, COS and CS(2) and physical processes during and upon regeneration of "spent" low-temperature H(2)S sorbents based on ZnO that were developed for desulfurization of fuel reformates, syngas and similar streams. Recent trends in research on the ultradeep H(2)S sorbents are discussed with an impetus on real-time in situ and Operando techniques of instrumental chemical analysis, and the challenges of direct determination of the structure of active sites and of the experimental mechanistic studies in general are described.     

Enzyme Activity by Design: An Artificial Rhodium Hydroformylase for Linear Aldehydes

Artificial metalloenzymes (ArMs) are hybrid catalysts that offer a unique opportunity to combine the superior performance of natural protein structures with the unnatural reactivity of transition‐metal catalytic centers. Therefore, they provide the prospect of highly selective and active catalytic chemical conversions for which natural enzymes are unavailable. Herein, we show how by rationally combining robust site‐specific phosphine bioconjugation methods and a lipid‐binding protein (SCP‐2L), an artificial rhodium hydroformylase was developed that displays remarkable activities and selectivities for the biphasic production of long‐chain linear aldehydes under benign aqueous conditions. Overall, this study demonstrates that judiciously chosen protein‐binding scaffolds can be adapted to obtain metalloenzymes that provide the reactivity of the introduced metal center combined with specifically intended product selectivity.