Smartphone-Based Chemiluminescence Glucose Biosensor Employing a Peroxidase-Mimicking, Guanosine-Based Self-Assembled Hydrogel.

Chemiluminescence is widely used for hydrogen peroxide detection, mainly exploiting the highly sensitive peroxidase-luminol-H2O2 system. Hydrogen peroxide plays an important role in several physiological and pathological processes and is produced by oxidases, thus providing a straightforward way to quantify these enzymes and their substrates. Recently, biomolecular self-assembled materials obtained by guanosine and its derivatives and displaying peroxidase enzyme-like catalytic activity have received great interest for hydrogen peroxide biosensing. These soft materials are highly biocompatible and can incorporate foreign substances while preserving a benign environment for biosensing events. In this work, a self-assembled guanosine-derived hydrogel containing a chemiluminescent reagent (luminol) and a catalytic cofactor (hemin) was used as a H2O2-responsive material displaying peroxidase-like activity. Once loaded with glucose oxidase, the hydrogel provided increased enzyme stability and catalytic activity even in alkaline and oxidizing conditions. By exploiting 3D printing technology, a smartphone-based portable chemiluminescence biosensor for glucose was developed. The biosensor allowed the accurate measurement of glucose in serum, including both hypo- and hyperglycemic samples, with a limit of detection of 120 µmol L-1. This approach could be applied for other oxidases, thus enabling the development of bioassays to quantify biomarkers of clinical interest at the point of care.

Taurine Stabilizing Effect on Lysozyme.

Taurine is an important organic osmolyte in mammalian cells, and it weakens inflammation and oxidative stress mediated injuries in some diseases. Recently, taurine has been demonstrated to play a therapeutic role against neurodegenerative disorders, although its parallel involvement in several biochemical mechanisms makes not clear taurine specific role in these diseases. Furthermore, the stabilizing effect of this molecule in terms of protein stability is known, but not deeply investigated. In this work we explore by Circular Dichroism the stabilizing impact of taurine in lysozyme thermal denaturation and its influence in lysozyme aggregation into amyloid fibrils. Taurine even at low concentration modifies protein-protein interactions in lysozyme native state, as revealed by Small Angle X-ray Scattering experiments, and alters the amyloid aggregation pattern without completely inhibiting it, as confirmed by UV/Vis spectroscopy with Congo Red and by Atomic Force Microscopy. Evaluation of the cytotoxicities of the amyloid fibrils grown in presence or in absence of taurine is investigated on SH-SY5Y neuroblastoma cells.

Self-Assembly of Functionalized Lipophilic Guanosines into Cation-Free Stacked Guanine-Quartets.

The hierarchical self-assembly of various lipophilic guanosines exposing either a phenyl or a ferrocenyl group in the C(8) position was investigated. In a solution, all the derivatives were found to self-assemble primarily into isolated guanine (G)-quartets. In spite of the apparent similar bulkiness of the two substituents, most of the derivatives form disordered structures in the solid state, whereas a specific 8-phenyl derivative self-assembles into an unprecedented, cation-free stacked G-quartet architecture.

Reversible Photoisomerization in Thin Surface Films from Azo-Functionalized Guanosine Derivatives.

Two novel azo-functionalized guanosine derivatives were synthesized, and their photoisomerization process was investigated in molecular monolayers at the air-water interface and in the Langmuir-Blodgett (LB) films on solid substrates. Measurements of surface pressure vs area isotherms, surface potential measurements, UV-visible (vis) absorption spectroscopy, Brewster angle microscopy (BAM), and atomic force microscopy (AFM) were performed. Despite not having a typical amphiphilic molecular structure, the derivatives formed stable films on the water surface. They could also undergo repeated photoisomerization in all of the investigated thin-film configurations. The observations suggest that in the films at the air-water interface, the molecules first exhibit a conformational change, and then they reorient to an energetically more favored orientation. In the LB films transferred onto solid substrates, the isomerization process occurs on a similar time scale as in solution. However, the isomerization efficiency is about an order of magnitude lower than that in solution. Our results show that DNA nucleobases functionalized with azobenzene moieties are suitable candidates for the fabrication of photoactive two-dimensional (2D) materials that can provide all beneficial functionalities of DNA-based compounds.

Trehalose Effect on the Aggregation of Model Proteins into Amyloid Fibrils.

Protein aggregation into amyloid fibrils is a phenomenon that attracts attention from a wide and composite part of the scientific community. Indeed, the presence of mature fibrils is associated with several neurodegenerative diseases, and in addition these supramolecular aggregates are considered promising self-assembling nanomaterials. In this framework, investigation on the effect of cosolutes on protein propensity to aggregate into fibrils is receiving growing interest, and new insights on this aspect might represent valuable steps towards comprehension of highly complex biological processes. In this work we studied the influence exerted by the osmolyte trehalose on fibrillation of two model proteins, that is, lysozyme and insulin, investigated during concomitant variation of the solution ionic strength due to NaCl. In order to monitor both secondary structures and the overall tridimensional conformations, we have performed UV spectroscopy measurements with Congo Red, Circular Dichroism, and synchrotron Small Angle X-ray Scattering. For both proteins we describe the effect of trehalose in changing the fibrillation pattern and, as main result, we observe that ionic strength in solution is a key factor in determining trehalose efficiency in slowing down or blocking protein fibrillation. Ionic strength reveals to be a competitive element with respect to trehalose, being able to counteract its inhibiting effects toward amyloidogenesis. Reported data highlight the importance of combining studies carried out on cosolutes with valuation of other physiological parameters that may affect the aggregation process. Also, the obtained experimental results allow to hypothesize a plausible mechanism adopted by the osmolyte to preserve protein surface and prevent protein fibrillation.

Metallo-responsive self-assembly of lipophilic guanines in hydrocarbon solvents: a systematic SAXS structural characterization.

Lipophilic guanines (LipoGs) in aprotic solvents undergo different self-assembly processes based on different H-bonded motifs. Cylindrical nanotubes made by π-π stacked guanine tetramers (G-quadruplexes) and flat, tape-like aggregates (G-ribbons) have been observed depending on the presence of alkali metal ions. To obtain information on the structural properties and stability of these LipoG aggregates, Small-Angle X-ray Scattering (SAXS) experiments have been performed in dodecane, both in the presence and in the absence of potassium ions. As a result, the occurrence of the two different metallo-responsive architectures (nanoribbons or columnar nanotubes) was confirmed and we reported here for the first time a systematic study on the dependence of the aggregate properties on composition, temperature and molecular unit structure. Even if dodecane was selected to favour LipoG solubility, a strong tendency to self-organize into ordered lyotropic phases was indeed detected.

Playing supramolecular dominoes with light: building and breaking a photoreversible G-quadruplex made from guanosine, boric acid and an azobenzene.

Addition of azobenzene-derivative 1 in its E configuration to an aqueous solution containing various guanosine borate esters induces a helical G-quartet based self-organization, stabilized by intercalation of the dye. The process is driven, in a domino fashion, by the initial host-guest interaction between the dye and a specific guanosine borate diester, whose structure can be thus assigned. This inclusion complex templates the formation of G-quartets. The quartets, in turn, pile up to form a supramolecular G-quadruplex structure, in which other G species present in solution are progressively included. The G-quadruplex can be reversibly broken and reformed by photoisomerization of the dye. This hierarchical and photosensitive self-assembly is unprecedented for simple guanosine derivatives.

Halloysite nanotubes for efficient loading, stabilization and controlled release of insulin.

HYPOTHESIS: Oral insulin administration is not actually effective due to insulin rapid degradation, inactivation and digestion by proteolytic enzymes which results in low bioavailability. Moreover insulin is poorly permeable and lack of lipophilicity. These limits can be overcome by the loading of protein in some nanostructured carrier such as halloysite nanotubes (HNTs). EXPERIMENTS: Herein we propose an easy strategy to obtain HNT hybrid materials for the delivery of insulin. We report a detailed description on the thermal behavior and stability of insulin loaded and released from the HNTs hybrid by the combination of several techniques. FINDINGS: Release experiments of insulin from the HNTs revealed the efficacy of the nanocarrier. Circular Dichroism data evidenced that the released insulin exhibits its native-like secondary structure confirming the suitability of HNT/insulin as delivery system for at least three months. The loaded nanotubes were filled into chitosan matrix with the aim to prepare bionanocomposite films that can be used for transdermal delivery. This work puts forward an efficient strategy to prepare halloysite based nanocarriers containing insulin that could be employed in several biomedical applications. The detailed description of the prepared HNT/insulin hybrid represents a fundamental point for designing advanced delivery systems.

A lipophilic "fully-anti" dodecamer from a (5'S)-5',8-cyclo-2'-deoxyguanosine.

The self-assembly of a lipophilic derivative of (5'S)-5',8-cyclo-2'-deoxyguanosine, a mutagenic product formed by hydroxyl radical attack against DNA, has been investigated. This derivative forms, with high fidelity, a dodecameric complex composed of three stacked G-quartets in the presence of strontium picrate. This is the first example of a fully-anti lipophilic G-quadruplex.

In-peptide synthesis of di-oxazolidinone and dehydroamino acid-oxazolidinone motifs as ß-turn inducers.

Small and easy-to-do mimetics of ß-turns are of great interest to interfere with protein-protein recognition events mediated by ß-turn recognition motifs. We propose a straightforward procedure for constraining the conformation of tetrapeptides lacking a pre-formed scaffold. According to the stereochemistry array, N-Ts tetrapeptides including Thr or PhSer (phenylserine) at the positions 2 or 3 gave rise in a single step to the sequences Oxd(2)-Oxd(3) or ΔAbu(2)-Oxd(3) (Oxd, oxazolidin-2-one; ΔAbu, 2,3-dehydro-2-aminobutyric). These pseudo-Pro residues displayed highly constrained ϕ, ψ, and χ dihedral angles, and induced clear ß-turns or inverse turns of type I or II, as determined by extensive spectroscopic and computational analyses.

Control of the helical chirality of enantiopure sulfinyl (Z)-azobenzene-based photoswitches.

A new class of enantiopure ortho,ortho-disubstituted azobenzene photoswitches has been synthesized from (S)-2-(p-tolylsulfinyl)benzoquinone and arylhydrazines. The sulfoxide acts as a unidirectional controller of the helical chirality that arises in the Z isomer after photoisomerization. Highly congested E-azobenzenes 5 c showed two atropisomeric diastereoconformers in the solid state that converged upon irradiation into a unique Z isomer with defined helicity (M), as evident in the X-ray structure. The chiroptical properties of this three-state enantiopure switch can be externally tuned both photochemically and/or thermally. Theoretical CD spectra calculated by using time-dependent DFT methods support the existence of two atropoisomeric E isomers and only one Z isomer with (M) helicity. Complementary to the classical azobenzene-based switches, the photoswiching event is promoted under green/blue light and do not occur under UV irradiation.

1,2-Di(phenylethynyl)ethenes with axially chiral, 2,2'-bridged 1,1'-binaphthyl substituents: potent cholesteric liquid-crystal inducers.

Axially chiral, 3,5-dihydro-4H-dinaphtho[2,1-c:1',2'-e]azepine (dinaphthazepine) and 1,1'-binaphthyl-2,2'-disulfonimide (dinaphthosulfonimide) moieties were rigidly connected via N-p-phenylene linkers to photochemically (E)/(Z)-isomerisable 1,2-diethynylethene scaffolds. The chemical stability of the resulting systems was found to be critically related to the other substituents on the central π-conjugated scaffold. High helical twisting power (HTP), up to 315 µm(-1), for the induction of a cholesteric liquid-crystalline phase through doping of a nematic phase was measured, resulting from the introduction of the chiral, mesogenic 1,1'-binaphthyl motifs. Single crystal X-ray analysis revealed that the phenylene spacer is in π-conjugation with the N-atom of the dinaphthazepine but not with the N-atom of the dinaphthosulfonimide moiety. This difference in orientation results in visible-transparency in the electronic absorption spectrum and higher (E)/(Z)-photoisomerisation quantum yields of the dinaphthosulfonimide-derived chiral dopants, as compared to the dinaphthazepine systems, which feature intramolecular charge-transfer absorption in the visible region.

The interplay of configuration and conformation in helical perylenequinones: Insights from chirality induction in liquid crystals and calculations.

The chirality transfer in liquid crystals induced by two helical perylenequinones (namely, the natural compounds cercosporin and phleichrome) was investigated by integrating measurements of helical twisting power with a conformational analysis by DFT calculations and with the prediction of their twisting ability by the surface-chirality method. The two quasi-enantiomeric derivatives induce oppositely handed cholesteric phases when introduced as dopants in nematic solvents. We evaluated the role of the different conformations of the chiral hydroxyalkyl side chains in determining the helical twisting power: They were found to affect the strength of the chirality transfer, although the handedness of the induced cholesteric phase is essentially determined by the axial chirality (helicity) of the core of the perylenequinones.

Central-to-axial chirality transfer revealed by liquid crystals: a combined experimental and computational approach for the determination of absolute configuration of carboxylic acids with an α chirality centre.

The conversion into 6,7-dihydro-5H-dibenz[c,e]azepine (DAZ) N-protected amides is a viable route for the determination of the absolute configuration of chiral 2-substituted carboxylic acids. The biphenyl moiety of DAZ, besides being a probe of chirality for the electronic circular dichroism (ECD) spectroscopy, makes these systems suitable for configuration assignment by exploiting the chirality amplification which occurs in nematic liquid crystals. To assess the reliability of the liquid crystal method in detecting the absolute stereochemistry of chiral amides bound to a biphenyl group, we measured the helical twisting power of a series of DAZ-N-protected amides and compared these data with the results obtained from ECD measurements. We will show that the liquid crystal method, corroborated by HTP predictions, is trustworthy with our biphenyl derivatives, even when ECD spectra are ambiguous for the presence of aryl moieties displaying strong UV absorptions in the same range of the biphenyl chromophore.

Identifying guanosine self assembly at natural isotopic abundance by high-resolution 1H and 13C solid-state NMR spectroscopy.

By means of the (1)H chemical shifts and the proton-proton proximities as identified in (1)H double-quantum (DQ) combined rotation and multiple-pulse spectroscopy (CRAMPS) solid-state NMR correlation spectra, ribbon-like and quartet-like self-assembly can be identified for guanosine derivatives without isotopic labeling for which it was not possible to obtain single crystals suitable for diffraction. Specifically, characteristic spectral fingerprints are observed for dG(C10)(2) and dG(C3)(2) derivatives, for which quartet-like and ribbon-like self-assembly has been unambiguously identified by (15)N refocused INADEQUATE spectra in a previous study of (15)N-labeled derivatives (Pham, T. N.; et al. J. Am. Chem. Soc.2005, 127, 16018). The NH (1)H chemical shift is observed to be higher (13-15 ppm) for ribbon-like self-assembly as compared to 10-11 ppm for a quartet-like arrangement, corresponding to a change from NH···N to NH···O intermolecular hydrogen bonding. The order of the two NH(2)(1)H chemical shifts is also inverted, with the NH(2) proton closest in space to the NH proton having a higher or lower (1)H chemical shift than that of the other NH(2) proton for ribbon-like as opposed to quartet-like self-assembly. For the dG(C3)(2) derivative for which a single-crystal diffraction structure is available, the distinct resonances and DQ peaks are assigned by means of gauge-including projector-augmented wave (GIPAW) chemical shift calculations. In addition, (14)N-(1)H correlation spectra obtained at 850 MHz under fast (60 kHz) magic-angle spinning (MAS) confirm the assignment of the NH and NH(2) chemical shifts for the dG(C3)(2) derivative and allow longer range through-space N···H proximities to be identified, notably to the N7 nitrogens on the opposite hydrogen-bonding face.

Chirality transfer across length-scales in nematic liquid crystals: fundamentals and applications.

When a chiral dopant is dissolved in an achiral liquid crystal medium, the whole sample organizes into a helical structure with a characteristic length-scale of the order of microns. The relation between chirality at these quite different length-scales can be rationalized by a relatively simple model, which retains the relevant factors coming into play: the molecular shape of the chiral dopant, which controls the chirality of short range intermolecular interactions, and the elastic properties of the nematic environment, which control the restoring torques opposing distortion of the director. In this tutorial review the relation between molecular and phase chirality will be reviewed and several applications of the chiral doping of nematic LCs will be discussed. These range from the exploitation of the amplified molecular chirality for stereochemical purposes (e.g., the determination of the absolute configuration or the enantiomeric excess), to newer applications in physico-chemical fields. The latter take advantage of the periodicity of the chiral field, with length-scales ranging from hundreds to thousands of nanometres, which characterise the cholesteric phase.

Nanopatterning the surface with ordered supramolecular architectures of N(9)-alkylated guanines: STM reveals.

STM study of the self-assembly at the solid-liquid interface of substituted guanines exposing in the N(9)-position alkyl side chains with different lengths revealed the formation of distinct crystalline nanopatterns.

A non-empirical chromophoric interpretation of CD spectra of DNA G-quadruplex structures.

G-quadruplex DNA (G4-DNA) structures are four-stranded helical DNA (or RNA) structures, comprising stacks of G-tetrads, which are the outcome of planar association of four guanines in a cyclic Hoogsteen hydrogen-bonding arrangement. In the last decade the number of publications where CD spectroscopy has been used to study G4-DNAs, is extremely high. However, with very few exceptions, these investigations use an empirical interpretation of CD spectra. In this interpretation two basic types of CD spectra have been associated to a single specific difference in the features of the strand folding, i.e. the relative orientation of the strands, "parallel" (all strands have the same 5' to 3' orientation) or "antiparallel". Different examples taken from the literature where the empirical interpretation is not followed or is meaningless are presented and discussed. Furthermore, the case of quadruplexes formed by monomeric guanosine derivatives, where there is no strand connecting the adjacent quartets and the definition parallel/antiparallel strands cannot apply, will be discussed. The different spectral features observed for different G-quadruplexes is rationalised in terms of chromophores responsible for the electronic transitions. A simplified exciton coupling approach or more refined QM calculations allow to interpret the different CD features in terms of different stacking orientation (head-to-tail, head-to-head, tail-to-tail) between adjacent G-quartets irrespectively of the relative orientation of the stands (parallel/antiparallel).

Solvent-induced switching between two supramolecular assemblies of a guanosine-terthiophene conjugate.

We report here our findings on a lipophilic guanosine derivative armed with a terthiophene unit that undergoes a pronounced variation of its supramolecular organisation by changing the polarity of the solvent. In chloroform the guanosine derivative, templated by alkali metal ions, assembles via H-bonding in G-quartet based D(4)-symmetric octamers; the polar guanine bases are located into the inner part of the assembly and act as a scaffold for the terthienyl pendants. On the other hand, in the more polar (and H-bond competing) acetonitrile, different aggregates are observed in which the terthiophene chains are pi-pi stacked in a helicoidal (left-handed) arrangement in the central core, and the guanine bases (free from hydrogen bonding) are located at the periphery and exposed to the solvent. The system can be switched back and forth by subsequent addition of chloroform and acetonitrile. The solvent-induced switching can be easily followed by circular dichroism spectroscopy: the CD exciton-couplet in the guanine chromophore absorption region observed in chloroform disappears after addition of acetonitrile, indicating the disassembly of the G-quartet based octameric structure, while an intense quasi-conservative exciton splitting in the 300-450 nm spectral region becomes predominant in the CD spectrum. This latter strong bisignate optical activity can be ascribed to the helical packing of conjugated terthiophene moieties stabilised by pi-pi interactions. NMR spectra and photophysical investigations confirm the structures of the guanine-directed and thiophene-directed assemblies in chloroform and acetonitrile, respectively.