Dakin–West reaction on 1-thyminyl acetic acid for the synthesis of 1,3-bis(1-thyminyl)-2-propanone, a heteroaromatic compound with nucleopeptide-binding properties

This work deals with the Dakin-West synthesis, starting from the nucleoamino acid 1-thyminyl acetic acid, as well the NMR, ESI MS, and X-ray characterization of a heteroaromatic compound denominated by us T(2)CO, comprising two thymine moieties anchored to a 2-propanonic unit, the spectroscopic properties of which were studied by UV as a function of temperature and ionic strength. Preliminary binding-studies with molecules of biomedical interest such as nucleic acids and proteins, performed on samples containing T(2)CO, suggested that this molecule is able to interact very weakly with double-stranded RNA, whereas it does not seem to bind other nucleic acids or proteins. Moreover, by studies with fresh human serum we found that T(2)CO is resistant to enzymatic degradation till 24?h, whereas UV metal binding-studies, performed using solutions of copper (II) chloride dihydrate and nickel (II) chloride hexahydrate, revealed a certain ability of T(2)CO to bind copper (II) cation. Finally, by CD spectroscopy we investigated the influence of T(2)CO on the already described supramolecular networks based on L-serine-containing nucleopeptides. More particularly, we found that T(2)CO is able to increase the level of structuration of the non-covalent supramolecular assembly of the chiral nucleopeptides, which is a feature of remarkable interest for the development of innovative drug delivery tools.     

Synthesis, biological evaluation and supramolecular assembly of novel analogues of peptidyl nucleosides

This work concerns the synthesis, the supramolecular assembly and the evaluation of some biological properties, such as DNA and RNA-binding ability and human serum stability, of novel nucleopeptides. These compounds are of potential interest for the well-known properties that similar compounds, such as natural peptidyl nucleosides, possess in biology and medicine and also for the possibility to realize nucleopeptide-based supramolecular systems useful for drug and gene delivery applications. More particularly, all four nucleobase-containing peptides were synthesized by solid phase synthesis, purified by HPLC and characterized by NMR and ESI-MS. Subsequently, nucleopeptide self-assembly as well as DNA and RNA-binding ability were investigated by CD spectroscopy and further information on the formation of molecular networks, based on the peptidyl nucleoside analogues and nucleic acids, was obtained by Laser Light Scattering. Finally, nucleopeptide enzymatic stability was studied and a half life of about 2 hours was found in the presence of 50% fresh human serum.     

Supramolecular assembly of collagen triblock peptides

The relationship between primary sequence and collagen triple-helix formation is relatively well characterized, while higher levels of structural assembly from these sequences is poorly understood. To address this gap, a new collagen-like triblock peptide design was used to study the relationship between amino acid sequence and supramolecular assembly. Four collagen-like peptides with the sequence (Glu)(5)(Gly-Xaa-Hyp-Gly-Pro-Hyp)(6)(Glu)(5) and corresponding to Xaa = alanine, proline, serine, or valine, and an analogous peptide without the glutamic acid end blocks, were solubilized in water at high concentrations (20-150 mg/mL) and analyzed in optical polarizing microscopy and transmission electron microscopy. Some of the peptides self-assembled into supramolecular structures, the nature of which was determined by the core collagen-like sequence. The globular end blocks appeared necessary for these short triple-helix-forming peptides to spontaneously organize into supramolecular structures in solution and also provided enhanced thermal stability based on CD analysis. The results indicate a strong dependence of the peptide triblock assembly behavior on the identity of the guest residue Xaa; nematic order when Xaa was valine, no organization when Xaa was serine, and banded spherulites displaying a cholesteric-like twist when Xaa was proline or alanine. According to these results, the identity of the amino acid in position Xaa of the triplet Gly-Xaa-Yaa dramatically determined the type of supramolecular assembly formed by short triple helices based on collagen-triblock like sequences. Moreover, the structural organization observed for these collagen-triblock peptides was analogous to some assemblies observed for native collagen in vivo and in vitro. The amino acid sequence in the native collagen proteins may therefore be a direct determinant of the different supramolecular architectures found in connective tissues.     

Synthesis of a thymine-functionalized nucleoamino acid for the solid phase assembly of cationic nucleopeptides

In this work, we report the synthesis of a thymine-functionalized nucleoamino acid suitable for the solid phase synthesis of nucleopeptides. The monomer was obtained in solution starting from commercial compounds and after NMR (¹H and ¹³C) and ESIMS (positive ions) characterization it was used for the assembly of a cationic nucleopeptide obtained by sequentially introducing underivatized l-lysine units and nucleoamino acid monomers. After detachment from the resin, performed in acidic conditions, the oligomer was purified by HPLC and characterized by LC-ESIMS (positive ions) which confirmed the identity of the thymine-based nucleopeptide. The cationic nucleobase-containing peptide, well soluble in water, was studied by CD spectroscopy which allowed us to exclude any helical pre-organization of the nucleopeptide in the experimental conditions used. Furthermore, CD behavior of the oligomer at different temperatures was also studied as described in this work.     

Synthesis and hybridization properties of polyamide based nucleic acid analogues incorporating pyrrolidine-derived nucleoamino acids

Ndelta-Fmoc protected nucleoamino acids of type I (Base = T, C, A) have been synthesized and employed as building blocks for the construction of novel polyamide based nucleic acid analogues. Homopyrimidine oligomer A binds to complementary RNA with significant affinity and in a sequence-specific fashion, while no binding was observed to complementary DNA.     

The Baseplate Wedges of Bacteriophage T4 Spontaneously Assemble into Hubless Baseplate-Like Structure In Vitro

The baseplate of phage T4 is an important model system in viral supramolecular assembly. The baseplate consists of six wedges surrounding the central hub. We report the first successful attempt at complete wedge assembly using an in vitro approach based on recombinant proteins. The cells expressing the individual wedge proteins were mixed in a combinatorial manner and then lysed. Using this approach, we could both reliably isolate the complete wedge along with a series of intermediate complexes as well as determine the exact sequence of assembly. The individual proteins and intermediate complexes at each step of the wedge assembly were successfully purified and characterized by sedimentation velocity and electron microscopy. Although our results mostly confirmed the hypothesized sequential wedge assembly pathway as established using phage mutants, interestingly, we also detected some protein interactions not following the specified order. It was found that association of gene product 53 to the immediate precursor complex induces spontaneous association of the wedges to form a six-fold star-shaped baseplate-like structure in the absence of the hub. The formation of the baseplate-like structure was facilitated by the addition of gene product 25. The complete wedge in the star-shaped supramolecular complex has a structure similar to the baseplate in the expanded “star” conformation found after infection. Based on the results of the present and previous studies, we assume that the strict order of wedge assembly is due to the induced conformational change caused by every new binding event. The significance of a 40-S star-shaped baseplate structure, which was previously reported and was also found in this study, is discussed in the light of a new paradigm for T4 baseplate assembly involving the star-shaped wedge ring and the central hub. Importantly, the methods described in this article suggest a novel methodology for future structural characterization of supramolecular protein assemblies.     

In situ synthesis of amylose/single-walled carbon nanotubes supramolecular assembly

A supramolecular assembly of amylose and single-walled carbon nanotubes (SWNTs) was synthesized in situ through vine-twining polymerization. Raman analysis indicated that the amylose-SWNTs supramolecular assembly was formed after the polymerization and SEM images displayed the twisted ribbons in the SWNTs wrapped by amylose. The dispersion stability of the SWNTs in aqueous solutions was improved by the wrapping of short-chain amylose molecules around the SWNTs.     

Thiophenyl-substituted triazolyl-thione l-alanine: asymmetric synthesis,aggregation and biological properties

In this work, we report the asymmetric synthesis and characterization of an artificial amino acid based on triazolyl-thione l-alanine, which was modified with a thiophenyl-substituted moiety, as well as in vitro studies of its nucleic acid-binding ability. We found, by dynamic light scattering studies, that the synthetic amino acid was able to form supramolecular aggregates having a hydrodynamic diameter higher than 200 nm. Furthermore, we demonstrated, by UV and CD experiments, that the heteroaromatic amino acid, whose enzymatic stability was demonstrated by HPLC analysis also after 24 h of incubation in human serum, was able to bind a RNA complex, which is a feature of biomedical interest in view of innovative antiviral strategies based on modulation of RNA–RNA molecular recognition.     

Studies of Thermal Stability of Multivalent DNA Hybridization in a Nanostructured System

A fundamental understanding of molecular self-assembly processes is important for improving the design and construction of higher-order supramolecular structures. DNA tile based self-assembly has recently been used to generate periodic and aperiodic nanostructures of different geometries, but there have been very few studies that focus on the thermodynamic properties of the inter-tile interactions. Here we demonstrate that fluorescently-labeled multihelical DNA tiles can be used as a model platform to systematically investigate multivalent DNA hybridization. Real-time monitoring of DNA tile assembly using fluorescence resonance energy transfer revealed that both the number and the relative position of DNA sticky-ends play a significant role in the stability of the final assembly. As multivalent interactions are important factors in nature's delicate macromolecular systems, our quantitative analysis of the stability and cooperativity of a network of DNA sticky-end associations could lead to greater control over hierarchical nanostructure formation and algorithmic self-assembly.     

Polyethylene glycol‐based low generation dendrimers functionalized with β‐cyclodextrin as cryo‐ and dehydro‐protectant of catalase formulations

Polyethylene glycol (PEG)‐based low generation dendrimers are analyzed as single excipient or combined with trehalose in relation to their structure and efficiency as enzyme stabilizers during freeze‐thawing, freeze‐drying, and thermal treatment. A novel functional dendrimer (DG_o‐CD) based on the known PEG's ability as cryo‐protector and β‐CD as supramolecular stabilizing agent is presented. During freeze‐thawing, PEG and β‐CD failed to prevent catalase denaturation, while dendrimers, and especially DG_o‐CD, offered the better protection to the enzyme. During freeze‐drying, trehalose was the best protective additive but DG_o‐CD provided also an adequate catalase stability showing a synergistic behavior in comparison to the activities recovered employing PEG or β‐CD as unique additives. Although all the studied dendrimers improved the enzyme remaining activity during thermal treatment of freeze‐dried formulations, the presence of amorphous trehalose was critical to enhance enzyme stability. The crystallinity of the protective matrix, either of PEG derivatives or of trehalose, negatively affected catalase stability in the freeze‐dried systems. When humidified at 52% of relative humidity, the dendrimers delayed trehalose crystallization in the combined matrices, allowing extending the protection at those conditions in which normally trehalose fails. The results show how a relatively simple covalent combination of a polymer such as PEG with β‐CD could significantly affect the properties of the individual components. Also, the results provide further insights about the role played by polymer–enzyme supramolecular interactions (host–guest crosslink, hydrogen bonding, and hydrophobic interactions) on enzyme stability in dehydrated models, being the effect on the stabilization also influenced by the physical state of the matrix. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:786–795, 2013     

Redesigning channel-forming peptides: amino acid substitutions that enhance rates of supramolecular self-assembly and raise ion transport activity

Three series of 22-residue peptides derived from the transmembrane M2 segment of the glycine receptor alpha1-subunit (M2GlyR) have been designed, synthesized, and tested to determine the plasticity of a channel-forming sequence and to define whether channel pores with enhanced conductive properties could be created. Sixteen sequences were examined for aqueous solubility, solution-association tendency, secondary structure, and half-maximal concentration for supramolecular assembly, channel activity, and ion transport properties across epithelial monolayers. All peptides interact strongly with membranes: associating with, inserting across, and assembling to form homooligomeric bundles when in micromolar concentrations. Single and double amino acid replacements involving arginine and/or aromatic amino acids within the final five C-terminal residues of the peptide cause dramatic effects on the concentration dependence, yielding a range of K1/2 values from 36 +/- 5 to 390 +/- 220 microM for transport activity. New water/lipid interfacial boundaries were established for the transmembrane segment using charged or aromatic amino acids, thus limiting the peptides' ability to move perpendicularly to the plane of the bilayer. Formation of discrete water/lipid interfacial boundaries appears to be necessary for efficient supramolecular assembly and high anion transport activity. A peptide sequence is identified that may show efficacy in channel replacement therapy for channelopathies such as cystic fibrosis.     

Novel intermolecular iterative mechanism for biosynthesis of mycoketide catalyzed by a bimodular polyketide synthase

In recent years, remarkable versatility of polyketide synthases (PKSs) has been recognized; both in terms of their structural and functional organization as well as their ability to produce compounds other than typical secondary metabolites. Multifunctional Type I PKSs catalyze the biosynthesis of polyketide products by either using the same active sites repetitively (iterative) or by using these catalytic domains only once (modular) during the entire biosynthetic process. The largest open reading frame in Mycobacterium tuberculosis, pks12, was recently proposed to be involved in the biosynthesis of mannosyl-beta-1-phosphomycoketide (MPM). The PKS12 protein contains two complete sets of modules and has been suggested to synthesize mycoketide by five alternating condensations of methylmalonyl and malonyl units by using an iterative mode of catalysis. The bimodular iterative catalysis would require transfer of intermediate chains from acyl carrier protein domain of module 2 to ketosynthase domain of module 1. Such bimodular iterations during PKS biosynthesis have not been characterized and appear unlikely based on recent understanding of the three-dimensional organization of these proteins. Moreover, all known examples of iterative PKSs so far characterized involve unimodular iterations. Based on cell-free reconstitution of PKS12 enzymatic machinery, in this study, we provide the first evidence for a novel "modularly iterative" mechanism of biosynthesis. By combination of biochemical, computational, mutagenic, analytical ultracentrifugation and atomic force microscopy studies, we propose that PKS12 protein is organized as a large supramolecular assembly mediated through specific interactions between the C- and N-terminus linkers. PKS12 protein thus forms a modular assembly to perform repetitive condensations analogous to iterative proteins. This novel intermolecular iterative biosynthetic mechanism provides new perspective to our understanding of polyketide biosynthetic machinery and also suggests new ways to engineer polyketide metabolites. The characterization of novel molecular mechanisms involved in biosynthesis of mycobacterial virulent lipids has opened new avenues for drug discovery.     

In vitro stability of alpha-helical peptide nucleic acids (alphaPNAs)

Alpha-helical peptide nucleic acids (alphaPNAs) are synthetic molecules that merge the alpha-helix secondary structure of peptides with the codified Watson-Crick base pairing capability of nucleic acids. It is now demonstrated that alphaPNAs made up of either L- or D-amino acids are resistant to degradation by the proteases present in human serum. The increased stability of alphaPNAs towards proteases may be attributable to the presence of unnatural nucleoamino acid residues [-NHCH(CH(2)OCH(2)B)CO-, where B=thymine or cytosine] since the replacement of these amino acids by serine yields a control peptide that does break down in human serum. The stability of alphaPNAs towards proteases makes them attractive candidates for further development as antisense agents.     

New polymer syntheses. IV. Polypeptides of lysine and ornithine with pending pyrimidine bases

Starting with β-methoxy methacryloylisocyanate, β-methoxy methacrylisothiocyanate, and β-isocyanatopropionyl chloride, on the one hand, and N^α-Z-lysine or N^α-Z-ornithine, on the other hand, N^α-Z-amino acids with pyrimidine bases in the side chain were synthesized. These Z-protected nucleoamino acids were converted to the corresponding N-carboxyanhydrides (NCAs) via the silylester method. In the case of 2-thiothymine derivatives, the reaction intermediate of the NCA synthesis caused benzylation of the thioxo- group, so that a new class of 2-mercaptopyrimidine derivatives was isolated unexpectedly. The poly(nucleoamino acids) obtained by polymerization of the nucleoamino acid NCAs were characterized by elemental analyses, optical rotations ¹H-nmr and ¹³C-nmr spectra. Vapor pressure osmometry revealed that the DP s were in the range of 20–30. Their spectra suggest a helical secondary structure. While all homopolypeptides are insoluble in water, copolypeptides containing L -lysine N^ε-hydrobromide possess good solubility in water.     

Functional Amyloids: Where Supramolecular Amyloid Assembly Controls Biological Activity or Generates New Functionality

Functional amyloids are a rapidly expanding class of fibrillar protein structures, with a core cross-β scaffold, where novel and advantageous biological function is generated by the assembly of the amyloid. The growing number of amyloid structures determined at high resolution reveal how this supramolecular template both accommodates a wide variety of amino acid sequences and also imposes selectivity on the assembly process. The amyloid fibril can no longer be considered a generic aggregate, even when associated with disease and loss of function. In functional amyloids the polymeric β-sheet rich structure provides multiple different examples of unique control mechanisms and structures that are finely tuned to deliver assembly or disassembly in response to physiological or environmental cues. Here we review the range of mechanisms at play in natural, functional amyloids, where tight control of amyloidogenicity is achieved by environmental triggers of conformational change, proteolytic generation of amyloidogenic fragments, or heteromeric seeding and amyloid fibril stability. In the amyloid fibril form, activity can be regulated by pH, ligand binding and higher order protofilament or fibril architectures that impact the arrangement of associated domains and amyloid stability. The growing understanding of the molecular basis for the control of structure and functionality delivered by natural amyloids in nearly all life forms should inform the development of therapies for amyloid-associated diseases and guide the design of innovative biomaterials.     

Supramolecular aggregation/disaggregation-based molecular sensing: a review focused on investigations from China

Supramolecular aggregation and disaggregation induced by external stimuli can impact the optical or electrical signals of the aggregates/constituting units (receptors). Therefore, manipulating supramolecular aggregation/disaggregation has recently been employed to construct novel and promising photoluminescence (PL)‐based sensing and recognition systems. The sensing systems were capable of substantially enhancing the sensitivity, relying on cooperative interactions occurring in the assembly/disassembly processes (mostly operating in emission turned‐on or emission‐enhanced mode). This review focuses mainly on recent advances in the new emerging PL‐based sensing platforms, based on manipulating the behaviours of supramolecular aggregation/disaggregation, including aggregation‐induced emission (AIE), metallophilic interactions‐related sensing (metallophilic interactions‐induced aggregation/disaggregation), metal coordination polymers‐related sensing, and other sensing systems involving supramolecular aggregation/disaggregation. In particular, those sensing systems developed by scientists in China are summarized and highlighted. Copyright © 2011 John Wiley & Sons, Ltd.     

Different assembly properties of cod, bovine, and rat brain microtubules.

Assembly properties of cod, bovine, and rat brain microtubules were compared. Estramustine phosphate, heparin, poly-L-aspartic acid, as well as NaCl, inhibited the assembly and disassembled both bovine and rat microtubules by inhibition of the binding between tubulin and MAPs. The assembly of cod brain microtubules was in contrast only marginally affected by these agents, in spite of a release of the MAPs. The results suggest that cod tubulin has a high intrinsic ability to assemble. This was confirmed by studies on phosphocellulose-purified cod tubulin, since the critical concentration for assembly was independent of the presence or absence of MAPs. The results show therefore that cod brain tubulin has, in contrast to bovine and rat brain tubulins, a high propensity to assembly under conditions which normally require the presence of MAPs. Even if cod MAPs, which have an unusual protein composition, were not needed for the assembly of cod microtubules, they were able to induce assembly of bovine brain tubulin. Both cod and bovine MAPs bound to cod microtubules, and bovine MAP1 and MAP2 bound to, and substituted at least the 400 kDa cod protein. This suggests that the tubulin-binding sites and the assembly-stimulatory ability of MAPs are common properties of MAPs from different species, independent of the tubulin assembly propensity.     

Does domain swapping improve the stability of RNase A?

Self-assembling complexes have potential as novel supramolecular biomaterials but domain swapped complexes have yet to investigated in this capacity. Bovine ribonuclease A (RNase A) is a useful model protein as it is able to form a range of three dimensional domain swapped structures, including dimers, trimers and tetramers that have similar catalytic ability. However, little work has been carried out investigating the physical characteristics of these complexes. In an effort to characterise the strength of these oligomeric interactions, analytical ultracentrifugation was carried out to measure the dissociation of higher order complexes, using fluorescent tags to test for dissociation at very low concentrations. Results of this work suggest that the oligomers form a very tight complex, with no evidence of dissociation down to 250 pM. RNase A oligomers also had similar thermal stability to that of monomeric enzyme, suggesting that the main limiting factor in RNase A stability is the tertiary, rather than quaternary structure. Following thermal unfolding of RNase A, the protein refolded upon cooling, but returned to the monomeric state. This latter result may limit the potential of domain swapping as a means of material assembly.     

G-quartet self-assembly under osmotic pressure: remote control by vesicle shrinking rather than stress

Does osmotic pressure stimulate assembly or disassembly of supramolecules in vesicles? Self‐assembly was conceivable as intravesicular response to osmotic shrinking upon application of extravesicular overpressure, whereas disassembly was conceivable as a response to bilayer stress in hyperosmotic vesicles. Self‐assembly of guanosine 5′‐monophosphates (GMPs) into G‐quartets was selected to investigate the nature of remote control of supramolecular chemistry within vesicles by osmotic pressure. Using circular dichroism spectroscopy to selectively detect G‐quartets, we found that extravesicular overpressure stimulates intravesicular self‐assembly, whereas underpressure stimulates disassembly. G‐quartet self‐assembly by osmotic pressure exhibited ion‐selective metal‐cation templation, as expected. The key conclusions are that supramolecular chemistry within vesicles is governed by vesicle shape rather than vesicle stress and that detection of osmotic pressure by CD spectroscopy is an interesting alternative to the commonly used methods based on fluorescence self‐quenching. Chirality 15:766–771, 2003. © 2003 Wiley‐Liss, Inc.     

Supramolecular photochemical synthesis of an unsymmetrical cyclobutane.

2-styrylbenzothiazole (1) and cinnamic acid (2) derivatives containing 15-crown-5 ether moieties form a supramolecular assembly in the presence of Ba(2+) cations in acetonitrile. The assembly is stabilized by hydrogen bonding between the heterocyclic N atom of 1 and the proton of the carboxylic group of 2, by sandwich Ba(2+) complex formation between the crown ether moieties of 1 and 2, and by pi-pi stacking interactions. Irradiation of solutions containing these supramolecular complexes leads to highly specific formation of an unsymmetrical cycloadduct. This investigation provides an interesting example of supramolecular control of [2 + 2]-photocyclization in solution.