Methyl Resonances Research Articles

1H, 13C, 15N backbone and IVL methyl group resonance assignment of the fungal \u03b2-glucosidase from Trichoderma reesei

β-glucosidases have received considerable attention due to their essential role in bioethanol production from lignocellulosic biomass. β-glucosidase can hydrolyse cellobiose in cellulose degradation and its low activity has been considered as one of the main limiting steps in the process. Large-scale conversions of cellulose therefore require high enzyme concentration which increases the cost. β-glucosidases with improved activity and thermostability are therefore of great commercial interest. The fungus Trichoderma reseei expresses thermostable cellulolytic enzymes which have been widely studied as attractive targets for industrial applications. Genetically modified β-glucosidases from Trichoderma reseei have been recently commercialised. We have developed an approach in which screening of low molecular weight molecules (fragments) identifies compounds that increase enzyme activity and are currently characterizing fragment-based activators of TrBgl2. A structural analysis of the 55 kDa apo form of TrBgl2 revealed a classical (α/β)8-TIM barrel fold. In the present study we present a partial assignment of backbone chemical shifts, along with those of the Ile (I)-Val (V)-Leu (L) methyl groups of TrBgl2. These data will be used to characterize the interaction of TrBgl2 with the small molecule activators.

Degradation and metabolic pathways of sulfamethazine and enrofloxacin in Chlorella vulgaris and Scenedesmus obliquus treatment systems

The degradation and metabolic pathways of sulfamethazine (SMZ) and enrofloxacin (ENR) via microalgal treatment systems were investigated in this study. SMZ and ENR applied at 1-25mgL-1 did not significantly inhibit the growth of Chlorella vulgaris or Scenedesmus obliquus. SMZ and ENR exposure did not significantly alter the maximum quantum efficiencies of C. vulgaris and S. obliquus. When cultured at light intensities of 45-50μmolphotonm-2s-1, the C. vulgaris and S. obliquus treatment systems achieved 24% and 11% degradation, respectively. The greatest removal of ENR was 52% and 43.3%, for C. vulgaris and S. obliquus treatment systems, respectively, after 15days. The results indicated that the degradation of SMZ and ENR occurred by a combination of biodegradation and photolysis. Kinetic investigations revealed that the removal of SMZ and ENR (5mgL-1) followed a first-order model, with apparent rate constants (k) ranging from 0.0141 to 0.0048day-1 and 0.0132 to 0.0086day-1, respectively. Fifteen metabolites of SMZ and five intermediates of ENR were identified by UPLC-MS, and degradation pathways for SMZ and ENR were proposed. SMZ transformation reactions included ring cleavage, hydroxylation, methylation, and oxidation, whereas ENR was degraded by dealkylation, decarboxylation, and defluorination. Graphical abstract.

Synthesis of internally alkylated azuliporphyrins

Examples of internally alkylated azuliporphyrins were prepared by MacDonald-type “3 + 1” condensations. 2-Methyl- and 2-ethylazulene reacted with an acetoxymethylpyrrole in the presence of an acid catalyst to give azulitripyrranes. Following cleavage of the terminal protective groups, condensation with a diformylpyrrole in the presence of hydrochloric acid and oxidation with ferric chloride afforded 21-alkylazuliporphyrins. An azulene dialdehyde similarly reacted with an [Formula: see text]-methyltripyrrane to generate a 23-methylazuliporphyrin. The products could only be isolated in protonated form and the free-base internally alkylated azuliporphyrins proved to be unstable. Nevertheless, the dications are highly diatropic and the internal alkyl group resonances were shifted upfield to beyond -3 ppm. Reaction of a 23-methylazuliporphyrin with palladium(II) acetate primarily afforded a palladium(II) complex with loss of the internal methyl substituent. However, two palladium(II) benzocarbaporphyrins were also identified that were formed by sequential oxidative ring contraction and methyl group migration. Internally alkylated azuliporphyrins provide new insights into the reactivity of the system and the results show that the introduction of alkyl substituents within porphyrinoid cavities greatly modifies the properties of these structures.

Mitochondria-targeted 1,4-naphthoquinone (SkQN) is a powerful prooxidant and cytotoxic agent

An increase in the production of reactive oxygen species (ROS) in mitochondria due to targeted delivery of redox active compounds may be useful in studies of modulation of cell functions by mitochondrial ROS. Recently, the mitochondria-targeted derivative of menadione (MitoK3) was synthesized. However, MitoK3 did not induce mitochondrial ROS production and lipid peroxidation while exerting significant cytotoxic action. Here we synthesized 1,4-naphthoquinone conjugated with alkyltriphenylphosphonium (SkQN) as a prototype of mitochondria-targeted prooxidant, and its redox properties, interactions with isolated mitochondria, yeast cells and various human cell lines were investigated. According to electrochemical measurements, SkQN was more active redox agent and, due to the absence of methyl group in the naphthoquinone ring, more reactive as electrophile than MitoK3. SkQN (but not MitoK3) stimulated hydrogen peroxide production in isolated mitochondria. At low concentrations, SkQN stimulated state 4 respiration in mitochondria, decreased membrane potential, and blocked ATP synthesis, being more efficient uncoupler of oxidative phosphorylation than MitoK3. In yeast cells, SkQN decreased cell viability and induced oxidative stress and mitochondrial fragmentation. SkQN killed various tumor cells much more efficiently than MitoK3. Since many tumors are characterized by increased oxidative stress, the use of new mitochondria-targeted prooxidants may be a promising strategy for anticancer therapy.

Local and global approaches to treat the torsional barriers of 4-methylacetophenone using microwave spectroscopy.

The Fourier transform microwave spectrum of 4-methylacetophenone recorded from 8 GHz to 18 GHz under jet-cooled conditions has revealed large tunneling splittings arising from a low barrier to internal rotation of the ring methyl group and small splittings from a high torsional barrier of the acetyl methyl group. The large splittings are especially challenging to model, while the small splittings are difficult to analyze due to the resolution limit of 120 kHz. The combination of two methyl groups undergoing internal rotations caused each rotational transition to split into five torsional species, which were resolved and fitted using a modified version of the XIAM code and the newly developed ntop code to a root-mean-square deviation close to measurement accuracy, providing an estimate of the V3 potential barriers of about 22 cm-1 and 584-588 cm-1 for the ring and the acetyl methyl groups, respectively. The assignment was aided by separately fitting the five torsional species using odd-power order operators. Only one conformer in which all heavy atoms are located on a symmetry plane could be identified in the spectrum, in agreement with results from conformation analysis using quantum chemical calculations.

Complete assignment of Ala, Ile, LeuProS, Met and ValProS methyl groups of the protruding domain from human norovirus GII.4 Saga

Attachment of human noroviruses to histo blood group antigens (HBGAs) is thought to be essential for infection, although how this binding event promotes infection is unknown. Recent studies have shown that 60% of all GII.4 epidemic strains may undergo a spontaneous post-translational modification (PTM) in an amino acid located adjacent to the binding pocket for HBGAs. This transformation proceeds with an estimated half-life of 1–2 days under physiological conditions, dramatically affecting HBGA recognition. The surface-exposed position of this PTM and its sequence conservation suggests a relevant role in immune escape and host-cell recognition. As a first step towards the understanding of the biological implications of this PTM at atomic resolution, we report the complete assignment of methyl resonances of a MILProSVProSA methyl-labeled sample of a 72 kDa protruding domain from a GII.4 Saga human norovirus strain. Assignments were obtained from methyl–methyl NOESY experiments combined with site-directed mutagenesis and automated assignment. This data provides the basis for a detailed characterization of the PTM-driven modulation of immune recognition in human norovirus on a molecular level.

8-Aminoquinoline-Assisted Synthesis and Crystal Structure Studies of Ferrocenyl Aryl Sulfones.

A copper-catalyzed 8-aminoquinoline-directed oxidative cross-coupling of the C-H bond of ferrocene with sodium arylsulfinates has been achieved. The robust copper catalyst tolerates a range of methyl, tert-butyl, bromo, chloro, iodo and nitro functional groups in the phenyl ring, and set the stage for the synthesis of substituted ferrocene sulfones. Furthermore, X-ray crystal structure study on several ferrocenyl sulfones reveals the tetrahedral geometry around sulfur; interestingly, the O-S-O angle is larger than the electropositive substituent C-S-C angle which could be explained by Bent's rule. Further, unusual intramolecular O(S)⋅⋅⋅N(amide) short contacts (2.925-3) and O(S)⋅⋅⋅C=O were also noticed in ferrocenyl sulfones.

Pyrolysis of Furan and Its Methylated Derivatives: A Shock-Tube/TOF-MS and Modeling Study.

In this study, the pyrolysis of furan (F) and its two methyl-substituted derivatives 2-methylfuran (2-MF) and 2,5-dimethylfuran (2,5-DMF) was investigated behind reflected shock waves at pressures near 1 bar in Ne as the bath gas. Concentration-time profiles of different stable species (reactants, intermediates, and products) were recorded simultaneously with high-repetition time-of-flight mass spectrometry and compared to results from simulations with a recently published, combined F/2-MF/2,5-DMF oxidation mechanism that was slightly modified already in another publication to describe the formation of H atoms in these pyrolysis systems. The temperature ranges covered in our experiments were chosen in line with the different thermal stabilities of the three reactants (F: T = 1050-1920 K; 2-MF: T = 1060-1900 K; 2,5-DMF: T = 1000-1800 K). In general, we obtained satisfactory agreement of the experimental and modeling results. To clarify the most important reaction routes for the formation of the detected species, we performed extensive sensitivity and rate-of-production analyses. The influence of increasing methylation of the furan ring on the formation and consumption of the different species is discussed in detail. Experimental concentration-time profiles are given in tabular form in the Supporting Information to enable tests of future mechanism developments.

Comparison of Solution Properties of Polymethylated DOTA-like Lanthanide Complexes with Opposite Chirality of the Pendant Arms.

Polymethylated lanthanide 4S4R-M4DOTMA complexes, bearing the ring methyl groups oriented in the SSSS position and the arm methyl groups in the RRRR configuration, exist exclusively as the SAP [Λ(δδδδ)] isomer in solution throughout the lanthanide series. This observation is in contrast to Ln-8S-M4DOTMA, which was recently reported to adopt the SAP [Λ(δδδδ)] isomer in the early lanthanides, while the late lanthanides adopt the TSAP [Δ(δδδδ)] isomer. The methyl groups on the ring and the arm are both oriented in the SSSS configuration for Ln-8S-M4DOTMA ( Dalton Trans. 2016 , 45 , 4673 - 4687 , DOI: 10.1039/C5DT03210E ). Quantum chemical calculations for Pr- and Yb-4S4R-M4DOTMA indicate that the SAP isomer is significantly more stable. The luminescence profiles of Eu-8S-M4DOTMA and Eu-4S4R-M4DOTMA showed similar profiles signifying identical coordination environments. The hydration state, q, of the Eu(III) complexes was q = 0.91-0.95, while Tb-8S-M4DOTMA had q = 0.86. A much lower q value was obtained for Tb-4S4R-M4DOTMA (q = 0.67), which indicates an elongation of the Ln-Ow bond. At 400 MHz, the relaxivity of Gd-8S-M4DOTMA is 5.1 ± 0.1 mM-1 s-1 and 3.9 ± 0.1 mM-1 s-1 at 25 and 37 °C, respectively, whereas the relaxivity of Gd-4S4R-M4DOTMA is 4.6 ± 0.1 mM-1 s-1 at 25 °C and 3.6 ± 0.1 mM-1 s-1 at 37 °C. At 45 MHz, the relaxivity of Gd-8S-M4DOTMA is 5.4 ± 0.1 mM-1 s-1, and the relaxivity of Gd-4S4R-M4DOTMA is 4.5 ± 0.1 mM-1 s-1 at 25 °C. The temperature dependence of the 17O NMR transverse relaxation rate of the Gd complexes revealed a 7-fold increase in the bound water residence lifetime of Gd-8S-M4DOTMA (1/kex = τM = 9.0 ± 0.5 ns and 1/kex = τM = 60 ± 3 ns). The Pr(III) complex of 8S-M4DOTMA crystallized as TSAP isomer with an apical water. The presence of the apical water for the TSAP of Pr-8S-M4DOTMA was further confirmed with the observation that the fluoride ion replaces the bound water from the TSAP isomer of Pr-8S-M4DOTMA. This was shown by the disappearance of the TSAP peaks and appearance of a new set of less shifted resonances, which exchange with the SAP isomer as confirmed by NMR exchange spectroscopy (EXSY).

Automatic structure-based NMR methyl resonance assignment in large proteins

Isotopically labeled methyl groups provide NMR probes in large, otherwise deuterated proteins. However, the resonance assignment constitutes a bottleneck for broader applicability of methyl-based NMR. Here, we present the automated MethylFLYA method for the assignment of methyl groups that is based on methyl-methyl nuclear Overhauser effect spectroscopy (NOESY) peak lists. MethylFLYA is applied to five proteins (28–358 kDa) comprising a total of 708 isotope-labeled methyl groups, of which 612 contribute NOESY cross peaks. MethylFLYA confidently assigns 488 methyl groups, i.e. 80% of those with NOESY data. Of these, 459 agree with the reference, 6 were different, and 23 were without reference assignment. MethylFLYA assigns significantly more methyl groups than alternative algorithms, has an average error rate of 1%, modest runtimes of 0.4–1.2 h, and can handle arbitrary isotope labeling patterns and data from other types of NMR spectra.

MAS dependent sensitivity of different isotopomers in selectively methyl protonated protein samples in solid state NMR.

Sensitivity and resolution together determine the quality of NMR spectra in biological solids. For high-resolution structure determination with solid-state NMR, proton-detection emerged as an attractive strategy in the last few years. Recent progress in probe technology has extended the range of available MAS frequencies up to above 100kHz, enabling the detection of resolved resonances from sidechain protons, which are important reporters of structure. Here we characterise the interplay between MAS frequency in the newly available range of 70-110kHz and proton content on the spectral quality obtainable on a 1GHz spectrometer for methyl resonances. Variable degrees of proton densities are tested on microcrystalline samples of the α-spectrin SH3 domain with selectively protonated methyl isotopomers (CH3, CH2D, CHD2) in a perdeuterated matrix. The experimental results are supported by simulations that allow the prediction of the sensitivity outside this experimental frequency window. Our results facilitate the selection of the appropriate labelling scheme at a given MAS rotation frequency.

Synthesis, Structural Elucidation and Anion Binding Studies of Isophthalamide Derivatives as Potential Receptor for Chromate Anions

This study highlights the synthesis and structural elucidation of two new isophthalamide isomers namely N,N’-1,3-bis(4-methylpyridin-2-yl)isophthalamide (4-MPI) and N,N’-1,3-bis(5-methylpyridin-2-yl)isophthalamide (5-MPI), prepared from reaction between isophthaloyl chloride with 2-amino-4-methylpyridine and 2-amino-5-methylpyridine, respectively. Their potential studies as anion receptors for chromate anions are also include in this paper. Characterization of these compounds were accomplished using common spectroscopic techniques such as Fourier Transform Infrared (FT-IR), 1H and 13C Nuclear Magnetic Resonance (NMR), UV-Vis spectrometer and mass spectrometry. FTIR analysis showed the presence of four significant peaks comprising ν(N-H), ν(C-H), ν(C=O) and δ(N-H) at range 3256-3274 cm-1, 2812-2928 cm-1, 1662-1686 cm-1 and 1533-1537 cm-1, respectively. Meanwhile, 1H NMR confirmed the presence of methyl, aromatic carbon and amide resonances at δH 2.983-2.994 ppm, 6.860-8.974 and 10.138-11.300 ppm, respectively. In the 13C NMR spectra, the appearance of methyl, aromatic and carbonyl carbon signals were determined at δC 18.45-20.59 ppm, 108.42-159.22 ppm and 167.04-167.20 ppm, respectively. UV-Vis study showed that 4-MPI and 5-MPI shows promising potential as anion receptors for chromate anions.

Effect of orientation of extra fused benzene ring and lateral methyl substituent on the mesophase behaviour of three-ring azo/ester molecules

ABSTRACTTwo homologous series of the three-ring azo/ester compounds 2-(or1-) naphthyl 4ʹ-(4ʹ’-alkoxy phenylazo) benzoates (Ina and IIna) were synthesised. A lateral methyl group was introduced in different positions of the alkoxy phenyl moiety, in position-2 to give series Inb and IInb and in position-3 to give series Inc and IInc. Molecular structures were characterised via elemental analyses, infrared and 1H-NMR. Their mesophase characteristics were investigated by differential scanning calorimetry (DSC) and their phases identified via polarised light microscopy (PLM). Transition temperatures were correlated with the alkoxy-chain length (n) that varies between 6, 8, 10, 12, 14, and 16 carbons. Comparative studies were first made to investigate the effect of including the extra fused benzene ring, and its orientation, into the previously investigated three-ring compounds, 4-phenyl 4ʹ-(4′′-alkoxyphenylazo) benzoates (IIIna). Investigation of the mesophase behaviour was extended to cover the effect of introducing the lateral methyl group and its position. The comparison between the present six series and their corresponding phenyl analogues IIIna,b,c, indicated that the 2-naphthyl analogues, Ina,b,c, exhibit the highest mesophase stability. Whereas, the steric effect of the protruded naphthalene group destabilises all mesophases and appear only monotropically. The results were discussed in terms of polarisability effect.

Removal and transformation pathways of benzothiazole and benzotriazole in membrane bioreactors treating synthetic municipal wastewater

Lab-scale membrane bioreactors (MBRs), with aerated activated sludge and internal microfiltration module, were used for the treatment of municipal wastewater containing high, yet environmentally relevant, concentrations of benzothiazole (BT) and benzotriazole (BTA). These high production volume compounds are commonly used in the industry and households, and therefore occur ubiquitously in municipal wastewater and the aquatic environment. The aim of this study was to assess the removal of BT and BTA from synthetic municipal wastewater in MBRs and to estimate the contribution of elimination processes and to identify potential biotransformation products. The overall removal of BT and BTA was high, and after the adaptation period, it reached 99.8% and 97.2%, respectively, but recurring periods of unstable BTA removal occurred. The removal due to biotransformation was 88% for BT and 84% for BTA and the disposal with waste sludge accounted for only <1% of the removed load. The remaining fraction of the removed load of BT and BTA was attributed to be retained by phenomena associated with membrane fouling. The adaptation process was reflected in multifold increase in biodegradation kinetic coefficient (kbiol) for BT (reported for the first time) and BTA. Biodegradation was attributed to catabolic mechanism rather than to cometabolism. Hydroxylation was observed to be the main transformation reaction for BT, whereas for BTA hydroxylation, methylation and cleavage of benzene ring were noted. This study has shown the feasibility of treating municipal wastewater with high concentrations of BT and BTA in MBRs and identified potential challenges for the removal of BTA.

Structural Aspects of Glycosylated Protein–Glycan Interactions by NMR

Characterization of glycosylated proteins and their response to interaction with glycans is challenging for any method, because of the heterogeneity of glycosylation and the extensive conformational sampling by most glycans. For NMR there are extra challenges because expression in mammalian cell cultures, which can preserve near‐native glycosylation, requires expensive substrates to achieve uniform isotopic labeling with 15N or 13C, and mammalian cells do not tolerate high levels of deuteration (something required to improve spectral resolution). We have developed a sparse‐labeling approach that preserves high resolution without deuteration, proves to be economical in most applications, and returns enough structural data to define geometries of multiple domain proteins and ligand protein complexes. The approach is based on labeling proteins with a single type of amino acid isotopically labeled in methyl groups with 13C (13C‐valine or 13C alanine). NMR signals from methyl groups are special in terms of the sensitivity and resolution that they provide. The addition of paramagnetic tags to specific sites in a protein provides long range distance and angular information on the location of labeled methyl groups. In combination with a newly developed approach to assign methyl resonances to specific sites in a protein, this information proves adequate to define relative domain orientations and facilitate the identification of ligand binding sites. Application to terminal domains from a cell surface signaling molecule, ROBO1, is used to illustrate the approach. It also provides an example of how binding of a heparan sulfate oligomer alters the relative orientation of domains.Support or Funding InformationThis work was supported by grants from the National Institutes of Health, P41GM103390 and R01GM033225.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Properties and Edition of NMR Spontaneous Maser Emission Spectra

Spontaneous maser emission provides the practical possibility for a spectral edition in methyl chloroacetate. Methyl and methylene resonances opposite to conventional nuclear magnetic resonance spectra are clearly separated within the time domain of free induction decay. Associated characteristic time delay, which represents the appropriate time from completion of the radiofrequency pulse to the signal maximum, can be properly broken on a random and a nonrandom part. Overall, the time delay is distinctively shorter about 0.296 s for methyl and longer about 0.489 s for methylene peaks, enabling spectra edition. Although maser emission reaches maximum for pulse rotation angle at 180°, it can be observed in a significantly broader range. The maser emission reveals random intensity, and its scattering range depends on the pulse rotation angle and is most limited when rotation angle is within direct vicinity of 180°. The spontaneous maser emission is triggered by thermal Nyquist noise through radiation damping. If the pulse rotation angle is sufficiently small, the amplified spin noise is observed.

Bioactivity and Structural Properties of Novel Synthetic Analogues of the Protozoan Toxin Climacostol.

Climacostol (5-[(2Z)-non-2-en-1-yl]benzene-1,3-diol) is a resorcinol produced by the protozoan Climacostomum virens for defence against predators. It exerts a potent antimicrobial activity against bacterial and fungal pathogens, inhibits the growth of several human and rodent tumour cells, and is now available by chemical synthesis. In this study, we chemically synthesized two novel analogues of climacostol, namely, 2-methyl-5 [(2Z)-non-2-en-1-yl]benzene-1,3-diol (AN1) and 5-[(2Z)-non-2-en-1-yl]benzene-1,2,3-triol (AN2), with the aim to increase the activity of the native toxin, evaluating their effects on prokaryotic and free-living protists and on mammalian tumour cells. The results demonstrated that the analogue bearing a methyl group (AN1) in the aromatic ring exhibited appreciably higher toxicity against pathogen microbes and protists than climacostol. On the other hand, the analogue bearing an additional hydroxyl group (AN2) in the aromatic ring revealed its ability to induce programmed cell death in protistan cells. Overall, the data collected demonstrate that the introduction of a methyl or a hydroxyl moiety to the aromatic ring of climacostol can effectively modulate its potency and its mechanism of action.

Invertion and methylation of pyrrole ring in tetrasulfophenylporphyrin: basicity, aggregation properties, chirality

Equilibria of methylated N-confused platform of 2-N-methyl-5,10,15,20-tetrakis-(4′-sulfophenyl)-2-aza-21-carbaporphyrin tetraanion HMeIP(PhSO3H)4 platform diprotonation by perchloric acid in water, H– and J-aggregates self-assembly were investigated using synchronous UV–Vis-fluorescence-pH titration and DFT/B3LYP/6-31++G(d,p) calculations. Methylation of inverted pyrrole ring in H2IP(PhSO3−)4 allows to fix the tautomer HMeIP(PhSO3−)4 with external NH-proton. Intramolecular tightness causes formation of two bifurcated NHN IMHB between pyrrole hydrogen and pyrrolenine nitrogen of platform HMeIP(PhSO3−)4. IMHB protect intramolecular centers of hydrogen bonding from intermolecular interactions. First stage of HМеIP(PhSO3−)4 protonation is accompanied by bifurcated IMHB type change from NHN to HNH. On the second protonation stage IMHB are broken and intermolecular hydrogen bonds with solvent molecules are formed. Diprotonated H3МеIP2+(PhSO3−)4 platform is a flexible 1,3-alternate, possessed molecular and anion receptor properties. Equilibrium in the second stage of protonation in water is completely shifted to the aquacomplex, which is formed due to hydrogen and electrostatic binding of the solvent molecules at the both receptor sites. Methylation and invertion of pyrrole ring leads to HMeIP(PhSO3−)4 proton affinity increasing and eliminated the phenomena of synchronous H2P(PhSO3−)4 porphyrin platform diprotonation, which is due to more stable aquacomplex [H4P2+(PhSO3−)4](H2O)2 formation. Initial tetraanion HMeIP(PhSO3−)4 and aquacomplex [H4IP2+(PhSO3−)4](H2O)2 are monomers of H- and J-aggregates self-assembly. Driving force of H-aggregates formation is the ionic self-assembly between sulfonate groups of monomers and spacer cations. J-aggregates self-assembly is due to formation of more stable anion complexes as a result of water molecules intermolecular substitution in [H3MeIP2+(PhSO3−)4](H2O)2 by monomers sulfonate groups. Protonated chitosan was used as a inducer and chiral scaffold of CW-H- and tubular CW-J-aggregates (porphyrin nanotubes) self-assembly. Parameters of CW-H- and CW-J-dimers geometry were calculated and 3D models of H-aggregates and porphyrin nanotubes were developed on their basis.

N-Methylation of ortho-substituted aromatic amines with methanol catalyzed by 2-arylbenzo[d]oxazole NHC-Ir(iii) complexes.

Seven new chelated cyclometalated Ir complexes of ABON,P, ABON,O, and ABON,C(carbene) based on a rigid and tunable 2-arylbenzo[d]oxazole backbone have been prepared for the N-methylation of amines. Among these three coordinated modes, ABON,C(carbene)-chelated iridium-based catalysts exhibited good performance in the monomethylation of aromatic amines with methanol (MeOH) as the green methylation reagent. The steric-modified synthesis of ABON,C(carbene) complexes was described. The most active ABON,C(carbene) complex with marginal steric hindrance as a catalyst was obtained from the benzoxazole ring without a substituent and methyl group of the benzimidazole ring on the N-heterocyclic carbene (NHC) ligand. A variety of amines including para- and meta-substituted aromatic amines, as well as heterocyclic amines, were formulated as suitable substrates. Importantly, this catalyst considerably promoted the yield of the N-methylation of ortho-substituted aromatic amines. Controlled kinetic experiments and deuterium-labeling reactions of these ortho-substituted amines were conducted under optimized conditions. On the basis of the experimental results, a plausible mechanism was proposed.

Combining Evolutionary Covariance and NMR Data for Protein Structure Determination.

Accurate protein structure determination by solution-state NMR is challenging for proteins greater than about 20kDa, for which extensive perdeuteration is generally required, providing experimental data that are incomplete (sparse) and ambiguous. However, the massive increase in evolutionary sequence information coupled with advances in methods for sequence covariance analysis can provide reliable residue-residue contact information for a protein from sequence data alone. These "evolutionary couplings (ECs)" can be combined with sparse NMR data to determine accurate 3D protein structures. This hybrid "EC-NMR" method has been developed using NMR data for several soluble proteins and validated by comparison with corresponding reference structures determined by X-ray crystallography and/or conventional NMR methods. For small proteins, only backbone resonance assignments are utilized, while for larger proteins both backbone and some sidechain methyl resonance assignments are generally required. ECs can be combined with sparse NMR data obtained on deuterated, selectively protonated protein samples to provide structures that are more accurate and complete than those obtained using such sparse NMR data alone. EC-NMR also has significant potential for analysis of protein structures from solid-state NMR data and for studies of integral membrane proteins. The requirement that ECs are consistent with NMR data recorded on a specific member of a protein family, under specific conditions, also allows identification of ECs that reflect alternative allosteric or excited states of the protein structure.