Synthesis of multiple antigenic peptides (MAPs)—strategies and limitations

Dendrimeric platforms such as MAPs can be synthesized either entirely by solid‐phase methods (SPPS, direct approach) or by conjugation in solution of preformed, SPPS‐made building blocks (indirect approach). Although MAPs and MAP‐like constructs have been extensively and successfully used for various biological (mainly immunological) applications, experimental reports are most often lacking in chemical detail about their preparation and characterization. Here, we provide complete accounts of the synthesis and analytical documentation of MAPs and similar dendrimers by either all‐SPPS (direct) or chemoselective thioether ligation (indirect) methods. We have chosen as model epitopes a 24‐residue sequence of the ectodomain of protein M2 from influenza virus (M2e), which is found to be a rather challenging peptide epitope, and a far more manageable, shortened (12‐residue) version of the same peptide. The advantages and shortcomings of both direct and indirect methods are discussed. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Polymer-supported biopolymer synthesis: 2. Phenolic poly(acryloylmorpholine)-based preparation of protected arginyl acylpeptide segments and derived arginyl peptides

A new poly(acryloylmorpholine)-based phenolic support matrix, Koch-Light Peptide Resin A, has been utilized for the solid (gel) phase assembly of protected or ginyl acylpeptide segments. Two methods, selective hydrazimolysis and autocatalysed transesterification with 2-dimethylaminoethanol, have been used to detach the peptide segments from the resin. Selective hydrazinolysis illustrates the use of the phenolic support matrix in the preparation of arginyl acylpeptide hydrazides bearing hydrazine-labile nitroguanidino and benylaxycarbonyl side chain protecting groups. Autocatalysed hydrolysis in trifluoroethanol/sodium trifluoroethanoate buffer has been used to convert the protected arginyl acylpeptide 2-dimethylaminoethyl esters to the corresponding protected arginyl acylpeptide acids. Total deprotection of the latter was effected by catalytic transfer hydrogenation in formic acid.     

Synthesis of peptides containing 5‐hydroxytryptophan,oxindolylalanine, N‐formylkynurenine and kynurenine

ROS, continuously produced in cells, can reversibly or irreversibly oxidize proteins, lipids, and DNA. At the protein level, cysteine, methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins in vitro and in vivo: 5‐HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then protected the ${\bf \alpha}$ ‐amino group of both amino acids, and the commercially available 5‐HTP, with Fmoc‐succinimide. High yields of Fmoc‐Kyn were obtained by acid hydrolysis of Fmoc‐NFK. All four Fmoc derivatives were successfully incorporated, at high yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M‐type), and ${\bf \beta}$ ‐enolase. The correct structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing 5‐HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion‐pair reagent on a C₁₈‐phase. Such synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

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

The 4‐methoxybenzyloxymethyl (MBom) group was introduced at the N^π‐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 N^π‐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. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and application of N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde)‐protected amino acids for optimization of solid‐phase peptide synthesis using gel‐phase 19F NMR spectroscopy

N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde) protected amino acids have been prepared and applied in solid‐phase peptide synthesis monitored by gel‐phase ¹⁹F NMR spectroscopy. The Fde protective group could be cleaved with 2% hydrazine or 5% hydroxylamine solution in DMF as determined with gel‐phase ¹⁹F NMR spectroscopy. The dipeptide Ac‐L ‐Val‐L ‐Val‐NH₂ 12 was constructed using Fde‐L ‐Val‐OH and no noticeable racemization took place during the amino acid coupling with N,N′‐diisopropylcarbodiimide and 1‐hydroxy‐7‐azabenzotriazole or Fde deblocking. To extend the scope of Fde protection, the hydrophobic nonapeptide LLLLTVLTV from the signal sequence of mucin MUC1 was successfully prepared using Fde‐L ‐Leu‐OH at diagnostic positions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of toxicological biomarkers of di(2‐ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis

The effects of di(2‐ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 μM) for 24 or 48 h. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 μM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose‐ and time‐dependent fashion. Proteomic analysis using two different pI ranges (4–7 and 6–9) and large size 2‐DE revealed the presence of 2776 protein spots. A total of 35 (19 up‐ and 16 down‐regulated) proteins were identified as biomarkers of DEHP by ESI‐MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up‐ or down‐regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin‐1, and haptoglobin‐related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.     

Synthesis and spectroscopic properties of 4-ethoxymethylene-2-(1)-naphthyl-5(4H)-oxazolone-labeled fluorescent peptides

Strategies for the preparation of new fluorescent oligopeptide conjugates labeled with 4-ethoxymethylene-2-[1]-naphthyl-5(4H)-oxazolone (naOx-OEt) at the N-terminal on solid support or in solution have been devised. These procedures are simple and easy to carry out by reacting naOx-OEt or N(alpha)-naOx-amino acid with side chain protected peptide chains attached to resins. The integrity of the N-alkyl bond was maintained even after the trifluoracetic acid or HF based cleavages procedures. Our data show that the naOx fluorophore is compatible with both Fmoc/tBu and Boc/Bzl methods and also suggest that naOx-amino acid could be utilized as building blocks for solid phase peptide synthesis. Comparative analysis of fluorescence properties of naOx-conjugates indicated that the spectral properties of the fluorophore do not change after incorporating into peptides. The compact size, the definite chemical reaction for its introduction in combination with the appropriate spectral features (e.g., intense emission, pH independent fluorescent characteristics, and beneficial photobleaching dose constant and rates) and with chemical and spectral stability, naOx-based labeling could be attractive for novel cellular fluorescent techniques (e.g., in laser scanning confocal FRET) to study peptide-protein and protein-protein interactions even in biological matrices.     

Comparison of patient‐derived high and low phosphatidylserine‐exposing colorectal carcinoma cells in their interaction with anti‐cancer peptides

Current cancer treatment is frequently compromised by severe adverse effects on healthy cells and tissues as well as by the increasing burden of (multi‐)drug resistances. Some representatives of small, amphipathic peptides known as host defense peptides possess the potential to overcome these limitations and to evolve as future anti‐cancer therapeutics. Peptide NK‐2, derived from porcine NK‐lysin, was originally discovered due to its broad‐spectrum antimicrobial activities. Today, also potent anti‐cancer activity is proven and accompanied by low toxicity towards normal human cells. The molecular basis underlying this target selectivity remains rather elusive. Nevertheless, it is presumptive that preferential peptide interactions with surface factors non‐abundant on healthy human cells play a key role. Here, we investigated the cytotoxicity of peptide NK‐2 and structurally improved anti‐cancer variants thereof against two patient‐derived colorectal cancer cell lines, exposing high and low levels of phosphatidylserine on their cell surfaces, respectively. Concluding from a range of in vitro tests involving cellular as well as lipid vesicle‐based methods, it is proposed that the magnitude of the accessible membrane surface charge is not a primarily decisive factor for selective peptide interactions. Instead, it is suggested that the level of membrane surface‐exposed phosphatidylserine is of crucial importance for the activity of peptide NK‐2 and enhanced variants thereof in terms of their cancer cell selectivity, the overall efficacy, as well as the underlying mode of action and kinetics. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Isolation and characterization of new poly(3HB)‐accumulating star‐shaped cell‐aggregates‐forming thermophilic bacteria

Aims: This study aimed at isolating thermophilic bacteria that utilize cheap carbon substrates for the economically feasible production of poly(3‐hydroxybutyrate), poly(3HB), at elevated temperatures. Methods and Results: Thermophilic bacteria were enriched from an aerobic organic waste treatment plant in Germany, and from hot springs in Egypt. Using the viable colony staining method for hydrophobic cellular inclusions with Nile red in mineral salts medium (MSM) containing different carbon sources, six Gram‐negative bacteria were isolated. Under the cultivation conditions used in this study, strains MW9, MW11, MW12, MW13 and MW14 formed stable star‐shaped cell‐aggregates (SSCAs) during growth; only strain MW10 consisted of free‐living rod‐shaped cells. The phylogenetic relationships of the strains as derived from 16S rRNA gene sequence comparisons revealed them as members of the Alphaproteobacteria. The 16S rRNA gene sequences of the isolates were very similar (>99% similarity) and exhibited similarities ranging from 93 to 99% with the most closely related species that were Chelatococcus daeguensis, Chelatococcus sambhunathii , Chelatococcus asaccharovorans, Bosea minatitlanensis, Bosea thiooxidans and Methylobacterium lusitanum. Strains MW9, MW10, MW13 and MW14 grew optimally in MSM with glucose, whereas strains MW11 and MW12 preferred glycerol as sole carbon source for growth and poly(3HB) accumulation. The highest cell density and highest poly(3HB) content attained were 4·8 g l^?l (cell dry weight) and 73% (w/w), respectively. Cells of all strains grew at temperatures between 37 and 55°C with the optimum growth at 50°C. Conclusions: New PHA‐accumulating thermophilic bacterial strains were isolated and characterized to produce poly(3HB) from glucose or glycerol in MSM at 50°C. SSCAs formation was reported during growth. Significance and Impact of the Study: To the best of our knowledge, this is the first report on the formation of SSCAs by PHA‐accumulating bacteria and also by thermophilic bacteria. PHA‐producing thermophiles can significantly reduce the costs of fermentative PHA production.     

Synthesis and biological characterization of human monocyte chemoattractant protein 1 (MCP-1) and its analogs.

Novel analogs of human monocyte chemoattractant protein 1 (MCP-1) were designed, synthesized and characterized to be used as tools to generate monoclonal antibodies as potential human therapeutics. MCP-1 and three analogs were synthesized by step-wise Fmoc solid phase synthesis. After oxidation to form the two-disulfide bonds, affinity chromatography using an immobilized mouse anti-human MCP-1 monoclonal antibody (mAb) was utilized for a simple and highly effective purification procedure for the proteins. The final products were extensively characterized and compared with recombinant human MCP-1 (rhMCP-1). All proteins showed identical binding with mouse anti-human MCP-1 mAbs as measured by surface plasmon resonance. Synthetic MCP-1 and the analogs were comparable to recombinant MCP-1 in competition radio-ligand binding to CCR2 receptors on THP-1 cells, and MCP-1-induced, calcium mobilization and chemotaxis assays.     

Assessing the correlation of microscopy‐based and volumetry‐based measurements for resin swelling in a range of potential greener solvents for SPPS

The degree of resin swelling in a particular solvent system is one of the critical parameters for solid‐phase peptide synthesis (SPPS) and for solid‐phase synthesis in general. Methods used for measuring the degree of resin swelling include microscopy‐based and volumetry‐based methods. This study describes and compares the use of both methods for a number of commercially available resins commonly used in SPPS, with a range of solvents, which have been identified in the literature as ‘greener’ than DCM, DMF and NMP. The results were analysed by statistical methods, and a significant correlation between the two distinct methods has been demonstrated for the first time. The results will likely be used, in conjunction with other literature methods, to help in choosing both the resin and solvent system for greener SPPS, as well as for continuous flow SPPS, which is of growing importance.     

Novel analogues of arginine vasopressin containing α‐2‐indanylglycine enantiomers in position 2

Continuing our efforts to obtain potent and selective analogues of AVP we synthesized and pharmacologically evaluated ten new compounds modified at position 2 with α‐2‐indanylglycine or its D ‐enantiomer (Igl or D ‐Igl, respectively). All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of these compounds to human OT receptor. The Igl² substitution resulted in a significant change of the pharmacological profile of the peptides. The new analogues were moderate or potent OT antagonists (pA₂ values ranging from 7.19 to 7.98) and practically did not interact with V_1a and V₂ receptors. It is worth emphasizing that these new peptides were exceptionally selective. On the other hand, the D ‐Igl² substituted counterparts turned out to be weak antagonists of the pressor response to AVP and displayed no antidiuretic activity. Some of the results were unexpected, e.g. dual activity in the rat uterotonic test in vitro: the D ‐Igl peptides showed a strong antioxytocic potency (pA₂ values ranging from 7.70 to 8.20) at low concentrations and full agonism at high concentrations. The results provided useful information about the SAR of AVP analogues. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Insights into the helix‐sense inversion of poly(β‐phenethyl‐L‐aspartate) by two‐dimensional Raman correlation spectroscopy

In this study, the transition process of the helix‐sense inversion of poly(β‐phenethyl‐L‐aspartate) was investigated by Raman scattering and 2‐dimensional correlation spectroscopy. Temperature‐dependent Raman spectra were obtained during the helix‐sense inversion. The results of 2‐dimensional correlation analysis in the spectral regions of 1600‐1800 and 3200‐3400 cm^?1 showed that the intensity changes of the side‐chain ester C═O stretching bands occurred prior to those of amide A and amide I bands in the unwinding process of αR‐helix on heating. The sequential order of the intensity changes for amide A, amide I, and the side‐chain ester C═O stretching bands during the inversion process was determined. It was found that the conformation change of the side chain occurred prior to that of the main chain for the αR‐helix on heating. Thus, we concluded that the transformation of the backbone chain from right‐handed to left‐handed is triggered by the conformational change of the side chains.     

Design,Synthesis, and Cytotoxic Activity of 3‐Aryl‐N‐hydroxy‐2‐(sulfonamido)propanamides in HepG2, HT‐1080, KB,and MCF‐7 Cells

A new series of (sulfonamido)propanamides ( 6a1 – 6a13 , 6b1 – 6b15 , 7c1 – 7c5 , 6d1 – 6d5 , 6e1 – 6e6 ) was designed and synthesized. All the synthesized compounds were characterized by NMR and mass spectrometry. The target compounds were evaluated for their in vitro cytotoxic activity against hepatocellular carcinoma (HepG2), fibrosarcoma (HT‐1080), mouth epidermal carcinoma (KB), and breast adenocarcinoma (MCF‐7) cell lines with the sulforhodamine B (SRB) assay, with gemcitabine and mitomycin C as positive controls. Most of these compounds exhibit a more potent cytotoxic effect than the positive control group on various cancer cell lines and the most potent compound, 6a7 , shows the IC₅₀ values of 29.78±0.516 μm , 30.70±0.61 μm , and 64.89±3.09 μm in HepG2, HT‐1080, KB, and MCF‐7 cell lines, respectively. Thus, these compounds with potent cytotoxic activity have potential for development as new chemotherapy agents.     

Fluorescence enhancement of europium (III) perchlorate by 1,10‐phenanthroline on the 1‐(naphthalen‐2‐yl)‐2‐(phenylsulthio)ethanone complex and luminescence mechanism

A novel ligand, 1‐(naphthalen‐2‐yl)‐2‐(phenylsulthio)ethanone was synthesized using a new method and its two europium (Eu) (III) complexes were synthesized. The compounds were characterized by elemental analysis, coordination titration analysis, molar conductivity, infrared, thermo gravimetric analyzer‐differential scanning calorimetry (TGA‐DSC), ¹H NMR and UV spectra. The composition was suggested as EuL₅ · (ClO₄)₃ · 2H₂O and EuL₄ · phen(ClO₄)₃ · 2H₂O (L = C₁₀H₇COCH₂SOC₆H₅). The fluorescence spectra showed that the Eu(III) displayed strong characteristic metal‐centered fluorescence in the solid state. The ternary rare earth complex showed stronger fluorescence intensity than the binary rare earth complex in such material. The strongest characteristic fluorescence emission intensity of the ternary system was 1.49 times as strong as that of the binary system. The phosphorescence spectra were also discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Synthesis of dendrimer‐type chiral stationary phases based on the selector of (1S,2R)‐(+)‐2‐Amino‐1,2‐diphenylethanol derivate and their enantioseparation evaluation by HPLC

In our recent work, a series of dendritic chiral stationary phases (CSPs) were synthesized, in which the chiral selector was L‐2‐(p‐toluenesulfonamido)‐3‐phenylpropionyl chloride (selector I), and the CSP derived from three‐generation dendrimer showed the best separation ability. To further investigate the influence of the structures of dendrimer and chiral selector on enantioseparation ability, in this work, another series CSPs ( CSPs 1‐4 ) were prepared by immobilizing (1S,2R)‐1,2‐diphenyl‐2‐(3‐phenylureido)ethyl 4‐isocyanatophenylcarbamate (selector II) on one‐ to four‐generation dendrimers that were prepared in previous work. CSPs 1 and 4 demonstrated the equivalent enantioseparation ability. CSPs 2 and 3 showed the best and poorest enantioseparation ability respectively. Basically, these two series of CSPs exhibited the equivalent enantioseparation ability although the chiral selectors were different. Considering the enantioseparation ability of the CSP derived from aminated silica gel and selector II is much better than that of the one derived from aminated silica gel and selector I, it is believed that the dendrimer conformation essentially impacts enantioseparation. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Cu(OBt)2 and Cu(OAt)2, copper(II)-based racemization suppressors ready for use in fully automated solid-phase peptide synthesis.

The complexes Cu(OBt)2 and Cu(OAt)2, which are derived from copper(II) and HOBt and HOAt, respectively, are shown to be more effective in suppressing racemization during solid-phase peptide synthesis (SPPS) than are those compounds currently being used for this purpose. These compounds can readily be used in conjunction with the commonly applied coupling reagents in fully automated systems for solid-phase peptide chemistry.     

Synthesis,characterization and luminescent properties of europium complexes with 2,4,6‐tris‐(2‐pyridyl)‐s‐triazine as highly efficient sensitizers

Using 2,4,6‐tris‐(2‐pyridyl)‐s‐triazine (TPTZ) as a neutral ligand, and p‐hydroxybenzoic acid, terephthalic acid and nitrate as anion ligands, five novel europium complexes have been synthesized. These complexes were characterized using elemental analysis, rare earth coordination titrations, UV/vis absorption spectroscopy and infrared spectroscopy. Luminescence spectra, luminescence lifetime and quantum efficiency were investigated and the mechanism discussed in depth. The results show that the complexes have excellent emission intensities, long emission lifetimes and high quantum efficiencies. The superior luminescent properties of the complexes may be because the triplet energy level of the ligands matches well with the lowest excitation state energy level of Eu³⁺. Moreover, changing the ratio of the ligands and metal ions leads to different luminescent properties. Among the complexes, Eu₂(TPTZ)₂(C₈H₄O₄)(NO₃)₄(C₂H₅OH)·H₂O shows the strongest luminescence intensity, longest emission lifetime and highest quantum efficiency. Copyright © 2015 John Wiley & Sons, Ltd.