Carbon nanotubes/(pLys/dsDNA) n layer-by-layer multilayer films for electrochemical studies of DNA damage

Carboxylic group-functionalized carbon nanotubes (c-CNT) were modified on the surface of carbon paste electrode to obtain a conducting precursor film. Positively charged poly-l-lysine (pLys) and negatively charged double-stranded DNA (dsDNA) were alternately adsorbed on the c-CNT-modified electrode, forming (pLys/dsDNA) _n layer-by-layer (LBL) films. Cyclic voltammetry and electrochemical impedance spectroscopy of the electroactive probe [Fe(CN)₆]^3−/4− could give the valuable dynamic information of multilayer films growth. The oxidative DNA damage induced by cadmium ion (Cd²⁺) in the LBL multilayer films was studied by differential pulse voltammetry (DPV) with methylene violet (MV) as the intercalation redox probe. The electrochemical signals of MV on the multilayer films were effectively amplified via LBL technology. The specific intercalation of MV into dsDNA base pairs and the amplified electrochemical response of MV, combined with the unique feature of loading reversibility of MV in the DNA layer-by-layer films, made the difference in DPV response between the intact, and damaged dsDNA films become pronounced. This biosensor exhibited that the (pLys/dsDNA) _n films could be utilized for investigations of DNA damage.     

Improved electrochemical performances of polyaniline nanotubes-poly-L-lysine composite for label-free impedance detection of DNA hybridization

A sensitive label-free DNA hybridization biosensing platform was fabricated based on the synergistic effect of polyaniline nanotubes (PANI_nt) and poly-L-lysine (pLys). The composite of pLys and PANI_nt was coated onto the carbon paste electrode (CPE) to form a uniform and very stable nanocomposite membrane. The pLys in the composite film not only acts as a membrane to retain good electron transfer capability of PANI_nt even at physiological pH, but also possesses fine biocompatibility for bio-analytes. DNA probes with negatively charged phosphate groups were readily linked to the positively charged pLys surface due to the strong electrostatic affinity. The synergistic effect of PANI_nt and pLys could significantly enhance the sensitivity of DNA hybridization recognition. The phosphinothricin acetyltransferase (PAT) gene fragment from transgenic corn and the polymerase chain reaction amplification of the terminator of nopaline synthase gene from the real sample of a kind of transgenic soybean were detected by this DNA electrochemical biosensor via label-free impedance method. This stable composite gives convenient permselectivity properties as a transducer material for the design of modern electrochemical impedance biosensor using [Fe(CN)₆]^3?/4? as an indicator.     

Development of stable phosphohistidine analogues

Protein phosphorylation is one of the most common and extensively studied posttranslational modifications (PTMs). Compared to the O-phosphorylation of Ser, Thr, and Tyr residues, our understanding of histidine phosphorylation is relatively limited, particularly in higher eukaryotes, due to technical difficulties stemming from the intrinsic instability and isomerism of phosphohistidine (pHis). We report the design and synthesis of stable and nonisomerizable pHis analogues. These pHis analogues were successfully utilized in solid-phase peptide synthesis and semi-synthesis of histone H4. Significantly, the first antibody that specifically recognizes pHis was obtained using the synthetic peptide as the immunogen.     

Emergent Catalytic Behavior of Self‐Assembled Low Molecular Weight Peptide‐Based Aggregates and Hydrogels

We report a series of short peptides possessing the sequence (FE)_n or (EF)_n and bearing l ‐proline at their N‐terminus that self‐assemble into high aspect ratio aggregates and hydrogels. We show that these aggregates are able to catalyze the aldol reaction, whereas non‐aggregated analogues are catalytically inactive. We have undertaken an analysis of the results, considering the accessibility of catalytic sites, pK_a value shifts, and the presence of hydrophobic pockets. We conclude that the presence of hydrophobic regions is indeed relevant for substrate solubilization, but that the active site accessibility is the key factor for the observed differences in reaction rates. The results presented here provide an example of the emergence of a new chemical property caused by self‐assembly, and support the relevant role played by self‐assembled peptides in prebiotic scenarios. In this sense, the reported systems can be seen as primitive aldolase I mimics, and have been successfully tested for the synthesis of simple carbohydrate precursors.     

β‐Sheet to Helical‐Sheet Evolution Induced by Topochemical Polymerization: Cross‐α‐Amyloid‐like Packing in a Pseudoprotein with Gly‐Phe‐Gly Repeats

Protein‐mimics are of great interest for their structure, stability, and properties. We are interested in the synthesis of protein‐mimics containing triazole linkages as peptide‐bond surrogate by topochemical azide‐alkyne cycloaddition (TAAC) polymerization of azide‐ and alkyne‐modified peptides. The rationally designed dipeptide N₃‐CH₂CO‐Phe‐NHCH₂CCH ( 1 ) crystallized in a parallel β‐sheet arrangement and are head‐to‐tail aligned in a direction perpendicular to the β‐sheet‐direction. Upon heating, crystals of 1 underwent single‐crystal‐to‐single‐crystal polymerization forming a triazole‐linked pseudoprotein with Gly‐Phe‐Gly repeats. During TAAC polymerization, the pseudoprotein evolved as helical chains. These helical chains are laterally assembled by backbone hydrogen bonding in a direction perpendicular to the helical axis to form helical sheets. This interesting helical‐sheet orientation in the crystal resembles the cross‐α‐amyloids, where α‐helices are arranged laterally as sheets.     

N,N,N‐Trimethyl‐5‐[(2,3,5,6‐tetrafluorophenoxy)carbonyl]pyridin‐2‐aminium trifluoromethanesulfonate a precursor for the synthesis of 2,3,5,6‐tetrafluorophenyl 6‐[18F]‐fluoronicotinate

The synthesis, recrystallization, and X‐ray deterimination of N,N,N‐trimethyl‐5‐[(2,3,5,6‐tetrafluorophenoxy)carbonyl]pyridin‐2‐aminium trifluoromethanesulfonate (PyTFP‐precursor), C₁₅H₁₃F₄N₂O₂⁺·CF₃SO₃⁻, is described. This triflate salt precursor is required for the synthesis of 2,3,5,6‐tetrafluorophenyl 6‐[¹⁸F]‐fluoronicotinate ([¹⁸F]FPyTFP), a prosthetic group used to radiolabel peptides for positron emission tomography (PET), as peptides are increasingly being used as PET‐imaging probes in nuclear medicine. Radiolabeling of peptides is typically done using a `prosthetic group', a small synthon to which the radioisotope is attached in the first step, followed by attachment to the peptide in the second step. During the synthesis of the PyTFP‐precursor, displacement of a Cl atom with trimethylamine gas and anion replacement with a triflate counter‐ion is critical, as incomplete replacement would hinder radioisotopic incorporation of nucleophilic fluorine‐18 and result in diminished radiochemical yields. The structural determination of the PyTFP‐precursor by X‐ray crystallography helped confirm the anion exchange of chloride with triflate.     

Visualizing the interaction between poly-L-lysine and poly(acrylic acid) microgels using microscopy techniques: effect of electrostatics and peptide size

The interaction between lightly cross-linked poly(acrylic acid) (pAA) microgels (50-150 microm in diameter) and poly-L-lysine (pLys) was studied as a function of pH, ionic strength, peptide size, and concentration. The swelling response and distribution of polypeptides within microgel particles was monitored by micromanipulator-assisted light microscopy and confocal laser scanning microscopy, while binding isotherms of pLys in the microgels were determined spectrophotometrically. Conformational changes of pLys were investigated by circular dichroism. The molecular weight of pLys was found to influence the degree of peptide-induced microgel deswelling, largely due to limitation of peptides larger than the effective network mesh size to penetrate the entire gel. Large peptides were concentrated within a surface layer of the gel particles, and at low ionic strength this dense surface layer was shown to act as a largely steric barrier for further penetration of compounds into the gel core. Small peptides, however, distributed evenly throughout the microgel particles and were able to create large microgel volume reductions. The deswelling of microgels increased with decreasing pH, while the uptake of pLys was significantly reduced at low pH. The effect of ionic strength on the interactions of pLys and oppositely charged pAA microgels was moderate and only pronounced for deswelling of gels at high pH. A significant increase in the alpha-helix content of pLys interacting with the oppositely charged microgels was observed for high molecular weight peptides, and the extent of alpha-helix formation was as expected more pronounced at high pH, i.e., at high charge density of the microgels but reduced charge density of the peptides.     

Fluorinated peptidomimetics: synthesis, conformational and biological features

Peptides modified with fluoroalkyl functions in key backbone positions have been scarcely studied so far. Thus, little is known about their synthesis, their structural and physico-chemical properties, and their biological features. Our interest in this field of research led to the development of stereocontrolled synthetic protocols, both in solution and in solid phase, for many different fluoroalkyl peptidomimetics, some of which are overviewed in this paper: (a) ψ[CH(CF₃)NH]-peptide mimics holding a great potential as hybrids between natural peptides and hydrolytic transition state analogs; (b) trifluoromethyl (Tfm) malic peptidomimetics as micromolar inhibitors of some matrix metalloproteinases; (c) bis-Tfm analogs of Pepstatin A, that are nanomolar and selective inhibitors of the protozoal aspartyl protease Plasmepsin II.     

Transport of poly-L-lysine into oppositely charged poly(acrylic acid) microgels and its effect on gel deswelling

The interaction between poly-L-lysine (pLys) and oppositely charged poly(acrylic acid) (pAA) microgels (? approximately 80-120 microm) was studied by micromanipulator-assisted light microscopy and confocal laser scanning microscopy. The aim of this study was to investigate effects of peptide size, pH, and salt concentration on binding, transport, and distribution of pLys in pAA microgel particles and thereby also to outline the details of the gel deswelling in response to pLys binding and incorporation. Both peptide distribution and gel deswelling kinetics were found to be strongly influenced by the pLys molecular weight, originating partly from limited entry of large peptides into the gel particle core. Also pH was shown to influence both deswelling and pLys incorporation kinetics, with a decreased deswelling rate observed with increasing pH. These effects are determined by a complex interplay between the pH-dependence of both pLys and the gel network, also influencing volume transitions of the latter. Finally, salt concentration was shown to have a significant effect on both gel deswelling rate and pLys transport, with an increased electrolyte concentration resulting in decreased deswelling rate but also in an increased peptide transport rate within the microgel particles.     

Sensitively Electrochemical Sensing for Sequence‐Specific Detection of Phosphinothricin Acetyltransferase Gene: Layer‐by‐Layer Films of Poly‐L‐Lysine and Au‐Carbon Nanotube Hybrid

An electrochemical DNA sensing film was constructed based on the multilayers comprising of poly‐L ‐lysine (pLys) and Au‐carbon nanotube (Au‐CNT) hybrid. A precursor film of mercaptopropionic acid (MPA) was firstly self‐assembled on the Au electrode surface. pLys and Au‐CNT hybrid layer‐by‐layer assembly films were fabricated by alternately immersing the MPA‐modified electrode into the pLys solution and Au‐CNT hybrid solution. Cyclic voltammetry was used to monitor the consecutive growth of the multilayer films by utilizing [Fe(CN)₆]^3?/4? and [Co(phen)₃]^3+/2+ as the redox indicators. The outer layer of the multilayer film was the positively charged pLys, on which the DNA probe was easily linked due to the strong electrostatic affinity. The hybridization detection of DNA was accomplished by using methylene blue (MB) as the indicator, which possesses different affinities to dsDNA and ssDNA. Differential pulse voltammetry was employed to record the signal response of MB and determine the amount of the target DNA sequence. The established biosensor has high sensitivity, a relatively wide linear range from 1.0×10^?10 mol/L to 1.0×10^?6 mol/L and the ability to discriminate the fully complementary target DNA from single or double base‐mismatched DNA. The sequence‐specific DNA related to phosphinothricin acetyltransferase gene from the transgenically modified plants was successfully detected.     

Streamlined Protein-Protein Interface Loop Mimicry

Cyclic peptides comprising endocyclic organic fragments, “cyclo-organopeptides”, can be probes for perturbing protein-protein interactions (PPIs). Finding loop mimics is difficult because of high conformational variability amongst targets. Backbone Matching (BM), introduced here, helps solve this problem in the illustrative cases by facilitating efficient evaluation of virtual cyclo-organopeptide core-structure libraries. Thus, 86 rigid organic fragments were selected to build a library of 602 cyclo-organopeptides comprising Ala and organic parts: “cyclo-{-(Ala)_n-organo-}”. The central hypothesis is “hit” library members have accessible low energy conformers corresponding to backbone structures of target protein loops, while library members which cannot attain this conformation are probably unworthy of further evaluation. BM thereby prioritizes candidate loop mimics, so that less than 10 cyclo-organopeptides are needed to be prepared to find leads for two illustrative PPIs: iNOS ⋅ SPSB2, and uPA ⋅ uPAR.     

A DFT study on 1,4‐dihydro‐1,4‐azaborinine annulated linear polyacenes: Absorption spectra,singlet‐triplet energy gap,aromaticity, and HOMO–LUMO energy modulation

Linear polyacene (LPA) mimics containing multiple heterocycles have been computationally designed by annulating 1,4‐dihydro‐1,4‐azaborinine moieties to benzene (aB₁–aB₅), naphthalene (aN₁–aN₅), anthracene (aA₁–aA₅), and tetracene (aT₁–aT₅) cores. DFT studies conducted on them using M06L/6‐311++G(d,p) method reveal a perfect planar structure for all and suggest the utilization of nitrogen lone pairs for aromatic π‐electron delocalization. The computed values of aromaticity indices such as HOMA, NICS, and dehydrogenation energy (E _dh) of heterocycles support strong aromatic character for each six‐membered ring in the LPA mimics. On the basis of the minimum value of the molecular electrostatic potential (V _min) observed on each LPA unit in the LPA mimics, the extended delocalization of π‐electrons is verified. The energetic parameter E _dh showed strong linear correlation with HOMA, NICS and V _min parameters, which strongly supports the multidimensional character of aromaticity in LPA mimics. The electronic property modification is shown by the theoretical absorption spectra data and singlet‐triplet energy gap (ΔE _ST). The bandgap and ΔE _ST tunings are achieved for LPA mimics by selecting appropriate number of azaborinine type units and the size of LPA core used for annulation. © 2017 Wiley Periodicals, Inc.     

Monofluoroalkene-Isostere as a 19F NMR Label for the Peptide Backbone: Synthesis and Evaluation in Membrane-Bound PGLa and (KIGAKI)3

Solid-state ¹⁹F NMR is a powerful method to study the interactions of biologically active peptides with membranes. So far, in labelled peptides, the ¹⁹F-reporter group has always been installed on the side chain of an amino acid. Given the fact that monofluoroalkenes are non-hydrolyzable peptide bond mimics, we have synthesized a monofluoroalkene-based dipeptide isostere, Val-Ψ[(Z)-CF=CH]-Gly, and inserted it in the sequence of two well-studied antimicrobial peptides: PGLa and (KIGAKI)₃ are representatives of an α-helix and a β-sheet. The conformations and biological activities of these labeled peptides were studied to assess the suitability of monofluoroalkenes for ¹⁹F NMR structure analysis.     

Synthesis and conformational analysis of new troponyl aromatic amino acid

Synthetic peptides are in huge demand in expansion of potential peptide mimics, which may have improved or comparable function as natural one. With these concerns, phenyl bearing aromatic amino acids and peptides has extensively explored, because phenyl residue has high probability in forming stable secondary structure, owing to the presence of an extra stabilizing factor as π–π non-covalent interactions. Apart from phenyl bearing benzenoid aromatic amino acids, a few non-benzenoid aromatic derivatives such as tropolone and related compounds are also occurred in nature, but troponyl containing amino acids and peptides are very poorly understood. Tropolonyl derivatives also contain carbonyl functional group, which may play an important role to provide stable conformation in peptide. Herein we report the synthesis, and conformational analysis of rationally designed new unnatural δ-amino acid, troponyl aminoethylglycine (Tr-aeg), which contains troponyl residue as side chain in flexible aminoethylglycine (aeg) amino acid backbone. We also demonstrate the role of troponyl carbonyl of Tr-aeg residue in hydrogen bonding with adjacent amide NH of their hybrid di/tri-peptides with NMR methods and DFT calculations. In future, Tr-aeg amino acid would be a potential building block in development of promisable peptide mimics.     

New Open‐Chain and Cyclic Tetrapeptides,Consisting of α‐, β2‐, and β3‐Amino‐Acid Residues,as Somatostatin Mimics – A Survey

Cyclo‐β‐tetrapeptides are known to adopt a conformation with an intramolecular transannular hydrogen bond in solution. Analysis of this structure reveals that incorporation of a β²‐amino‐acid residue should lead to mimics of ‘α‐peptidic β‐turns’ (cf. A, B, C ). It is also known that short‐chain mixed β/α‐peptides with appropriate side chains can be used to mimic interactions between α‐peptidic hairpin turns and G protein‐coupled receptors. Based on these facts, we have now prepared a number of cyclic and open‐chain tetrapeptides, 7 – 20 , consisting of α‐, β²‐, and β³‐amino‐acid residues, which bear the side chains of Trp and Lys, and possess backbone configurations such that they should be capable of mimicking somatostatin in its affinity for the human SRIF receptors (hsst_1–5). All peptides were prepared by solid‐phase coupling by the Fmoc strategy. For the cyclic peptides, the three‐dimensional orthogonal methodology (Scheme 3) was employed with best success. The new compounds were characterized by high‐resolution mass spectrometry, NMR and CD spectroscopy, and, in five cases, by a full NMR‐solution‐structure determination (in MeOH or H₂O; Fig. 4). The affinities of the new compounds for the receptors hsst_1–5 were determined by competition with [¹²⁵I]LTT‐SRIF₂₈ or [¹²⁵I] [Tyr¹⁰]‐CST₁₄. In Table 1, the data are listed, together with corresponding values of all β‐ and γ‐peptidic somatostatin/Sandostatin^® mimics measured previously by our groups. Submicromolar affinities have been achieved for most of the human SRIF receptors hsst_1–5. Especially high, specific binding affinities for receptor hsst₄ (which is highly expressed in lung and brain tissue, although still of unknown function!) was observed with some of the β‐peptidic mimics. In view of the fact that numerous peptide‐activated G protein‐coupled receptors (GPCRs) recognize ligands with turn structure (Table 2), the results reported herein are relevant far beyond the realm of somatostatin: many other peptide GPCRs should be ‘reached’ with β‐ and γ‐peptidic mimics as well, and these compounds are proteolytically and metabolically stable, and do not need to be cell‐penetrating for this purpose (Fig. 5).     

Aminoacylphosphonates-Novel Modified Amino Acid Derivatives

Abstract

Modified amino acids and peptides are in the focus of medicinal chemistry as potential mimics of natural compounds that may act as inhibitors and modulators of natural processes. Along with many other types of analogs, considerable effort was invested into the synthesis of phospha amino acids. Presently phospha analogs for all amino acids and for many peptides are known and some of these are of considerable practical importance.     

A clickable,highly soluble oligopeptide that easily forms organogels

An artificial peptide, N₃-GVGV-OMe (G, glycine; V, valine), which mimics the repeating GAGA (A, alanine) sequence in Bombix Mori silk, was synthesised via solution-phase synthesis. Compared with N₃-GAGA-OMe sequence, N₃-GVGV-OMe showed high solubility in common organic solvents (such as CHCl₃, THF and CH₂Cl₂), and easily formed organogels simply by adding poor solvents (such as toluene or ether) to the peptide solution at room temperature. The hierarchical nanostructure of N₃-GVGV-OMe organogel was dependent on the nature of the poor solvents, although in all cases, β-sheets were formed exclusively. Gels formed in ether showed higher level hierarchical assembly, as evidenced by AFM and CD studies. Solution-state FT-IR analysis showed that the pre-organisation of the peptides in solution was not significant, and well-defined antiparallel β-sheets were formed after the addition of the poor solvent. The high solubility and strong tendency for self-assembly of N₃-GVGV-OMe, together with its terminal azide group, might facilitate the modification of functional organic molecules even macromolecules for better nanostructure control.     

Combining free energy calculations with tailored enzyme activity assays to elucidate substrate binding of a phospho-lysine phosphatase

Studying enzymes that are involved in the regulation of dynamic post-translational modifications (PTMs) is of key importance in proteomics research. Such investigations can be particularly challenging when the modification itself is intrinsically labile. In this article, we elucidate the enzymatic activity of Phospholysine Phosphohistidine Inorganic Pyrophosphate Phosphatase (LHPP) towards different O- and N-phosphorylated peptides by a combined experimental and computational approach. LHPP has been previously described to hydrolyze the phosphoramidate bonds in different small molecule substrates, including phosphorylated lysine (pLys). Taking the instability of the phosphoramidate bond into account, we conducted a carefully adjusted enzymatic assay with various pLys pentapeptides to confirm enzymatic phosphatase activity with LHPP. Molecular docking was employed to explore possible binding poses of the substrates in complex with the enzyme. Molecular dynamics based free energy calculations, which are unique in their accuracy and solid theoretical basis, were further applied to predict relative binding affinity of different substrates. Comparison of simulations with experiments clearly suggested a distinct binding motif of pLys peptides as well as a very narrow promiscuity of LHPP. We believe this integrated approach can be widely adopted to study the structure and interaction of poorly characterized enzyme–substrate complexes, in particular with synthetically challenging or labile substrates.

Combining phosphatase activity assays with molecular docking and free energy calculations reveals low promiscuity and substrate binding of intrinsically labile phospho-lysine peptides to the enzyme LHPP.     

Multimeric Presentation of RGD Peptidomimetics Enhances Integrin Binding and Tumor Cell Uptake

The use of multimeric ligands is considered as a promising strategy to improve tumor targeting for diagnosis and therapy. Herein, tetrameric RGD (Arg-Gly-Asp) peptidomimetics were designed to target α_vβ₃ integrin-expressing tumor cells. These compounds were prepared by an oxime chemoselective assembly of cyclo(DKP-RGD) ligands and a cyclodecapeptide scaffold, which allows a tetrameric presentation. The resulting tetrameric RGD peptidomimetics were shown to improve α_vβ₃ integrin binding compared with the monomeric form. Interestingly, these compounds were also able to enhance tumor cell endocytosis in the same way as tetrameric RGD peptides. Altogether, the results show the potential of the tetrameric cyclo(DKP-RGD) ligands for in vivo imaging and drug delivery.     

Study of Nanocrystalline BiMnO3‐‐PbTiO3: Synthesis,Structural Elucidation,and Magnetic Characterization of the Whole Solid Solution

In the last ten years, the study and the search for new multiferroic materials have been a major challenge due to their potential applications in electronic technology. In this way, bismuth‐containing perovskites (BiMO₃), and particularly those in which the metal M position is occupied by a magnetically active cation, have been extensively investigated as possible multiferroic materials. From the point of view of synthesis, only a few of the possible bismuth‐containing perovskites can be prepared by conventional methods but at high pressures. Herein, the preparation of one of these potential multiferroic systems, the solid solution xBiMnO₃‐(1?x)PbTiO₃ by mechanosynthesis is reported. Note that this synthetic method allows the oxides with high x values, and more particularly the BiMnO₃ phase, to be obtained as nanocrystalline phases, in a single step and at room temperature without the application of external pressure. These results confirm that, in the case of Bi perovskites, mechanosynthesis is a good alternative to high‐pressure synthesis. These materials have been studied from the point of view of their structural characteristics by precession electron diffraction and magnetic property measurements.