Advanced Insulin Synthesis by One-pot/Stepwise Disulfide Bond Formation Enabled by S-Protected Cysteine Sulfoxide.

An advanced insulin synthesis is presented that utilizes one-pot/stepwise disulfide bond formation enabled by acid-activated S-protected cysteine sulfoxides in the presence of chloride anion. S-chlorocysteine generated from cysteine sulfoxides reacts with an S-protected cysteine to afford S-sulfenylsulfonium cation, which then furnishes the disulfide or reversely returns to the starting materials depending on the S-protection employed and the reaction conditions. Use of S-acetamidomethyl cysteine (Cys(Acm)) and its sulfoxide (Cys(Acm)(O)) selectively give the disulfide under weak acid conditions in the presence of MgCl2 even if S-p-methoxybenzyl cysteine (Cys(MBzl)) and its sulfoxide (Cys(MBzl)(O)) are also present. In contrast, the S-MBzl pair yields the disulfide under more acidic conditions in the presence of a chloride anion source. These reaction conditions allowed a one-pot insulin synthesis. Additionally, lipidated insulin was prepared by a one-pot disulfide-bonding/lipidation sequence.

Cysteinylprolyl ester-mediated drug release from a lipid-drug conjugate.

For small-molecule drugs, lipidation via a cleavable linkage can extend half-life in circulation through interaction with albumin. Here we modified the cysteinylprolyl ester (CPE) system used in peptide thioester synthesis, which normally requires basic conditions, for use as an self-immolative linker and release device for a lipid-gemcitabine conjugate. To improve release under physiological conditions for medical application, a methyl group at the α-position of cysteine on the CPE unit was incorporated in anticipation of the Thorpe-Ingold effect. As a result, Ac-Gly-(α-Me)Cys(SH)-Pro-gemcitabine 11 drastically promoted the release of gemcitabine in comparison with Ac-Gly-Cys(SH)-Pro-gemcitabine 10. Furthermore, in the presence of bovine serum albumin and/or 2-mercaptoethanesulfonic acid, the gentle and continuous release of gemcitabine from the lipid-gemcitabine conjugate 16 was achieved. In addition to gemcitabine, this method could allow high clearance drugs, including nucleic acid and prostacyclin derivatives, to maintain their biological activity long enough to become effective.

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.

Enhanced tumor specific drug release by hypoxia sensitive dual-prodrugs based on 2-nitroimidazole.

Hypoxia in cancer is important in the development of cancer-selective medicines. Here, a novel hypoxia-responsible dual-prodrug is described. We designed and synthesized 2-nitroimidazole derivatives which spontaneously release both a PYG inhibitor and gemcitabine under hypoxic conditions. One such derivative, a prodrug 9 was found to be stable against chemical and enzymatic hydrolysis, and upon chemical reduction of the nitro group on imidazole, successfully releases both drugs. In an in vitro proliferation assay using human pancreatic cells, compound 9 exhibited significant anti-proliferative effects in hypoxia but fewer effects in normoxia. Consequently, prodrug 9 should be useful for cancer treatment due to its improved cancer selectivity and potential to overcome drug resistance.

CA9 and PRELID2; hypoxia-responsive potential therapeutic targets for pancreatic ductal adenocarcinoma as per bioinformatics analyses.

A strong hypoxic environment has been observed in pancreatic ductal adenocarcinoma (PDAC) cells, which contributes to drug resistance, tumor progression, and metastasis. Therefore, we performed bioinformatics analyses to investigate potential targets for the treatment of PDAC. To identify potential genes as effective PDAC treatment targets, we selected all genes whose expression level was related to worse overall survival (OS) in The Cancer Genome Atlas (TCGA) database and selected only the genes that matched with the genes upregulated due to hypoxia in pancreatic cancer cells in the dataset obtained from the Gene Expression Omnibus (GEO) database. Although the extracted 107 hypoxia-responsive genes included the genes that were slightly enriched in angiogenic factors, TCGA data analysis revealed that the expression level of endothelial cell (EC) markers did not affect OS. Finally, we selected CA9 and PRELID2 as potential targets for PDAC treatment and elucidated that a CA9 inhibitor, U-104, suppressed pancreatic cancer cell growth more effectively than 5-fluorouracil (5-FU) and PRELID2 siRNA treatment suppressed the cell growth stronger than CA9 siRNA treatment. Thus, we elucidated that specific inhibition of PRELID2 as well as CA9, extracted via exhaustive bioinformatic analyses of clinical datasets, could be a more effective strategy for PDAC treatment.

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.

Peptide probes containing a non-hydrolyzable phosphotyrosine-mimetic residue for enrichment of protein tyrosine phosphatases.

We developed peptide probes containing a non-hydrolyzable phosphotyrosine mimetic, 4-[difluoro(phosphono)methyl]-L-phenylalanine (F2 Pmp) for the enrichment of protein tyrosine phosphatases (PTPs). We found that different F2 Pmp probes can enrich different PTPs, depending on the probe sequence. Furthermore, proteins containing a Src homology 2 (SH2) domain were enriched together. Importantly, probes containing phosphotyrosine instead of F2 Pmp failed to enrich PTPs due to dephosphorylation during the pulldown step. This enrichment approach using peptides containing F2 Pmp could be a generic tool for tyrosine phosphatome analysis without the use of antibodies.

Development of Naturally Inspired Peptide and Protein Chemistry.

Diverse naturally occurring events relevant to proteins, including processing and post-translational modification, provide significant clues enabling advances in peptide/protein chemistry. Our motivation to synthesize large proteins prompted us to seek scientific bases utilized in synthetic experiments on the intein-mediated protein processing system. This account describes peptide/protein thioester-producing protocols whose design is based on acyl transfer reactions observed in the intein system, and the development of the stimulus-responsive amide cleavage and its application to the modulation of peptide function. Finally, several findings derived from nature-inspired research efforts, including peptide mimetic synthesis and S-protected cysteine chemistry, are described.

Advances in Preparation of Peptide and Protein Thioesters Aiming to Use in Medicinal Sciences.

The growing interest in artificial proteins modified by synthetic functional units has fueled the demand for their facile preparation. Native chemical ligation (NCL) enables the chemoselective condensation of peptide thioesters with a cysteine-installed synthetic partner and has enjoyed great success in the production of artificial proteins with up to 100-150 residues. A practical method for converting expressed proteins to the corresponding thioesters should lead to significant progress in the NCL-mediated technology. This account describes our recent contributions to the conversion of naturally occurring peptides to the corresponding thioesters by chemical or chemoenzymatic protocols aiming at their future prevalent use in the preparation of sophisticated protein biologics.

Peptide Cyclization Mediated by Metal-Free S-Arylation: S-Protected Cysteine Sulfoxide as an Umpolung of the Cysteine Nucleophile.

Covalent linking of side chains provides a method to produce cyclic or stapled peptides that are important in developing peptide-based drugs. A variety of crosslinking formats contribute to fixing the active conformer and prolonging its biological activity under physiological conditions. One format uses the cysteine thiol to participate in crosslinking through nucleophilic thiolate anions or thiyl radicals to form thioether and disulfide bonds. Removal of the S-protection from an S-protected Cys derivative generates the thiol, which functions as a nucleophile. S-Oxidation of a protected Cys allows the formation of a sulfoxide that operates as an umpolung electrophile. Herein, the applicability of S-p-methoxybenzyl Cys sulfoxide (Cys(MBzl)(O)) to the formation of a thioether linkage between tryptophan and Cys has been investigated. The reaction of peptides containing Cys(MBzl)(O) and Trp with trifluoromethanesulfonic acid (TFMSA) or methanesulfonic acid (MSA) in TFA in the presence of guanidine hydrochloride (Gn ⋅ HCl) proceeded to give cyclic or stapled peptides possessing the Cys-Trp thioether linkage. In this reaction, strong acids such as TFMSA or MSA are necessary to activate the sulfoxide. Additionally, Gn ⋅ HCl plays a critical role in producing an electrophilic Cys derivative that combines with the indole by aromatic electrophilic substitution. The findings led us to conclude that the less-electrophilic Cys(MBzl)(O) serves as an acid-activated umpolung of a Cys nucleophile and is useful for S-arylation-mediated peptide cyclization.

Discovery of a Macropinocytosis-Inducing Peptide Potentiated by Medium-Mediated Intramolecular Disulfide Formation.

Macropinocytosis is a ubiquitous cellular uptake mechanism of peptide-based intracellular delivery. This entry pathway shows promise as a route for the intracellular uptake of biomacromolecules and nanoparticles. In this work, we obtained the 8-residue analogue P4A bearing higher macropinocytosis induction ability. P4A contains vital cysteine residues in its sequence, which immediately reacts with cystine in culture medium to convert into its oxidized forms, including the intramolecularly oxidized form (oxP4A) as the dominant and active species. The conjugate of oxP4A and the membrane lytic peptide LK15 delivered bioactive proteins into cells; notably, this peptide delivered functional proteins fused with a negatively charged protein tag at a significantly reduced amount (up to nanomolar range) without compromising the delivery efficiency and the cellular activities of delivered proteins.

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.

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.

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.

An aromatic amino acid within intracellular loop 2 of the prostaglandin EP2 receptor is a prerequisite for selective association and activation of Gαs.

We previously demonstrated that the aromatic moiety of Tyr143 within the intracellular loop 2 (ICL2) region of the prostaglandin EP2 receptor plays a crucial role in Gs coupling. Here we investigated whether the ICL2 of the EP2 receptor directly binds to Gαs and whether an aromatic moiety affects this interaction. In Chinese hamster ovary cells, mutations of Tyr143 reduced the ability of the EP2 receptor to interact with G proteins as demonstrated by GTPγS sensitivity, as well as the ability of agonist-induced cAMP formation, with the rank order of Phe>Tyr (wild-type)=Trp>Leu>Ala (=0). We found that the wild-type ICL2 peptide (i2Y) and its mutant with Phe at Tyr143 (i2F) inhibited receptor-G protein complex formation of wild-type EP2 in membranes, whereas the Ala-substituted mutant (i2A) did not. Specific interactions between these peptides and the Gαs protein were detected by surface plasmon resonance, but Gαs showed different association rates, with a rank order of i2F>i2Y≫i2A, with similar dissociation rates. Moreover, i2F and i2Y, but not i2A activated membrane adenylyl cyclase. These results indicate that the ICL2 region of the EP2 receptor is its potential interaction site with Gαs, and that the aromatic side chain moiety at position 143 is a determinant for the accessibility of the ICL2 to the Gαs protein.

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.