Residue-Selective C-H Sulfenylation Enabled by Acid-Activated S-Acetamidomethyl Cysteine Sulfoxide with Application to One-Pot Stapling and Lipidation Sequence.

A tyrosine (Tyr)- or tryptophan (Trp)-selective metal-free C-H sulfenylation reaction using an acid-activated S-acetamidomethyl cysteine (Cys) sulfoxide, Cys(Acm)(O), has been achieved. The dually protonated intermediate produced from Cys(Acm)(O) under acidic conditions allows the sulfenylation of Tyr. Significantly, the reaction in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) mainly affords a Cys-Tyr-linked peptide even in the presence of Trp residues. In contrast, a Cys-Trp-linked peptide was selectively obtained from the reaction in the presence of guanidine hydrochloride (Gn ⋅ HCl) under acidic conditions. Established Tyr- and Trp-selective sulfenylation methods were used in the Cys-Tyr stapling and Trp lipidation of glucagon-like peptides 1 in a one-pot/stepwise manner. Investigation of the mechanism showed that orbital- and charge-controlled reactions are responsible for the Trp and Tyr selectivity, respectively.

Theoretical study to gain fundamental insight into reaction mechanism of N-S acyl transfer of N-sulfanylethylanilide-based protein labeling reagent on protein surface.

Elucidation of protein function is one of the central issues in the field of life sciences. To study the function of proteins not in isolation, but in a cell or its lysate, thus, it is necessary to selectively label the target protein in a mixture. Affinity labeling is one of several widely used methods for selective labeling; however, this method has the disadvantage that the labeling reagent is always activated, albeit weakly. Therefore, fine-tuning of the reactivity and/or reaction conditions is generally required for successful target-selective labeling. We previously developed a new affinity labeling reagent with N-sulfanylethylanilide (SEAlide) as a key reactive unit. It was designed based on the following hypotheses. SEAlide is less reactive and does not label in the absence of a target protein. Upon target binding, amino acid side-chain functional groups on the target surface convert SEAlide into a thioester form via N-S acyl transfer, allowing the target to be labeled. However, no evidence has been obtained so far to directly prove the hypothesis. In this study, we examine whether amino acid side-chain functional groups can activate SEAlide from the viewpoint of theoretical chemistry. The theoretical studies show that the activation free energy and enthalpy of the acyl transfer of SEAlide are reduced in the presence of methylammonium, which is a model for the protonated side chain of Lys, and acetate, which is a model for the deprotonated side chain of Asp/Glu. It suggests that Lys and Asp/Glu side chains could potentially stabilize the activation transition states to accelerate the thioester formation. Furthermore, the significant decrease in the activation enthalpy indicates that the contribution of entropy to the transition state is large. This result supports the original hypothesis that the SEAlide-based labeling reagent is efficiently activated by binding to the target protein.

Identification of Functional Domains of CXCL14 Involved in High-Affinity Binding and Intracellular Transport of CpG DNA.

Some CXC chemokines, including CXCL14, transport CpG oligodeoxynucleotides (ODNs) into dendritic cells (DCs), thereby activating TLR9. The molecular basis of this noncanonical function of CXC chemokines is not well understood. In this study, we investigated the CpG ODN binding and intracellular transport activities of various CXC chemokines and partial peptides of CXCL14 in mouse bone marrow-derived dendritic cells. CXCL14, CXCL4, and CXCL12 specifically bound CpG ODN, but CXCL12 failed to transport it into cells at low dose. CXCL14 N-terminal peptides 1-47, but not 1-40, was capable of transporting CpG ODN into the cell, resulting in an increase in cytokine production. However, both the 1-47 and 1-40 peptides bound CpG ODN. By contrast, CXCL14 peptides 13-50 did not possess CpG ODN binding capacity or transport activity. The chimeric peptides CXCL12 (1-22)-CXCL14 (13-47) bound CpG ODN but failed to transport it. These results suggest that amino acids 1-12 and 41-47 of CXCL14 are required for binding and intracellular transport of CpG ODN, respectively. We found that an anti-CXCL14 Ab blocked cell-surface binding and internalization of the CpG ODN/CXCL14 complex. On the basis of these findings, we propose that CXCL14 has two functional domains, one involved in DNA recognition and the other in internalization of CXCL14-CpG DNA complex via an unidentified CXCL14 receptor, which together are responsible for eliciting the CXCL14/CpG ODN-mediated TLR9 activation. These domains could play roles in CXCL14-related diseases such as arthritis, obesity-induced diabetes, and various types of carcinoma.

Copper-Mediated Deprotection of Thiazolidine and Selenazolidine Derivatives Applied to Native Chemical Ligation.

Cupric sulfate efficiently opens thiazolidine and selenazolidine rings, producing a protected N-terminal cysteine or selenocysteine derivative without the use of inert gas or solvent. This is a clear advantage over methods that use water-soluble palladium salts, which fail to react with the selenazolidine ring. This copper-mediated reaction proceeds with monovalent or divalent copper ions, and disulfide bond formation followed by ring-opening promotes the process. This copper-mediated reaction, which is compatible with the standard native chemical ligation conditions, was applied to the synthesis of the 77-mer CXCL14 protein.

Theoretical study on reaction mechanism of phosphate-catalysed N-S acyl transfer of N-sulfanylethylanilide (SEAlide).

C-Terminally thioesterificated peptides are essential building blocks for chemical protein synthesis. To date, many acyl transfer auxiliaries have been developed to enable facile preparation of peptide thioesters. We previously developed an N-sulfanylethylanilide (SEAlide) auxiliary, which causes an N-S acyl transfer reaction upon addition of phosphate salt to convert a C-terminal amide to a thioester. The mechanism of how phosphate triggers the reaction is speculative, and the details are unknown. In this study, the mechanism by which phosphate promotes acyl transfer is discussed based on density functional theory (DFT) calculations and non-covalent interaction (NCI) analysis. As a result, although the notion that phosphate acts as an acid-base catalyst, as speculated in our previous study, was correct, it became clear that two competing reaction pathways exist: a previously proposed stepwise pathway and a concerted one. Furthermore, calculation was performed in the presence of various additives other than phosphate to uncover the effect of the additives on the stability of transition states.

Deprotection of S-acetamidomethyl cysteine with copper(ii) and 1,2-aminothiols under aerobic conditions.

Ring-opening by CuSO4 of a 1,3-thiazolidine carbonyl structure (Thz) as an N-terminal cysteine (Cys) residue revealed that an intramolecular S-acetamidomethyl cysteine (Cys(Acm)) can also be deprotected with concomitant formation of a disulphide bond connecting the two Cys residues. A mechanistic study on the disulphide formation led to a general protocol for deprotection of the S-Acm group by CuSO4 and a 1,2-aminothiol under aerobic conditions. Application of this new deprotection reaction allowed for the synthesis of Apamin, a peptide with two-disulphides in a one-pot/stepwise disulphide-bridging procedure.

Development of a Turn-On Fluorescent Traceable Linker Employing N-Sulfanylethylcoumarinyl Amide for the Enrichment and Visualization of Target Proteins.

A turn-on fluorescent traceable linker based on N-sulfanylethylcoumarinyl amide (SECmide) has been developed as an advanced cleavable linker. It was successfully employed for the enrichment and selective visualization of a target protein in cell lysate. The results demonstrated that the SECmide-based traceable linker is potentially applicable to the identification of low molecular weight target proteins, a factor which has been problematic for a previously developed N-sulfanylethylanilide-based traceable linker.

Sequence-Independent Traceless Method for Preparation of Peptide/Protein Thioesters Using CPaseY-Mediated Hydrazinolysis.

Proteins incorporating artificial moieties such as fluorophores and drugs have enjoyed increasing use in chemical biology and drug development research. Preparation of such artificial protein derivatives has relied mainly on native chemical ligation in which peptide/protein thioesters chemoselectively react with N-terminal cysteine (Cys) peptides to afford protein molecules. The protein thioesters derived from expressed proteins represent thioesters that are very useful for the preparation of artificial proteins by native chemical ligation with synthetic peptides with N-terminal Cys. We recently have developed a traceless thioester-producing protocol using carboxypeptidase Y (CPaseY) which is compatible with an expressed protein. The traceless character is ensured by CPaseY-mediated hydrazinolysis of C-terminal Xaa (X)-Cys-proline (Pro)-leucine (Leu)-OH sequence followed by an auto-processing of the Cys-Pro (CP) dipeptide unit, affording the corresponding X-thioester (X-SR). However, hydrazinolysis of the amide bond in the prolyl leucine junction depends significantly on the nature of X. In the case of hydrophobic X residues, the hydrazinolysis overreacts to give several hydrazides while the reaction of hydrophilic X residues proceeds slowly. In this research, we attempted to develop an X-independent CPaseY-mediated protocol and found that the incorporation of a triple CP sequence into the C-terminal end (X-(CP)3-Leu-OH) allows for efficient X-SR formation in a manner that is independent of X.

Asymmetric Synthesis of α-Amino Phosphonic Acids using Stable Imino Phosphonate as a Universal Precursor.

A practical method for synthesizing chiral α-amino phosphonic acid derivatives was developed. Readily available and stable N-o-nitrophenylsulfenyl (Nps) imino phosphonate was utilized as a substrate for a highly enantioselective Friedel-Crafts-type addition of indole or pyrrole nucleophiles catalyzed by chiral phosphoric acid. The resulting adduct was easily converted into N-9-fluorenylmethyloxycarbonyl (Fmoc) amino phosphonic acid, which is useful for synthesizing peptides containing an amino phosphonic acid.

A Photo-Activatable Peptide Mimicking Functions of Apolipoprotein A-I.

Apolipoprotein A-I (apoA-I) plays a critical role in high-density lipoprotein (HDL) biogenesis, function and structural dynamics. Peptides that mimic apoA-I have a short amphipathic α-helical structure that can functionally recapitulate many of the same biologic properties of full-length apoA-I in HDL. Hence, they might be expected to have clinical applications in the reduction of atherosclerosis. However, nonspecific cellular efflux of cholesterol induced by apoA-I mimetic peptides might cause side effects that are, as yet, unidentified. In this study, we developed a photo-activatable peptide, 2F*, which is an 18 amino acid peptide mimicking apoA-I bearing an internal photocleavable caging group that is designed to assume an α-helical structure in response to a light stimulus and trigger efflux of cholesterol from cells. Without light irradiation, 2F* peptide showed a low tendency for the formation of α-helices, and therefore did not associate with lipids and failed to induce efflux of cholesterol. In addition, 2F* did not cause hemolysis under our experimental condition. Mass spectrometry indicated that, after light exposure, the caging group detached from 2F* and it assumed the α-helical structure in the presence of lipids, and enhanced cholesterol efflux from cells. Photo-activatable peptides such as 2F* that control cholesterol efflux following light stimulus may be useful for future atherosclerosis-reducing therapies.

Development of Chemical Biology Tools Focusing on Peptide/Amide Bond Cleavage Reaction.

Peptides and proteins are involved in almost all biological events. In this review, three chemical biology tools, which were developed for peptide/protein sciences from a viewpoint of peptide/amide bond cleavage, are overviewed. First, study on an artificial amino acid that enables stimulus-responsive functional control of peptides/proteins is briefly described. Two N-S acyl transfer reaction-based tools, one a linker molecule for facile identification of target proteins of bioactive compounds and the other a reagent for selective labeling of proteins of interest, are then discussed.

Enthalpy-driven interactions with sulfated glycosaminoglycans promote cell membrane penetration of arginine peptides.

The first step of cell membrane penetration of arginine peptides is thought to occur via electrostatic interactions between positive charges of arginine residues and negative charges of sulfated glycosaminoglycans (GAGs) on the cell surface. However, the molecular interaction of arginine peptides with GAG still remains unclear. Here, we compared the interactions of several arginine peptides of Tat, R8, and Rev and their analogues with heparin in relation to the cell membrane penetration efficiency. The high-affinity binding of arginine peptides to heparin was shown to be driven by large favorable enthalpy contributions, possibly reflecting multidentate hydrogen bondings of arginine residues with sulfate groups of heparin. Interestingly, the lysine peptides in which all arginine residues are substituted with lysine residues exhibited negligible binding enthalpy despite of their considerable binding to heparin. In CHO-K1 cells, arginine peptides exhibited a great cell-penetrating ability whereas their corresponding lysine peptides did not penetrate into cells. The degree of cell penetration of arginine peptides markedly decreased by the chlorate treatment of cells which prevents the sulfation of GAG chains. Significantly, the cell penetration efficiency of arginine peptides was found to be correlated with the favorable enthalpy of binding to heparin. These results suggest that the enthalpy-driven strong interaction with sulfated GAGs such as heparan sulfate plays a critical role in the efficient cell membrane penetration of arginine peptides.

Elucidation of inhibitor-binding pockets of d-amino acid oxidase using docking simulation and N-sulfanylethylanilide-based labeling technology.

Because of the relevance of d-serine (d-Ser) to schizophrenia, inhibitors of d-amino acid oxidase (DAO), which catalyzes degradation of d-Ser in the presence of flavin adenine dinucleotide (FAD), are expected to be anti-schizophrenia therapeutics. In this study, binding pockets of DAO to its inhibitor 4-bromo-3-nitrobenzoic acid were searched by combining in silico docking simulation and labeling experiments employing an N-sulfanylethylanilide-based labeling technology that we have developed. The results clearly demonstrated that there are two binding pockets: one is shared with d-Ser and FAD, and the other is an unexpected cleft between the subunits of a DAO dimer. These findings will provide insight to aid the development of new DAO inhibitors. In addition, it was also proved that our labeling technology could be applicable to elucidate the binding pockets of proteins.

Invention of stimulus-responsive peptide-bond-cleaving residue (Spr) and its application to chemical biology tools.

Elucidation of biological functions of peptides and proteins is essential for understanding peptide/protein-related biological events and developing drugs. Caged peptides and proteins that release a parent active peptide/protein by photo-irradiation have successfully been employed to elucidate the functions. Whereas the usual caged peptide/protein enables conversion of an inactive form to an active form (OFF-to-ON conversion) by photo-induced deprotection, photo-triggered main chain cleavage is reported to be applicable to ON-to-OFF conversion. These peptides and proteins are photo-responsive; however, if peptides and proteins could respond to other stimuli such as disease-related environment or enzymes, their range of application should be widened. To convert the photo-responsive peptide/protein into other stimulus-responsive peptide/protein, quite laborious de novo design and synthesis of the stimulus-responsive unit are required. In this context, we designed a stimulus-responsive peptide-bond-cleaving residue (Spr) in which the stimuli available for the main chain cleavage vary according to the choice of protecting groups on the residue. In this review, design and synthesis of Spr are introduced, and challenges to apply Spr to other fields to enable, for example, functional control, localization control, delivery of cargos, labeling of a protein of interest in living cells, and identification of target proteins of bioactive ligands are discussed. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Chemical Synthetic Platform for Chlorpromazine Oligomers That Were Reported as Photo-degradation Products of Chlorpromazine.

A synthetic platform for chlorpromazine (CPZ) oligomers, which could be generated via photo-reaction of CPZ, is essential to promote their biological and structural studies. In this paper, the first synthetic platform for CPZ oligomers is described. A photo-irradiation experiment of CPZ to confirm whether the structure of the CPZ dimer generated by the photo-irradiation was identical to that prepared by our synthetic method is also reported.

A Convenient Method for Preparation of α-Imino Carboxylic Acid Derivatives and Application to the Asymmetric Synthesis of Unnatural α-Amino Acid Derivative.

We describe herein a manganese(IV) oxide-mediated oxidation of N-p-methoxyphenyl (PMP)-protected glycine derivatives for the synthesis of α-imino carboxylic acid derivatives. Using this methodology, utilization of unstable glyoxic acid derivatives was avoided. Furthermore, using this methodology we synthesized novel α-imino carboxylic acid derivatives such as α-imino phenyl ester, perfluoroalkyl etsers, imides, and thioester. The asymmetric Mannich reaction of those novel imine derivatives with 1,3-dicarbonyl compound is also described, and the novel α-imino imide gave improved chemical yield and stereoselectivity compared with those obtained by the use of the conventional α-imino ester-type substrate.

Amyloidogenic Mutation Promotes Fibril Formation of the N-terminal Apolipoprotein A-I on Lipid Membranes.

The N-terminal amino acid 1-83 fragment of apolipoprotein A-I (apoA-I) has a strong propensity to form amyloid fibrils at physiological neutral pH. Because apoA-I has an ability to bind to lipid membranes, we examined the effects of the lipid environment on fibril-forming properties of the N-terminal fragment of apoA-I variants. Thioflavin T fluorescence assay as well as fluorescence and transmission microscopies revealed that upon lipid binding, fibril formation by apoA-I 1-83 is strongly inhibited, whereas the G26R mutant still retains the ability to form fibrils. Such distinct effects of lipid binding on fibril formation were also observed for the amyloidogenic prone region-containing peptides, apoA-I 8-33 and 8-33/G26R. This amyloidogenic region shifts from random coil to α-helical structure upon lipid binding. The G26R mutation appears to prevent this helix transition because lower helical propensity and more solvent-exposed conformation of the G26R variant upon lipid binding were observed in the apoA-I 1-83 fragment and 8-33 peptide. With a partially α-helical conformation induced by the presence of 2,2,2-trifluoroethanol, fibril formation by apoA-I 1-83 was strongly inhibited, whereas the G26R variant can form amyloid fibrils. These findings suggest a new possible pathway for amyloid fibril formation by the N-terminal fragment of apoA-I variants: the amyloidogenic mutations partially destabilize the α-helical structure formed upon association with lipid membranes, resulting in physiologically relevant conformations that allow fibril formation.

Tailored Synthesis of 162-Residue S-Monoglycosylated GM2-Activator Protein (GM2AP) Analogues that Allows Facile Access to a Protein Library.

A synthetic protocol for the preparation of 162-residue S-monoglycosylated GM2-activator protein (GM2AP) analogues bearing various amino acid substitutions for Thr69 has been developed. The facile incorporation of the replacements into the protein was achieved by means of a one-pot/N-to-C-directed sequential ligation strategy using readily accessible middle N-sulfanylethylanilide (SEAlide) peptides each consisting of seven amino acid residues. A kinetically controlled ligation protocol was successfully applied to the assembly of three peptide segments covering the GM2AP. The native chemical ligation (NCL) reactivities of the SEAlide peptides can be tuned by the presence or absence of phosphate salts. Furthermore, NCL of the alkyl thioester fragment [GM2AP (1-31)] with the N-terminal cysteinyl prolyl thioester [GM2AP (32-67)] proceeded smoothly to yield the 67-residue prolyl thioester, with the prolyl thioester moiety remaining intact. This newly developed strategy enabled the facile synthesis of GM2AP analogues. Thus, we refer to this synthetic protocol as "tailored synthesis" for the construction of a GM2AP library.

Preparation of peptide thioesters from naturally occurring sequences using reaction sequence consisting of regioselective S-cyanylation and hydrazinolysis.

The vital roles of peptide/protein thioesters in protein chemistry, including chemical or semi-synthesis of proteins, have encouraged studies on the development of methods for the preparation of such chemical units. Biochemical protocols using intein or sortase have proved to be useful in protein chemistry as methods suitable for naturally occurring sequences, including recombinant proteins. Although chemical protocols are potential options for thioester preparation, only a few are applicable to naturally occurring sequences, because standard chemical protocols require an artificial chemical device for producing thioesters. In this context, the chemical preparation of thioesters based on a reaction sequence consisting of regioselective S-cyanylation and hydrazinolysis was investigated. Regioselective S-cyanylation, which is required for cysteine-containing thioesters, was achieved with the aid of a zinc-complex formation of a CCHH-type zinc-finger sequence. Free cysteine residues that are not involved in complex formation were selectively protected with a 6-nitroveratryl group followed by S-cyanylation of the zinc-binding cysteine. Hydrazinolysis of the resulting S-cyanopeptide and subsequent photo-removal of the 6-nitroveratryl group yielded the desired peptide hydrazide, which was then converted to the corresponding thioester. The generated thioester was successfully used in N-to-C-directed one-pot/sequential native chemical ligation using an N-sulfanylethylanilide peptide to give a 64-residue peptide toxin. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 531-546, 2016.

Development of an Intein-Inspired Amide Cleavage Chemical Device.

A photoresponsive amide cleavage device was developed based on the asparagine imidation-mediated cleavage of peptide bonds during intein-mediated protein splicing. The chemical environment of the protein splicing process was mimicked by the incorporation of geminal dimethyl groups and a secondary amine unit in asparagine scaffold. Furthermore, the resulting photoresponsive device could induce the phototriggered cleavage of an amide bond by the protection of the secondary amine unit with an o-nitrobenzyloxycarbonyl group.