Long‐Range Chemical Ligation from N→N Acyl Migrations in Tryptophan Peptides via Cyclic Transition States of 10‐ to 18‐Members

Chemical ligations to form native peptides from N→N acyl migrations in Trp‐containing peptides via 10‐ to 18‐membered cyclic transition states are described. In this study, a statistical, predictive model that uses an extensive synthetic and computational approach to rationalize the chemical ligation is reported. N→N acyl migrations that form longer native peptides without the use of Cys/Ser/Tyr residues or an auxiliary group at the ligation site were achieved. The feasibility of these traceless chemical ligations is supported by the N?C bond distance in N‐acyl isopeptides. The intramolecular nature of the chemical ligations is justified by using competitive experiments and theoretical calculations.     

Histidine‐Containing Peptide Catalysts Developed by a Facile Library Screening Method

Although peptide catalysts have a high potential for the use as organocatalysts, the optimization of peptide sequences is laborious and time‐consuming. To address this issue, a facile screening method for finding efficient aminocatalysts from a peptide library has been developed. In the screening for the Michael addition of a malonate to an enal, a dye‐labeled product is immobilized on resin‐bound peptides through reductive amination to visualize active catalysts. This procedure allows for the monitoring of the reactivity of entire peptides without modifying the resin beads beforehand. Peptides containing histidine at an appropriate position were identified by this method. A novel function of the histidyl residue, which enhances the binding of a substrate to the catalyst by capturing an iminium intermediate, was indicated.     

Histidine‐Rich Oligopeptides To Lessen Copper‐Mediated Amyloid‐β Toxicity

Brain copper imbalance plays an important role in amyloid‐β aggregation, tau hyperphosphorylation, and neurotoxicity observed in Alzheimer's disease (AD). Therefore, the administration of biocompatible metal‐binding agents may offer a potential therapeutic solution to target mislocalized copper ions and restore metallostasis. Histidine‐containing peptides and proteins are excellent metal binders and are found in many natural systems. The design of short peptides showing optimal binding properties represents a promising approach to capture and redistribute mislocalized metal ions, mainly due to their biocompatibility, ease of synthesis, and the possibility of fine‐tuning their metal‐binding affinities in order to suppress unwanted competitive binding with copper‐containing proteins. In the present study, three peptides, namely HWH , HK^CH , and HAH , have been designed with the objective of reducing copper toxicity in AD. These tripeptides form highly stable albumin‐like complexes, showing higher affinity for Cu^II than that of Aβ(1‐40). Furthermore, HWH , HK^CH , and HAH act as very efficient inhibitors of copper‐mediated reactive oxygen species (ROS) generation and prevent the copper‐induced overproduction of toxic oligomers in the initial steps of amyloid aggregation in the presence of Cu^II ions. These tripeptides, and more generally small peptides including the sequence His‐Xaa‐His at the N‐terminus, may therefore be considered as promising motifs for the future development of new and efficient anti‐Alzheimer drugs.     

Electrochemical Difunctionalization of Alkenes by a Four‐Component Reaction Cascade Mumm Rearrangement: Rapid Access to Functionalized Imides

An electrochemical four‐component reaction cascade Mumm rearrangement was developed. It is a rare example of in situ generation of O‐acyl isoamides for 1,3‐(O→N) acyl transfer. Inexpensive, commercially available arylethylenes, aryl or heterocyclic acids, acetonitrile, and alcohols were used as substrates. A wide range of aryl acids and alcohols were tolerated and provided imides in satisfactory yields. Subsequent hydrolysis of imides could be utilized to synthesize valuable amides and β‐amino alcohol derivatives.     

Aziridine‐Mediated Ligation at Phenylalanine and Tryptophan Sites

An efficient approach towards peptide synthesis that allows easy access to variety of small peptides via one‐pot aziridine‐mediated ligation/desulfurization strategy has been described. The protocol afforded a library of phenylalanine‐ and tryptophan‐containing α‐peptides in good yields by regioselective ring‐opening of aziridine‐3‐aryl‐2‐carboxylates with peptide thioacids, followed by desulfurization.     

Peptide‐Templated Acyl Transfer: A Chemical Method for the Labeling of Membrane Proteins on Live Cells

The development of a method is described for the chemical labeling of proteins which occurs with high target specificity, proceeds within seconds to minutes, and offers a free choice of the reporter group. The method relies upon the use of peptide templates, which align a thioester and an N‐terminal cysteinyl residue such that an acyl transfer reaction is facilitated at nanomolar concentrations. The protein of interest is N‐terminally tagged with a 22 aa long Cys‐E3 peptide (acceptor), which is capable of forming a coiled‐coil with a reporter‐armed K3 peptide (donor). This triggers the transfer of the reporter to the acceptor on the target protein. Because ligation of the two interacting peptides is avoided, the mass increase at the protein of interest is minimal. The method is exemplified by the rapid fluorescent labeling and fluorescence microscopic imaging of the human Y₂ receptor on living cells.     

One‐Pot Four‐Segment Ligation Using Seleno‐ and Thioesters: Synthesis of Superoxide Dismutase

The synthesis of a peptide selenoester was efficiently carried out by the 9‐fluorenylmethoxycarbonyl (Fmoc) method using N‐alkylcysteine, at the C‐terminus of the peptide, as the N‐to‐S acyl shift device. The selenoester selectively reacted with the terminal amino group of the peptide aryl thioester in the presence of N ,N ‐diisopropylethylamine and dipyridyldisulfide, thus leaving the aryl thioester intact. Combined with silver‐ion‐promoted and silver‐ion‐free thioester activation methods, a one‐pot four‐segment ligation was realized. The method was successfully used to assemble the entire sequence of superoxide dismutase (SOD), which is composed of 153 amino‐acid residues, in one pot. After the folding reaction, the fully active SOD was obtained.     

Selenocysteine‐Mediated Native Chemical Ligation

C‐Terminal peptide thioesters are shown to react efficiently with peptide fragments containing an N‐terminal selenocysteine to give selenoproteins. In analogy to the native chemical ligation of thioesters and peptides containing N‐terminal cysteines, the selenol presumably attacks the thioester nucleophilically to give a selenoester intermediate that subsequently rearranges to give a native chemical bond. The utility of this procedure was demonstrated by the synthesis of a selenium‐containing derivative of bovine pancreatic trypsin inhibitor (BPTI) in which Cys³⁸ is replaced by selenocysteine. The artificial selenoprotein folds into a conformation similar to that of wild‐type BPTI and inhibits trypsin and chymotrypsin with unaltered affinity.     

Long-range intramolecular S → N acyl migration: a study of the formation of native peptide analogues via 13-, 15-, and 16-membered cyclic transition states

The intramolecular long-range S → N acyl migration via 13-, 15-, and 16-membered cyclic transition states to form native tetra- and pentapeptide analogues was studied on S-acylcysteine peptides containing β- or γ-amino acids. The pH-dependency study of the acyl migration via a 15-membered cyclic transition state indicated that the reaction is favored at a pH range from 7.0 to 7.6. Experimental observations are supported by structural and computational investigations.     

One‐Pot/Sequential Native Chemical Ligation Using N‐Sulfanylethylanilide Peptide

N‐Sulfanylethylanilide (SEAlide) peptides were developed with the aim of achieving facile synthesis of peptide thioesters by 9‐fluorenylmethyloxycarbonyl (Fmoc)‐based solid‐phase peptide synthesis (Fmoc SPPS). Initially, SEAlide peptides were found to be converted to the corresponding peptide thioesters under acidic conditions. However, the SEAlide moiety was proved to function as a thioester in the presence of phosphate salts and to participate in native chemical ligation (NCL) with N‐terminal cysteinyl peptides, and this has served as a powerful protein synthesis methodology. The reactivity of a SEAlide peptide (anilide vs. thioester) can be easily tuned with or without the use of phosphate salts. This interesting property of SEAlide peptides allows sequential three‐fragment or unprecedented four‐fragment ligation for efficient one‐pot peptide/protein synthesis. Furthermore, dual‐kinetically controlled ligation, which enables three peptide fragments simultaneously present in the reaction to be ligated in the correct order, was first achieved using a SEAlide peptide. Beyond our initial expectations, SEAlide peptides have served in protein chemistry fields as very useful crypto‐peptide thioesters. DOI 10.1002/tcr.201200007     

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.     

Visible‐Light‐Promoted Iminyl‐Radical Formation from Acyl Oximes: A Unified Approach to Pyridines,Quinolines, and Phenanthridines

A unified strategy involving visible‐light‐induced iminyl‐radical formation has been established for the construction of pyridines, quinolines, and phenanthridines from acyl oximes. With fac‐[Ir(ppy)₃] as a photoredox catalyst, the acyl oximes were converted by 1 e^? reduction into iminyl radical intermediates, which then underwent intramolecular homolytic aromatic substitution (HAS) to give the N‐containing arenes. These reactions proceeded with a broad range of substrates at room temperature in high yield. This strategy of visible‐light‐induced iminyl‐radical formation was successfully applied to a five‐step concise synthesis of benzo[c]phenanthridine alkaloids.     

Visible‐Light‐Promoted Iminyl‐Radical Formation from Acyl Oximes: A Unified Approach to Pyridines,Quinolines, and Phenanthridines

A unified strategy involving visible‐light‐induced iminyl‐radical formation has been established for the construction of pyridines, quinolines, and phenanthridines from acyl oximes. With fac‐[Ir(ppy)₃] as a photoredox catalyst, the acyl oximes were converted by 1 e⁻ reduction into iminyl radical intermediates, which then underwent intramolecular homolytic aromatic substitution (HAS) to give the N‐containing arenes. These reactions proceeded with a broad range of substrates at room temperature in high yield. This strategy of visible‐light‐induced iminyl‐radical formation was successfully applied to a five‐step concise synthesis of benzo[c]phenanthridine alkaloids.     

Copper(I)‐Catalyzed Intramolecular N‐N Coupling of Cyclopropyl O‐Acyl Ketoximes: Synthesis of Spiro‐fused Pyrazolin‐5‐ones

A convenient copper‐catalyzed approach has been developed for the synthesis of substituted spiro‐fused pyrazolin‐5‐ones from readily available cyclopropyl O‐acyl ketoximes via an intramolecular N? N bond formation reaction. These catalytic reactions proceed in excellent yields with a broad scope.     

Synthesis of O‐Acyl Isopeptides

O‐Acyl isopeptides, in which the N‐acyl linkage on the hydroxyamino acid residue (e.g., Ser and Thr) is replaced with an O‐acyl linkage, generally possess superior water‐solubility to their corresponding native peptides, as well as other distinct physicochemical properties. In addition, O‐acyl isopeptides can be rapidly converted into their corresponding native peptide under neutral aqueous conditions through an O‐to‐N acyl migration. By exploiting these characteristics, researchers have applied the O‐acyl isopeptide method to various peptide‐synthesis fields, such as the synthesis of aggregative peptides and convergent peptide synthesis. This O‐acyl‐isopeptide approach also serves as a means to control the biological function of the peptide in question. Herein, we report the synthesis of O‐acyl isopeptides and some of their applications.     

Supramolecular Self‐Assembly of Histidine‐Capped‐Dialkoxy‐Anthracene: A Visible‐Light‐Triggered Platform for Facile siRNA Delivery

Supramolecular self‐assembly of histidine‐capped‐dialkoxy‐anthracene (HDA) results in the formation of light‐responsive nanostructures. Single‐crystal X‐ray diffraction analysis of HDA shows two types of hydrogen bonding. The first hydrogen bond is established between the imidazole moieties while the second involves the oxygen atom of one amide group and the hydrogen atom of a second amide group. When protonated in acidic aqueous media, HDA successfully complexes siRNA yielding spherical nanostructures. This biocompatible platform controllably delivers siRNA with high efficacy upon visible‐light irradiation leading up to 90 % of gene silencing in live cells.     

Chemical Synthesis of Native S‐Palmitoylated Membrane Proteins through Removable‐Backbone‐Modification‐Assisted Ser/Thr Ligation

The preparation of native S‐palmitoylated (S‐palm) membrane proteins is one of the unsolved challenges in chemical protein synthesis. Herein, we report the first chemical synthesis of S‐palm membrane proteins by removable‐backbone‐modification‐assisted Ser/Thr ligation (RBM^GABA‐assisted STL). This method involves two critical steps: 1) synthesis of S‐palm peptides by a new γ‐aminobutyric acid based RBM (RBM^GABA) strategy, and 2) ligation of the S‐palm RBM‐modified peptides to give the desired S‐palm product by the STL method. The utility of the RBM^GABA‐assisted STL method was demonstrated by the synthesis of rabbit S‐palm sarcolipin (SLN) and S‐palm matrix‐2 (M2) ion channel. The synthesis of S‐palm membrane proteins highlights the importance of developing non‐NCL methods for chemical protein synthesis.     

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.     

Catalytic Enantioselective α‐Fluorination of 2‐Acyl Imidazoles via Iridium Complexes

The first highly enantioselective α‐fluorination of 2‐acyl imidazoles utilizing iridium catalysis has been accomplished. This transformation features mild conditions and a remarkably broad substrate scope, providing an efficient and highly enantioselective approach to obtain a wide range of fluorine‐containing 2‐acyl imidazoles which are found in a variety of bioactive compounds and prodrugs. A large scale synthesis has also been tested to demonstrate the potential utility of this fluorination method.     

Quantitative analysis of complex amino acids and RGD peptides by X‐ray photoelectron spectroscopy (XPS)

The C 1 s, N 1 s, and O 1 s core level binding energies (BEs) of the functional groups in amino acids (glycine, aspartic acid, glutamic acid, arginine, and histidine) with varied side‐chains and cell‐binding RGD‐based peptides have been determined and characterized by X‐ray photoelectron spectroscopy with a monochromatic Al K_α source. The zwitterionic nature of the amino acids in the solid state is unequivocally evident from the N 1 s signals of the protonated amine groups and the C 1 s signature of carboxylate groups. Significant adventitious carbon contamination is evident for all samples but can be quantitatively accounted for. No intrinsic differences in the XP spectra are evident between two polymorphs (α and γ) of glycine, indicating that the crystallographic differences have a minor influence on the core level BEs for this system. The two nitrogen centers in the imidazole group of histidine exhibit an N 1 s BE shift that is in line with previously reported data for theophylline and aqueous imidazole solutions, while the nitrogen and carbon chemical shifts reflect the unusual guanidinium chemical environment in arginine. It is shown that the complex envelopes of C 1 s and O 1 s photoemission spectra for short‐chain peptides can be analyzed quantitatively by reference to the less complex XP spectra of the constituent amino acids, provided the peptides are of high enough purity. The distinctive N 1 s photoemission from the amide linkages provides an indicator of peptide formation even in the presence of common impurities, and variations in the relative intensities of N 1 s were found to be diagnostic for each of the three peptides investigated (RGD, RGDS, and RGDSC). Copyright © 2013 John Wiley & Sons, Ltd.