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.

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.

Development of a novel tocopheryl ester for suppression of lipid accumulation without cytotoxicity by optimization of dicarboxylic ester moiety.

Tocopheryl succinate (Tsuc) is a succinic acid ester of the well-known antioxidant α-tocopherol (T). Tsuc exhibits various biological activities, including tumor growth suppression via activation of cell signaling and prevention of lipid accumulation in mouse adipocyte 3T3-L1 cells. The latter findings suggest that Tsuc may be a drug candidate for the treatment of obesity. However, Tsuc was found to induce apoptosis of normal cells (in addition to cancer cells), demonstrating the need to reduce the cytotoxicity of Tsuc without losing the suppression effect on lipid accumulation. Based on our previous findings, we focused on the ester structure of Tsuc for controlling cytotoxicity. Herein, we examined the cytotoxicity and lipid accumulation suppression effect of various T ester derivatives. We found that the terminal carboxylic group is necessary for suppression of lipid accumulation. We synthesized tocopheryl glutarate (Tglu) and tocopheryl adipate (Tadi) by elongation of carbon atoms 1 and 2 of the dicarboxylic moiety, respectively. Tglu and Tadi did not show any cytotoxicity, and both esters suppressed lipid accumulation, although their suppression activities were weaker than that of Tsuc. Tadi showed a more potent lipid accumulation inhibitory effect than Tglu. Although Tadi inhibited lipogenesis and promoted lipolysis, lipolysis was induced at lower concentrations than inhibition of lipogenesis, suggesting that Tadi mainly affects lipolysis. Taken together, we succeeded in the reduction of cytotoxicity, without loss of the suppression effect on lipid accumulation, by elongation of the dicarboxylic moiety of Tsuc. Tadi may be a promising candidate as an anti-obesity drug.

Identification of an endoplasmic reticulum proteostasis modulator that enhances insulin production in pancreatic ß cells.

Perturbation of endoplasmic reticulum (ER) proteostasis is associated with impairment of cellular function in diverse diseases, especially the function of pancreatic ß cells in type 2 diabetes. Restoration of ER proteostasis by small molecules shows therapeutic promise for type 2 diabetes. Here, using cell-based screening, we report identification of a chemical chaperone-like small molecule, KM04794, that alleviates ER stress. KM04794 prevented protein aggregation and cell death caused by ER stressors and a mutant insulin protein. We also found that this compound increased intracellular and secreted insulin levels in pancreatic ß cells. Chemical biology and biochemical approaches revealed that the compound accumulated in the ER and interacted directly with the ER molecular chaperone BiP. Our data show that this corrector of ER proteostasis can enhance insulin storage and pancreatic ß cell function.

Late-stage macrolactonisation enabled by tandem acyl transfers followed by desulphurisation.

Intramolecular S-acylation of a thiol-installed threonine with a thioester unit, followed by S-O acyl transfer and subsequent desulphurisation, allows the synthesis of lactone peptides. A protocol has been developed enabling the cyclisation of a linear peptide, a reaction which has not been achieved by conventional methods.

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.

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.

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.

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.

Sulfanylmethyldimethylaminopyridine as a Useful Thiol Additive for Ligation Chemistry in Peptide/Protein Synthesis.

Sulfanylmethyl-installed dimethylaminopyridine, 2-sulfanylmethyl-4-dimethylaminopyridine (2), has an acidic thiol group comparable to that in aryl thiols due to the formation of a zwitterion consisting of a thiolate anion and a pyridinium cation. It can be used as an additive for native chemical ligation. The alkyl thiol in 2 allows it to be used for the one-pot native chemical ligation-desulfurization protocol in peptide synthesis. The utility of 2 in the synthesis of cyclic peptides is demonstrated.

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.

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.

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.

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.

Traceless synthesis of protein thioesters using enzyme-mediated hydrazinolysis and subsequent self-editing of the cysteinyl prolyl sequence.

A traceless thioester-producing protocol featuring carboxypeptidase Y-mediated hydrazinolysis of cysteinyl prolyl leucine-tagged peptides has been developed. The hydrazinolysis followed by thioesterification affords cysteinyl prolyl thioesters. Self-editing of the tag and subsequent trans-thioesterification yields peptide thioesters. The developed protocol was successfully applied to the conversion of recombinant proteins to thioesters.

[Looking Back on Study Abroad at The Scripps Research Institute].

Just after receiving my Ph.D. degree in 2004 from Tokushima University, under the supervision of Professor Masayuki Shibuya, I had the opportunity to work as a Research Associate in the laboratory of Professor Kim D. Janda at The Scripps Research Institute in the U.S., for about a year. Since it has already been more than 10 years since my time at Scripps, the specific research performed at that time may no longer be of interest to readers, but the benefit of working in a different research environment is timeless. Therefore, this paper describes not only details of the research conducted, but also the significance of working in a foreign country as a postdoc, and the subsequent influence those experiences at The Scripps Research Institute have had on my career.

Resin-Bound Crypto-Thioester for Native Chemical Ligation.

The resin-bound N-sulfanylethylanilide (SEAlide) peptide was found to function as a crypto-thioester peptide. Exposure of the peptide resin to an aqueous solution under neutral conditions in the presence of thiols affords thioesters without accompanying racemization of C-terminal amino acids. Furthermore, the resin-bound SEAlide peptides react with N-terminal cysteinyl peptides in the absence of phosphate salts to afford ligated products, whereas soluble SEAlide peptides do not. This unexpected difference in reactivity of the SEAlide peptides allows for a one-pot/three-fragment ligation using resin-bound and unbound peptides.

Light-induced propulsion of a giant liposome driven by peptide nanofibre growth.

Light-driven nano/micromotors are attracting much attention, not only as molecular devices but also as components of bioinspired robots. In nature, several pathogens such as Listeria use actin polymerisation machinery for their propulsion. Despite the development of various motors, it remains challenging to mimic natural systems to create artificial motors propelled by fibre formation. Herein, we report the propulsion of giant liposomes driven by light-induced peptide nanofibre growth on their surface. Peptide-DNA conjugates connected by a photocleavage unit were asymmetrically introduced onto phase-separated giant liposomes. Ultraviolet (UV) light irradiation cleaved the conjugates and released peptide units, which self-assembled into nanofibres, driving the translational movement of the liposomes. The velocity of the liposomes reflected the rates of the photocleavage reaction and subsequent fibre formation of the peptide-DNA conjugates. These results showed that chemical design of the light-induced peptide nanofibre formation is a useful approach to fabricating bioinspired motors with controllable motility.

Effect of Phosphatidylserine and Cholesterol on Membrane-mediated Fibril Formation by the N-terminal Amyloidogenic Fragment of Apolipoprotein A-I.

Here, we examined the effects of phosphatidylserine (PS) and cholesterol on the fibril-forming properties of the N-terminal 1‒83 fragment of an amyloidogenic G26R variant of apoA-I bound to small unilamellar vesicles. A thioflavin T fluorescence assay together with microscopic observations showed that PS significantly retards the nucleation step in fibril formation by apoA-I 1‒83/G26R, whereas cholesterol slightly enhances fibril formation. Circular dichroism analyses demonstrated that PS facilitates a structural transition from random coil to α-helix in apoA-I 1‒83/G26R with great stabilization of the α-helical structure upon lipid binding. Isothermal titration calorimetry measurements revealed that PS induces a marked increase in capacity for binding of apoA-I 1‒83/G26R to the membrane surface, perhaps due to electrostatic interactions of positively charged amino acids in apoA-I with PS. Such effects of PS to enhance lipid interactions and inhibit fibril formation of apoA-I were also observed for the amyloidogenic region-containing apoA-I 8‒33/G26R peptide. Fluorescence measurements using environment-sensitive probes indicated that PS induces a more solvent-exposed, membrane-bound conformation in the amyloidogenic region of apoA-I without affecting membrane fluidity. Since cell membranes have highly heterogeneous lipid compositions, our findings may provide a molecular basis for the preferential deposition of apoA-I amyloid fibrils in tissues and organs.