Total Synthesis and Functional Characterization of Selenoneine.

The N-α-trimethyl 2-selenohistidine selenoneine is the selenium isolog of the natural antioxidant ergothioneine. Sulfur-to-selenium substitutions are known to endow proteins and nucleic acids with special activities. In contrast, secondary metabolites that exploit selenium-specific chemistry are rare. Selenoneine therefore provides a unique opportunity to study how natural organoselenides interact with cellular processes. In this report we describe the chemical synthesis of selenoneine and other 2-selenoimidazoles. With synthetic selenoneine at hand we discovered a set of reactivities that distinguish selenoneine from ergothioneine, showing that the two compounds can fill distinct functional niches. Synthetic access to 2-selenoimidazoles should pave the way to explore the pharmaceutical potential and physiological function of this heretofore inaccessible class of compounds.

Development of Organelle Replacement Therapy Using a Stearyl-Polyhistidine Peptide against Lysosomal Storage Disease Cells.

We previously reported on a polyhistidine peptide, His16 peptide, as a new cell-penetrating peptide. This peptide is anticipated to be a new carrier for drug delivery systems (DDSs) for targeting intracellular lysosomes because it can transport macromolecules (e.g., liposomes) into these organelles. In the present study, we examined the application of His16 peptide as a DDS carrier against lysosomal storage disease (LSD) cells. LSDs are metabolic disorders caused by loss of specific lysosomal enzymes. For the treatment of LSD cells, we devised a system designated organelle replacement therapy (ORT). ORT is a strategy for transporting exogenous lysosomes containing all kinds of lysosomal enzymes from normal cells into endogenous lysosomes in LSD cells using His16 peptide. To develop the ORT system, we prepared His16 peptide-modified healthy lysosomes (His16-Lyso) by insertion of a stearyl-His16 peptide into a hydrophobic region in the lysosomal membrane. His16-Lyso showed cellular uptake and localization to endogenous lysosomes in LSD cells. His16-Lyso also restored the proliferation of LSD cells, which otherwise showed slower proliferation than normal cells. These results suggested that His16-Lyso replenished deficient lysosomal enzymes in LSD cells. The results further suggest that His16-Lyso are promising candidates as a treatment tool for LSD cells and to establish a foundation for ORT.

Total Synthesis and Structural Revision of the Antibiotic Tetrapeptide GE81112A.

The total synthesis of the naturally occurring antibiotic GE81112A, a densely functionalized tetrapeptide, is reported. Comparison of spectral data with those of the natural product and the lack of biological activity of the synthesized compound led us to revise the published configuration of the 3-hydroxypipecolic acid moiety. This hypothesis was fully validated by the synthesis of the corresponding epimer.

Biomimetic synthesis and evaluation of histidine-derivative templated chiral mesoporous silica for improved oral delivery of the poorly water-soluble drug, nimodipine.

In this study, spherical shaped chiral mesoporous silica nanoparticles (CMS) was biomimetic synthesized using histidine derivatives (C16-L-histidine) as template via the sol-gel reaction and employed as poorly water-soluble drug nimodipine (NMP) carrier. Characteristics of CMS and its application as drug carrier were intensively investigated and compared with MCM41. Then NMP was respectively loaded into CMS and MCM41 with the drug: carrier weight ratio of 2:1. Structural features of NMP before and after drug loading were systemically characterized. The results demonstrated that hydrogen bonds were formed between NMP and carriers during the drug loading process. After drug loading, crystalline state of NMP effectively converted into modification L and amorphous state, and the first form turned out to be easily removed by washing. On the other hand, drug dissolution rate was significantly improved after drug loading, and the best result came from NMP-C3 sample. It was able to release 17.83% of drug within 60 min, which was 6.8-fold higher than the release amount of pure NMP. Undoubtedly, NMP-C3 presented the highest relative bioavailability (386.22%), and the best therapeutic effect. Meanwhile, CMS improved the brain distribution of NMP in vivo.

Short protecting-group-free synthesis of 5-acetylsulfanyl-histidines in water: novel precursors of 5-sulfanyl-histidine and its analogues.

The discovery of a non-enzymatic oxidative introduction of sulfur to the 5-position of histidine is reported, by activation with bromine or NBS followed by reaction with thioacetic acid forming novel 5-acetylsulfanyl-histidine. Complementing the previously developed regioselective oxidative S-introduction to the 2-position of histidine by reaction with cysteine, this surprising finding provides straightforward access in multi-gram quantities to naturally occurring 5-sulfanyl-histidine and its N-methylated analogues, including a hitherto unknown regioisomer of l-ergothioneine.

Regioselective copper-catalyzed N(1)-(hetero)arylation of protected histidine.

We report regioselective N(1)-arylation of protected histidine using copper(i) iodide as a catalyst, trans-N,N'-dimethylcyclohexane-1,2-diamine as a ligand and readily available aryl iodides as coupling partners under microwave irradiation at 130 °C for 40 min. The reaction provides rapid access to electron-donating, electron-withdrawing and bulky group substituted N-arylated histidines in high yields, including previously inaccessible N-heteroaryl histidines. These N(1)-(hetero)aryl histidines are promising building blocks in peptide-based drug design and discovery.

Probing the self-assembly and stability of oligohistidine based rod-like micelles by aggregation induced luminescence.

The synthesis and self-assembly of a new C2-symmetric oligohistidine amphiphile equipped with an aggregation induced emission luminophore is reported. We observe the formation of highly stable and ordered rod-like micelles in phosphate buffered saline, with a critical aggregation concentration below 200 nM. Aggregation induced emission of the luminophore confirms the high stability of the anisotropic assemblies in serum.

A peptide-based fluorescent chemosensor for multianalyte detection.

A novel multifunctional peptide fluorescent chemosensor (DP-3) with a lysine backbone and double sides conjugated with histidine and dansyl groups has been designed and synthesized by solid phase synthesis. This chemosensor is a promising analytical tool for detecting Zn(2+), Cu(2+), and S(2-) based on different mechanisms in 100% aqueous solutions, and intracellular biosensing has been successfully actualized. The peptide beacon structure of DP-3 makes it more stable and capable of achieving multianalyte detection, especially for sulfide ions. Until now, there have been few examples of using a peptide fluorescent chemosensor to detect anions with a continuous method. As designed, DP-3 exhibits excellent cell permeation and low biotoxicity and displays high selectivity and sensitivity, with Zn(2+) and Cu(2+) detection limits of 82 nM and 78 nM, respectively. This study raises the new possibility of a highly selective peptide fluorescent chemosensor for multifunctional detection, including cation and anions, by different mechanisms in environmental and biological systems. We expect that this work will inspire the development of a multifunctional beacon peptide-based fluorescent chemosensor library using modifiable lateral and terminal groups for a variety of practical applications in physiological and pathological events.

Metallacrowns as products of the aqueous medium self-assembly of histidinehydroxamic acid-containing polypeptides.

Self-assembly is a widely studied, spontaneous, and reversible phenomenon leading to the formation of the ordered structures by non-covalent specific interactions among starting molecules. In this work, a new template for the self-assembly of polypeptides based on peptides containing the C-terminal histidinehydroxamic acid moiety and Cu(2+) ions is characterized. Two peptide (tripeptide and pentadecapeptide) hydroxamic acid systems were synthesized and their interactions with Cu(2+) ions were investigated, revealing a high stability of the supramolecular assemblies formed. The supramolecular metallacrown-based L4Cu5 complexes exist at physiological pH in the presence of Cu(2+) ions as is evidenced from the spectroscopic methods, ESI mass spectrometry, and physicochemical techniques.

Antibody purification from human plasma by metal-chelated affinity membranes.

Immobilized metal ion affinity chromatography (IMAC) has been used for purification of proteins. IMAC introduces a new approach for selectively interacting biomolecules on the basis of their affinities for metal ions. The separation is based on different binding abilities of the proteins to the chelated metal ions on support. Here, N-methacryloyl-(L)-histidine methyl ester (MAH) is used as the metal-chelating ligand. Poly(hydroxyethyl methacrylate) Poly(HEMA) based membranes were prepared by photo-polymerization technique. Then, Zn(2+), Ni(2+), Co(2+), and Cu(2+) ions were chelated directly on the poly(HEMA-MAH) membranes for purification of immunoglobulin G (IgG) from human plasma.

Design, synthesis and evaluation of XZH-5 analogues as STAT3 inhibitors.

Inhibition of the signaling pathways of signal transducer and activator of transcription 3 (STAT 3) has shown to be a promising strategy to combat cancer. In this paper we report the design, synthesis and evaluation of a novel class of small molecule inhibitors, that is, XZH-5 and its analogues, as promising leads for further development of STAT3 inhibitors. Preliminary SARs was established for XZH-5 and its derivatives; and the binding modes were predicted by molecular docking. Lead compounds with IC50 as low as 6.5µM in breast cancer cell lines and 7.6µM in pancreatic cancer cell lines were identified.

Synthesis of peptides containing 2-oxohistidine residues and their characterization by liquid chromatography-tandem mass spectrometry.

Protein oxidation by reactive oxygen species has been associated with aging and neurodegenerative disorders, and histidine is one of the major oxidation targets due to its metal-chelating property and susceptibility to metal-catalyzed oxidation. 2-Oxohistidine, the major product of histidine oxidation, has been recently identified as a stable marker of oxidative damage in biological systems, but its biophysical and biochemical properties are understudied, partly because of difficulties in its chemical synthesis. We developed an efficient method to generate a 2-oxohistidine side chain using metal-catalyzed oxidation, applicable to both monomers and peptides. By optimizing reagent ratios and pH buffering in Cu(2+) /ascorbate/O2 reaction system, we improved the yield more than tenfold compared to reported conditions, which allowed us to obtain homogeneously modified 2-oxohisidine peptides for further studies. Analysis of 2-oxohistidine-containing model peptides by liquid chromatography-tandem mass spectrometry demonstrated increased retention time in reverse-phase chromatography and general stability of 2-oxohistidine under electrospray ionization and collision-induced dissociation. Thus, large-scale analysis of 2-oxohistidine-modified proteome should be feasible using shotgun protein mass spectrometry, and we were able to observe such peptides in proteomics datasets. The feasibility of acquiring purified peptide probes and peptide antigens containing 2-oxohistidine will help advance the study of this non-enzymatic posttranslational modification.

Mono- and di-halogenated histamine, histidine and carnosine derivatives are potent carbonic anhydrase I, II, VII, XII and XIV activators.

Mono- and di-halogenated histamines, l-histidine methyl ester derivatives and carnosine derivatives incorporating chlorine, bromine and iodine were prepared and investigated as activators of five carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic hCA I, II and VII, and the transmembrane hCA XII and XIV. All of them were activated in a diverse manner by the investigated compounds, with a distinct activation profile.

Molecularly imprinted cryogels for carbonic anhydrase purification from bovine erythrocyte.

Molecularly imprinted PHEMAH cryogels were synthesized and used for purification of carbonic anhydrase from bovine erythrocyte. Cryogels were prepared with free radical cryopolymerization of 2-hydroxyethyl methacrylate and methacryloylamido histidine and characterized by swelling degree, macroporosity, FTIR, SEM, surface area and elemental analysis. Maximum carbonic anhydrase adsorption of molecularly imprinted PHEMAH cryogel was found to be 3.16 mg/g. Selectivity of the molecularly imprinted cryogel was investigated using albumin, hemoglobin, IgG, γ-globulin, and lysozyme as competitor proteins and selectivity ratios were found to be 15.26, 60.05, 21.88, 17.61, and 17.42, respectively. Carbonic anhydrase purity was demonstrated by SDS-PAGE and zymogram results.

Preparation of 2'-13C-L-histidine starting from 13C-thiocyanate: synthetic access to any site-directed stable isotope enriched L-histidine.

1-Benzyl-2-(methylthio)-imidazole-5-ketone is obtained in a few simple steps starting from thiocyanate and glycine amide (glycin). Subsequent treatment with diethyl phosphorocyanidate and functional group manipulations gives 1-benzyl-5-chloromethyl-imidazolium chloride. This compound is converted under mild O'Donnell conditions into the corresponding L-histidine derivative. After deprotection L-histidine is obtained in good yield and 99% enantiomeric excess. 2'-13C-L-Histidine has been obtained via this new scheme with high (99%) 13C incorporation starting with commercially available 13C- thiocyanate. This synthetic scheme allows access to any isotopomer of L-histidine and many other biologically important imidazole derivatives.

In vitro Characterization of a small molecule inhibitor of the alanine serine cysteine transporter -1 (SLC7A10).

NMDA receptor hypofunction is hypothesized to contribute to cognitive deficits associated with schizophrenia. Since direct activation of NMDA receptors is associated with serious adverse effects, modulation of the NMDA co-agonists, glycine or D-serine, represents a viable alternative therapeutic approach. Indeed, clinical trials with glycine and D-serine have shown positive results, although concerns over toxicity related to the high-doses required for efficacy remain. Synaptic concentrations of D-serine and glycine are regulated by the amino acid transporter alanine serine cysteine transporter-1 (asc-1). Inhibition of asc-1 would increase synaptic D-serine and possibly glycine, eliminating the need for high-dose systemic D-serine or glycine treatment. In this manuscript, we characterize Compound 1 (BMS-466442), the first known small molecule inhibitor of asc-1. Compound 1 selectively inhibited asc-1 mediated D-serine uptake with nanomolar potency in multiple cellular systems. Moreover, Compound 1 inhibited asc-1 but was not a competitive substrate for this transporter. Compound 1 is the first reported selective inhibitor of the asc-1 transporter and may provide a new path for the development of asc-1 inhibitors for the treatment of schizophrenia.

Self-assembled pH-responsive hyaluronic acid-g-poly((L)-histidine) copolymer micelles for targeted intracellular delivery of doxorubicin.

Hyaluronic acid (HA) was conjugated with hydrophobic poly(l-histidine) (PHis) to prepare a pH-responsive and tumor-targeted copolymer, hyaluronic acid-g-poly(l-histidine) (HA-PHis), for use as a carrier for anti-cancer drugs. The effect of the degree of substitution (DS) on the pH-responsive behaviour of HA-PHis copolymer micelles was confirmed by studies of particles of different sizes. In vitro drug release studies demonstrated that doxorubicin (DOX) was released from HA-PHis micelles in a pH-dependent manner. In vitro cytotoxicity assays showed that all the blank micelles were nontoxic. However, MTT assay against Michigan Cancer Foundation-7 (MCF-7) cells (overexpressed CD44 receptors) showed that DOX-loaded micelles with a low PHis DS were highly cytotoxic. Cellular uptake experiments revealed that these pH-responsive HA-PHis micelles taken up in great amounts by receptor-mediated endocytosis and DOX were efficiently delivered into cytosol. Moreover, micelles with the lowest DS exhibited the highest degree of cellular uptake, which indicated that the micelles were internalized into cells via CD44 receptor-mediated endocytosis and the carboxylic groups of HA are the active binding sites for CD44 receptors. Endocytosis inhibition experiments and confocal images demonstrated that HA-PHis micelles were internalized into cells mainly via clathrin-mediated endocytosis and delivered to lysosomes, triggering release of DOX into the cytoplasm. These results confirm that the biocompatible pH-responsive HA-PHis micelles are a promising nanosystem for the intracellular targeted delivery of DOX.

Thermo- and pH-responsive copolymers based on PLGA-PEG-PLGA and poly(L-histidine): synthesis and in vitro characterization of copolymer micelles.

A series of novel thermo- and pH-responsive block copolymers of PHis-PLGA-PEG-PLGA-PHis composed of poly(ethylene glycol) (PEG), poly(D,L-lactide-co-glycolide) (PLGA) and poly(L-histidine) (PHis) were synthesized and used for the construction of stimuli-responsive copolymer micelles. The starting polymers of PLGA-PEG-PLGA and PHis were synthesized by ring-opening polymerization of dl-lactide and glycolide with PEG as an initiator and L-histidine N-carboxylanhydride with isopropylamine as an initiator, respectively. The final copolymer was obtained by the coupling reaction of PHis with PLGA-PEG-PLGA. The copolymer micelles were constructed to have an inner core consisting of two hydrophobic blocks (PLGA and deprotonated PHis) and an outer hydrophilic PEG shell. The temperature- and pH-induced structure changes of the micelles were characterized by an alteration in particle size, a decrease in pyrene fluorescence intensity, and a variation of (1)H NMR spectra in D2O. It was speculated that the hydrophobic-hydrophilic transitions of PEG and PHis in response to temperature and pH variations accounted for the destabilization of micelles. In vitro release profiles, cell cytotoxicity and intracellular location studies further confirmed the temperature- and pH-responsive properties of the copolymer micelles. These results demonstrate the potential of the developed copolymers to be stimuli-responsive carriers for targeted delivery of anti-cancer drugs.

A ratiometric fluorescent detection of Zn(II) in aqueous solutions using pyrene-appended histidine.

A fluorescent chemosensor, Py-His, based on histidine was easily synthesized in solid phase synthesis. Py-His displayed a highly sensitive ratiometric response to Zn(II) with potent binding affinity (Ka = 1.17 × 10(13)M(-2)) in aqueous solutions. The detection limit of Py-His for Zn(II) was calculated as 80.8 nM. Moreover, Py-His distinguished Zn(II) and Hg(II) by different ratiometric response type; the chemosensor showed a more enhanced increase of excimer emission intensity to Zn(II) than Hg(II). Upon addition of Ag(I) and Cu(II), Py-His showed a turn-off response mainly due to the quenching effect of these metal ions. The binding stoichiometry (2:1 or 1:1) of Py-His to target metal ions played a critical role in the fluorescent response type (ratiometric and turn off response) to target metal ions. The role of imidazole group of Py-His for ratiometric detection of Zn(II) was proposed by pH titration experiments.

Synthesis and application of Nα-Fmoc-Nπ-4-methoxybenzyloxymethylhistidine in solid phase peptide synthesis.

The 4-methoxybenzyloxymethyl (MBom) group was introduced at the Nπ-position of the histidine (His) residue by using a regioselective procedure, and its utility was examined under standard conditions used for the conventional and the microwave (MW)-assisted solid phase peptide synthesis (SPPS) with 9-fluorenylmethyoxycarbonyl (Fmoc) chemistry. The Nπ-MBom group fulfilling the requirements for the Fmoc strategy was found to prevent side-chain-induced racemization during incorporation of the His residue even in the case of MW-assisted SPPS performed at a high temperature. In particular, the MBom group proved to be a suitable protecting group for the convergent synthesis because it remains attached to the imidazole ring during detachment of the protected His-containing peptide segments from acid-sensitive linkers by treatment with a weak acid such as 1% trifluoroacetic acid in dichloromethane. We also demonstrated the facile synthesis of Fmoc-His(π-MBom)-OH with the aid of purification procedure by crystallization to effectively remove the undesired τ-isomer without resorting to silica gel column chromatography. This means that the present synthetic procedure can be used for large-scale production without any obstacles.