Restricted Rotational Freedom Research Articles

Aggregation-Induced Emission-Based Chemodosimeter Approach for Selective Sensing and Imaging of Hg(II) and Methylmercury Species.

Methylmercury (CH3Hg+) is the common form of organic mercury and is more toxic than its inorganic or elemental forms. Mercury is emanated in the course of various natural events and human activities and converts to methylmercury by anaerobic organisms. CH3Hg+ are ingested by fish and subsequently bioaccumulated in their tissue and, eventually, enter the human diet, causing serious health issues. Therefore, selective and sensitive detection of bioaccumulated CH3Hg+ in fish samples is essential. Herein, the development of a simple, highly sensitive and selective aggregation-induced emission (AIE)-based turn-on probe for both inorganic mercury ions and organicmercury species is reported. The probe's function is based on mercury ion-promoted transmetalation reaction of aryl boronic acid. The probe, a tetraphenylethylene (TPE)-monoboronic acid (1), was successfully utilized for AIE-based fluorescence imaging study on methylmercury-contaminated live cells and zebrafish for the first time. Both Hg(II) and CH3Hg+ ensued a fast transmetalation of TPE-boronic acid causing drastic reduction in the solubility of the resulting product (TPE-HgCl/TPE-HgMe) in the working solvent system. At the dispersed phase, the aggregated form of TPE-mercury ions recovers planarity because of restricted rotational freedom promoting aggregation-induced emission. Simple design, cost-effective synthesis, high selectivity, inexpensive instrumentation, fast signal transduction, and low limit of detection (0.12 ppm) are some of the key merits of this analytical tool.

Amphiphilic Ditopic Bis-Aqua Gd-AAZTA-like Complexes Enhance Relaxivity of Lipidic MRI Nanoprobes.

Two amphiphilic mono- and dimeric GdAAZTA-like chelates composed of stable bis-aquo Gd(III) complexes (q=2) linked to one (for the monomer) or two dodecyl aliphatic chains (for the dimer) were synthesized. Both chelates showed high relaxivity when incorporated into the lipid bilayer of liposomes or after interaction with human serum albumin (HSA). The ditopic complex shows a significantly decreased internal motion relative to the monomeric complex, associated with an enhanced relaxivity (r1 ≈60 mm(-1) s(-1) , at 30 MHz and 310 K). The presence of two metal-bound water molecules in fast exchange and the restricted rotational freedom make the relaxivity of this system the highest measured for paramagnetic liposomes.

Geminally Substituted Tris(acenaphthyl) and Bis(acenaphthyl) Arsines, Stibines, and Bismuthine: A Structural and Nuclear Magnetic Resonance Investigation.

Tris(acenaphthyl)- and bis(acenaphthyl)-substituted pnictogens (iPr2P-Ace)3E (2-4) (E = As, Sb, or Bi; Ace = acenaphthene-5,6-diyl) and (iPr2P-Ace)2EPh (5 and 6) (E = As or Sb) were synthesized and fully characterized by multinuclear nuclear magnetic resonance (NMR), high-resolution mass spectrometry, elemental analysis, and single-crystal X-ray diffraction. The molecules adopt propeller-like geometries with the restricted rotational freedom of the sterically encumbered iPr2P-Ace groups resulting in distinct NMR features. In the tris(acenaphthyl) species (2-4), the phosphorus atoms are isochronous in the (31)P{(1)H} NMR spectra, and the rotation of the three acenaphthyl moieties around the E-Cipso bond is locked. On the other hand, the bis(acenaphthyl) species show a fluxional behavior, resulting in an AX to A2 spin system transition in the (31)P{(1)H} variable-temperature NMR spectra. This allowed elucidation of remarkable through-space couplings ((8TS)JPP) of 11.5 Hz (for 5) and 25.8 Hz (for 6) at low temperatures. In addition, detailed line shape analysis of the thermodynamic parameters of the restricted rotation of the "propeller blades" in 5 was performed in the intermediate temperature region and also at coalescence. The lone pairs on the pnictogen atoms in 2-6 are oriented such that they form a bowl-shaped area that is somehow buried within the molecule.

Di-μ-oxo Dimetal Core of MnIV and TiIV as a Linker Between Two Chiral Salen Complexes Leading to the Stereoselective Formation of Different M- and P-Helical Structures

Because of restricted rotational freedom along the metal-metal axis, a di-μ-oxo dimetal core could be an excellent building block to create dinuclear compounds with well-defined stereochemistry, but their stereoselective synthesis remains a challenge. We herein report the formation of di-μ-oxo dimanganese(IV) complexes with tetradentate salen ligands bearing different degrees of steric bulk, in order to study stereochemical aspects of the dimerization reaction that potentially generates multiple stereoisomers. X-ray crystallography shows that the di-μ-oxo dimanganese(IV) complex with salen, where salen is (R,R)-N,N'-bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexanediamine, adopts a unique structure in which two salen complexes are arranged in an M-helical fashion. According to the solution study using (1)H, (2)H NMR, and circular dichroism spectroscopies, the dimerization reaction is highly diastereoselective in the presence of the tert-butyl group at the 3/3' position as a determinant steric factor. In contrast, the di-μ-oxo dititanium(IV) complex with the same salen ligand was previously reported to afford an opposite P-helical dimer. The present DFT study clarifies that a less-covalent Ti-O bonding causes a distortion of the di-μ-oxo dititanium(IV) core structure, generating a completely different framework for interligand interaction. The present study provides a solid basis to understand the stereochemistry for the formation of the di-μ-oxo dimetal core.

N‐Heterocyclic Carbenes Functioning as Monoligating Clamps

AbstractBenzimidazolium salts N,N′‐disubstituted with 9‐alkylfluorenyl groups (3a–e, alkyl = methyl, ethyl, propyl, butyl, benzyl) have been synthesised in high yields in three steps from o‐phenylenediamine. This amine was treated with fluorenone in the presence of TiCl4 and tetramethylethylenediamine (TMEDA) to form N,N′‐bis(9H‐fluoren‐9‐ylidene)benzene‐1,2‐diamine (1) in 91 % yield. Diamines 2a–e were then obtained in yields superior or equal to 77 % by reacting diimine 1 with the appropriate organolithium reagent. In the final step, diamines 2a–e were treated with ethylorthoformate under acidic conditions to afford benzimidazolium salts 3a–e. These were readily converted into the PEPPSI palladium complexes 4a–e (PEPPSI = pyridine‐enhanced precatalyst preparation stabilisation and initiation). NMR and X‐ray diffraction studies revealed that the flat fluorenylidene moiety orientates the alkyl groups towards the metal centre and because of its restricted rotational freedom makes the ligand bulkiness time independent. Thus, the metal centre is permanently confined between the two alkyl groups, and thereby forms a monoligating clamp with the carbenic centre. The CH2 groups close to the palladium ion give rise to anagostic C–H···Pd interactions. Catalytic tests revealed that the palladium complexes 4a–e are highly efficient in Suzuki–Miyaura cross‐coupling reactions; their activity is equal or superior to the best PEPPSI catalysts reported to date.

3,4-Ethylenedioxythiophene in planarizable push–pull oligothiophenes

We report design, synthesis and evaluation of push-pull quaterthiophene amphiphiles containing one 3,4-ethylenedioxythiophene (EDOT) and a single strong twist in the scaffold. Planarizable push-pull oligothiophene amphiphiles have been introduced recently as conceptually innovative fluorescent probes that sense the fluidity and the potential of lipid bilayer membranes. The "hyper-twisted" EDOT probes respond to planarization and restricted rotational freedom with a red shift and changes in vibrational finestructure in the excitation spectrum, respectively. In solution, comparably weak solvatochromism and significant thermochromism are found. Planarization and restricted rotational freedom afford exquisite sensitivity toward nature and fluidity of lipid bilayer membranes, including ratiometric detection of phase transitions. The sensing of membrane potentials is weakened by these unique properties but remains possible.

Application of the Nano-Positioning System to the Analysis of Fluorescence Resonance Energy Transfer Networks

Single-molecule fluorescence resonance energy transfer (sm-FRET) has been recently applied to distance and position estimation in macromolecular complexes. Here, we generalize the previously published Nano-Positioning System (NPS), a probabilistic method to analyze data obtained in such experiments, which accounts for effects of restricted rotational freedom of fluorescent dyes, as well as for limited knowledge of the exact dye positions due to attachment via flexible linkers. In particular we show that global data analysis of complete FRET networks is beneficial and that the measurement of FRET anisotropies in addition to FRET efficiencies can be used to determine accurately both position and orientation of the dyes. This measurement scheme improves localization accuracy substantially, and we can show that the improvement is a consequence of the more precise information about the transition dipole moment orientation of the dyes obtained by FRET anisotropy measurements. We discuss also rigid body docking of different macromolecules by means of NPS, which can be used to study the structure of macromolecular complexes. Finally, we combine our approach with common FRET analysis methods to determine the number of states of a macromolecule.

Photoisomerization of a Capped Azobenzene in Solution Probed by Ultrafast Time-Resolved Electronic Absorption Spectroscopy

Ultrafast time-resolved electronic absorption spectroscopy has been used to study the photochemistry of trans-azobenzene and trans-1, a derivative in which azobenzene is capped by an azacrown ether, on UV excitation to the S2(ππ*) state. Excitation of trans-1 results in transient absorption which decays with a dominant component of lifetime ca. 2.6 ps and in bleaching of the ground-state UV absorption band which recovers on a similar time scale. In contrast, excitation of trans-azobenzene results in transient absorption which decays with a dominant component with a shorter lifetime of ca. 1 ps, and in bleaching which recovers on a much longer time scale of ca. 18 ps. The recovery of the ground-state UV absorption band is not complete in either case, and the ultrafast data indicate that the quantum yield of trans-to-cis photoisomerization of 1 is approximately twice that of azobenzene. These observations demonstrate that the restricted rotational freedom of the phenyl groups in trans-1 has a significant ef...

Spectroscopic studies of the interfacial binding of Humicola lanuginosa lipase†

The interaction of Humicola lanuginosa lipase (HLL) with small unilamellar vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG) and in the presence of tributyrin (TB) as a substrate was studied by the use of steady-state fluorescence techniques. An inactive mutant with the serine from the catalytic triad changed by alanine (S146A) was used in experiments with TB to avoid interferences from product formation. HLL binds to POPG vesicles in an active or open form for the catalytic turnover, and therefore POPG provides a suitable system for studying the conformational changes involving the movement of the loop of amino acids that covers the active site of the enzyme in solution. Tryptophan (Trp) fluorescence shows that HLL binding to POPG occurs with a change in the environment of Trp residue(s) and that there is only one type of bound form, even in the presence of TB. Accessibility to aqueous quenchers indicates shielding of Trp in the membrane. Fluorescence anisotropy of the enzyme increases on binding to the vesicles, indicating restricted rotational freedom for the Trp due to penetration in the bilayer. Resonance energy transfer experiments using an interfacial membrane probe, 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene p-toluenesulfonate (TMA-DPH), and an internal membrane probe, 1,6-diphenylhexa-1,3,5-triene (DPH), indicate that HLL does not penetrate very deeply in the hydrophobic core of the membrane, but preferentially stays close to the lipid interface. Addition of substrate (TB) does not result in any additional changes in the spectroscopic properties of HLL. It is suggested that the observed changes are due to the ‘opening of the lid’ on binding to POPG vesicles, leaving the active site accessible for the substrate to bind.

Fluorescent labeling of the nucleotide site in cytosolic rat liver phosphoenolpyruvate carboxykinase

Reaction of rat liver phosphoenolpyruvate carboxykinase (GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) with the alkylating fluorescent probe N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (1,5-I-AEDANS), results in complete loss of enzymatic activity. One mole of the fluorescent reagent is incorporated per mole of the inactivated enzyme. When the modification is carried out in the presence of GDPMn, the enzyme retains 97% of its activity with almost no incorporation of label. The specificity of the reaction is further supported by the detection of a unique fluorescent peptide from the trypsin-treated modified enzyme. Fluorescence emission of enzyme-bound AEDANS shows a broad band centered at 470 nm and presents a monoexponential decay with a lifetime of 19 ns. These data indicate that the probe-binding site is considerably less polar than water and similar in polarity to ethanol. Anisotropy determinations give evidence for restricted rotational freedom for AEDANS bound to the rat carboxykinase, while acrylamide quenching studies reveal limited accessibility to the probe site. The results are consistent with specific labeling of rat liver phosphoenolpyruvate carboxykinase at or near the GDP site. The characteristics of the nucleotide-binding sites of rat liver and yeast (ATP) phosphoenolpyruvate carboxykinase are compared.

Synthesis, n.m.r., and conformational studies of some 3,4-di-O-glycopyranosyl- substituted methyl α-D-galactopyranosides

Methyl α-D-galactopyranosides substituted in the 3- and 4-positions with one L-fucopyranosyl and one D-glucopyranosyl group have been synthesized, with all anomeric combinations. The trisaccharides were used for 1H and 13C n.m.r. studies. All 1H and 13C n.m.r, resonances were assigned and comparison was made between the observed glycosylation shifts of the trisaccharides and those calculated by using the glycosylation shifts for each disaccharide element. Large deviations, in most cases upfield shifts, were found in the 13C n.m.r. spectra for signals from some of the linkage carbons. Conformational analysis has been performed using the HSEA and GESA approaches. This analysis indicated restricted rotational freedom around the glycosidic bonds of the trisaccharides relative to those of the disaccharides. A number of proton-oxygen and proton-proton interactions in the trisaccharides was indicated which could be correlated to downfield and upfield shifts, respectively, of the proton signals.

N.m.r. and conformational studies of some 3-O-, 4-O-, and 3,4-di-O-glycopyranosyl-substituted methyl α-D-galactopyranosides

Both 1H and 13C n.m.r. studies and conformational analyses have been performed on methyl α-D-galactopyranosides substituted in the 3-, 4-, and 3,4-positions with different anomeric forms of either L-fucopyranosyl or D-glucopyranosyl groups. Conformational analysis using the HSEA and GESA approaches indicated restricted rotational freedom around the glycosidic bonds of the trisaccharides compared to those of the disaccharides. It also indicated a number of proton-oxygen and proton-proton interactions which could be correlated to downfield and upfield glycosylation shifts, respectively, of the proton signals in both the di- and the tri-saccharides. Similar inter-residue atomic interactions could also be correlated to the 13C n.m.r. glycosylation shifts observed for the disaccharides and to some extent for the trisaccharides. Comparison was made between the observed glycosylation shifts of the trisaccharides and those calculated by using the glycosylation shifts for each disaccharide element. Large deviations, in most cases upfield shifts, were found for the signals for many linkage carbons.

Diastereotopic effect of the methylene protons in N-ortho-substituted-N-β-cyanoethylanilides: X-ray structure of N-(β-cyanoethyl)-2′-chloromaleanilic acid

Some N-β-cyanoethyl-ortho-substituted anilides show a diastereotopic coupling effect of the methylene protons of the N-β-cyanoethyl chain in the 1H n.m.r. spectra. Resolution of those proton coupling systems have been done by computational methods and their analyses indicate that in chloroform solution the gauche conformation is preferred in all those compounds. The solid-state X-ray analysis of (2) also shows that this is in a special gauche conformation. The diasterotopic effect can be seen as restricted rotational freedom of the alkyl chain in ortho-substituted compounds. However, an internal linkage between an N-α-proton of the chain and the CO amide group seems to be the common anchorage of the α-methylene, while the β-methylene protons are fixed by steric and dipole effects.

Phosphorus 31 nuclear magnetic resonance study of tryptophanase. Pyridoxal phosphate-binding site.

The pyridoxal phosphate-dependent enzyme tryptophanase has been investigated using 31P nuclear magnetic resonance at 72.86 MHz. In the native enzyme, the pyridoxal-P 31P chemical shift was found to be 3.55 ppm and independent of pH, indicating that the dianionic phosphate group of the cofactor is not accessible to solvent. Binding of the competitive inhibitor, beta-phenyl-DL-serine, results in the formation of the transaldimination complex. This complex is fixed to the enzyme via the dianionic phosphate group of the cofactor: again the observed shift is independent of pH. In both cases, restricted rotational freedom of the phosphate group around the C-O bond linking the phosphate ester to the pyridine moiety of the cofactor could be asserted from line width data. Addition of the competitive inhibitor, L-alanine, to tryptophanase produces the quinonoid intermediate. The phosphate group of this complex has lost its specific interaction (probably a salt bridge) with the protein, as indicated by the pH dependence of the chemical shift.

Isomerization of n-butenes on A-type zeolites studied by infrared spectroscopy. Part 1.—n-butene adsorption on zeolites containing alkali and alkaline earth cations

Adsorption of n-butenes on highly activated type A-zeolites containing alkali and alkaline earth cations is studied by infrared spectroscopy. The rate of adsorption is determined by the butene–zeolite interaction, depending on the dipole moment and the steric requirements of the butene molecules, and the occupancy of the cation sites.The n-butenes are strongly physisorbed without fragmentation with preservation of the CC bond. The adsorption is accompanied by the appearance of forbidden i.r. fundamentals, small frequency shifts, a change in band intensities and a decrease in band widths. Narrow bands point to a largely restricted rotational freedom. The frequency shift of the CC band is cation sensitive and shows a direct polarizing power dependence. Under formation of a π-complex the butenes are attached to the cations in a “side on” mode. In the case of the alkali and alkaline earth forms isomerization is not affected. In the far infrared region phonon induced absorptions are observable.

A Conformational Study of Porcine Thyrocalcitonin

Abstract The structure of porcine thyrocalcitonin has been evaluated by circular dichroism, optical rotatory dispersion, infrared spectroscopy, and fluorescence. The degree of helical structure was estimated by circular dichroism (222 mµ) and optical rotatory dispersion (233 mµ) at the n → π* transition of the α helix. The optical activity at this transition suggests that thyrocalcitonin contains approximately 10% α-helical structure in aqueous solution. The spectrum of thyrocalcitonin in 6 m guanidine was similar to that of a randomly coiled polypeptide. In 2-chloroethanol, thyrocalcitonin formed a structure containing approximately 50% α helix. The near ultraviolet circular dichroic spectrum of thyrocalcitonin revealed a major band at 288 mµ, indicating that the tryptophanyl chromophore had restricted rotational freedom. Reduction and alkylation of the amino-terminal heptapeptide ring of thyrocalcitonin produced no significant change in the circular dichroic or optical rotatory spectra. In addition, the effects of alkaline pH and temperature on tryptophanyl emission were similar to the unmodified hormone. These findings preclude a high degree of organized structure in the heptapeptide ring, which is in marked contrast to the amino-terminal hexapeptide ring of oxytocin. These studies indicate that porcine thyrocalcitonin exists predominately in a random coil in aqueous solution. The polypeptide, however, does not appear to possess a rigid conformation and a coil ⇌ helix equilibrium may exist in water with guanidine shifting the equilibrium in favor of the coil and 2-chloroethanol in favor of the helix. The latter type of transition may occur at the receptor site of the hormone in lipid layers or cellular membranes where the water concentration is significantly reduced.

Structural Studies on Polypeptide Hormones: II. POLARIZATION OF FLUORESCENCE

The rotational characteristics of polypeptides containing a tryptophanyl residue have been evaluated by polarization of fluorescence measurements in concentrated glycerol solutions. A curve has been obtained showing the dependence of relaxation time on chain size based on the behavior of two series of peptides, i.e. Glyx−Trp−Glyy and adrenocorticotropic hormone and some of its fragments. If these molecules are structureless polypeptides then it can be ascertained, from the relaxation times of molecules with unknown structures, whether the indole group possesses unrestricted or restricted rotational freedom. In the latter case, one can conclude that there exist some elements of structure in the molecule. Several hormones and polypeptides (gastrin, glucagon, parathyroid hormone, and mellitin) and some of their fragments have been evaluated for tertiary interactions by this procedure.