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Inherently chiral compounds, such as calixarenes, are chiral due to a nonplanar three-dimensional (3D) structure. Determining their conformation is essential to understand their properties, with nuclear magnetic resonance (NMR) spectroscopy being one applicable method. Using alignment media to measure residual dipolar couplings (RDCs) to obtain structural information is advantageous when classical NMR parameters like the nuclear Overhauser effect (NOE) or J-couplings fail. Besides providing more accurate structural information, the alignment media can induce different orientations ofâ¯enantiomers. In this study, we examined the ability of polyglutamates with different side-chain moietiesâpoly-γ-benzyl-l-glutamate (PBLG) and poly-γ-p-biphenylmethyl-l-glutamate (PBPMLG) âto enantiodifferentiate the inherently chiral phenoxathiin-based thiacalix[4]arenes. Both media, in combination with two solvents, allowed for enantiodiscrimination, which was, to the best of our knowledge, proved for the first time on inherently chiral compounds. Moreover, using the experimental RDCs, we investigated the calix[4]arenes conformational preferences in solution, quantitatively analyzed the differences in the alignment of the enantiomers, and discussed the pitfalls of the use of the RDC analysis.
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We performed mutagenesis on a regular isoprenyl diphosphate synthase (IDS), neryl diphosphate synthase from Solanum lycopersicum (SlNPPS), that has a structurally related analogue performing non-head-to-tail coupling of two dimethylallyl diphosphate (DMAPP) units, lavandulyl diphosphate synthase from Lavandula x intermedia (LiLPPS). Wild-type SlNPPS catalyses regular coupling of isopentenyl diphosphate (IPP) and DMAPP in cis-orientation resulting in the formation of neryl diphosphate. However, if the enzyme is fed with DMAPP only, it is able to catalyse the coupling of two DMAPP units and synthesizes two irregular monoterpene diphosphates; their structures were elucidated by the NMR analysis of their dephosphorylation products. One of the alcohols is lavandulol. The second compound is the trans-isomer of planococcol, the first example of an irregular cyclobutane monoterpene with this stereochemical configuration. The irregular activity of SlNPPS constitutes 0.4 % of its regular activity and is revealed only if the enzyme is supplied with DMAPP in the absence of IPP. The exchange of asparagine 88 for histidine considerably enhanced the non-head-to-tail coupling. While still only observed in the absence of IPP, irregular activity of the mutant reaches 13.1 % of its regular activity. The obtained results prove that regular IDS are promising starting points for protein engineering aiming at the development of irregular activities and leading to novel monoterpene structures.
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Alquil e Aril Transferases/metabolismo , Monoterpenos/metabolismo , Engenharia de Proteínas , Solanum lycopersicum/enzimologia , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Monoterpenos/químicaRESUMO
Residual dipolar couplings (RDCs) become increasingly important as additional NMR parameters in the structure elucidation of organic compounds but are usually used in fitting procedures to discriminate between (computed) structures that are in accordance with RDCs and others that can be ruled out. Thus, the determination of configurations requires prior structural information. The direct use of RDCs as restraints to construct structures based on RDCs has only recently begun also in organic compounds. No protocol has been published though that uses the vector and dynamics information available in multialignment data sets directly for the joint determination of conformation and configuration of organic compounds. This is proposed in the current study. We show that by employing these data, even a flat or random start structure converges into the correctly configured structure when employing multiple alignment data sets in our iterative procedure. The requirements in terms of the number of RDCs and alignment media necessary are discussed in detail.
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Compostos Orgânicos , Espectroscopia de Ressonância Magnética , Conformação Molecular , Ressonância Magnética Nuclear Biomolecular , TitânioRESUMO
Residual dipolar couplings (RDCs) contain information on the relative arrangement and dynamics of internuclear spin vectors in chemical compounds. Classically, RDC data is analyzed by fitting to structure models, while model-free approaches (MFA) directly relate RDCs to the corresponding internuclear vectors. The recently introduced software TITANIA implements the MFA and extracts structure and dynamics parameters directly from RDCs to facilitate de novo structure refinement for small organic compounds. Encouraged by our previous results on simulated data, we herein focus on the prerequisites and challenges faced when using purely experimental data for this approach. These concern mainly the fact that not all couplings are accessible in all media, leading to voids in the RDC matrix and the concomitant effects on the structure refinement. It is shown that RDC data sets obtained experimentally from currently available alignment media and measurement methods are of sufficient quality to allow relative configuration determination even when the relative configuration of the analyte is completely unknown.
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Residual dipolar couplings (RDCs) are amongst the most powerful NMR parameters for organic structure elucidation. In order to maximize their effectiveness in increasingly complex cases such as flexible compounds, a maximum of RDCs between nuclei sampling a large distribution of orientations is needed, including sign information. For this, the easily accessible one-bond 1 H-13 C RDCs alone often fall short. Long-range 1 H-1 H RDCs are both abundant and typically sample highly complementary orientations, but accessing them in a sign-sensitive way has been severely obstructed due to the overflow of 1 H-1 H couplings. Here, we present a generally applicable strategy that allows the measurement of a large number of 1 H-1 H RDCs, including their signs, which is based on a combination of an improved PSYCHEDELIC method and a new selective constant-time ß-COSY experiment. The potential of 1 H-1 H RDCs to better determine molecular alignment and to discriminate between enantiomers and diastereomers is demonstrated.
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Plants are versatile chemists producing a tremendous variety of specialized compounds. Here, we describe the engineering of entirely novel metabolic pathways in planta enabling generation of halogenated indigo precursors as non-natural plant products. Indican (indolyl-ß-D-glucopyranoside) is a secondary metabolite characteristic of a number of dyers plants. Its deglucosylation and subsequent oxidative dimerization leads to the blue dye, indigo. Halogenated indican derivatives are commonly used as detection reagents in histochemical and molecular biology applications; their production, however, relies largely on chemical synthesis. To attain the de novo biosynthesis in a plant-based system devoid of indican, we employed a sequence of enzymes from diverse sources, including three microbial tryptophan halogenases substituting the amino acid at either C5, C6, or C7 of the indole moiety. Subsequent processing of the halotryptophan by bacterial tryptophanase TnaA in concert with a mutant of the human cytochrome P450 monooxygenase 2A6 and glycosylation of the resulting indoxyl derivatives by an endogenous tobacco glucosyltransferase yielded corresponding haloindican variants in transiently transformed Nicotiana benthamiana plants. Accumulation levels were highest when the 5-halogenase PyrH was utilized, reaching 0.93⯱â¯0.089â¯mg/g dry weight of 5-chloroindican. The identity of the latter was unambiguously confirmed by NMR analysis. Moreover, our combinatorial approach, facilitated by the modular assembly capabilities of the GoldenBraid cloning system and inspired by the unique compartmentation of plant cells, afforded testing a number of alternative subcellular localizations for pathway design. In consequence, chloroplasts were validated as functional biosynthetic venues for haloindican, with the requisite reducing augmentation of the halogenases as well as the cytochrome P450 monooxygenase fulfilled by catalytic systems native to the organelle. Thus, our study puts forward a viable alternative production platform for halogenated fine chemicals, eschewing reliance on fossil fuel resources and toxic chemicals. We further contend that in planta generation of halogenated indigoid precursors previously unknown to nature offers an extended view on and, indeed, pushes forward the established frontiers of biosynthetic capacity of plants.
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Índigo Carmim/metabolismo , Nicotiana/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/genética , Nicotiana/genéticaRESUMO
Poly(aspartic acid esters) are known to form either right-or left-handed α-helices depending on the ester group in the side chain, on solvent and/or on temperature. Polyphenethyl-l-aspartates (PPLA) exhibit a helix reversal from the right- to the left-handed form with increasing temperature. We have recently reported the application of polyphenethylaspartates as helically chiral alignment media. The thermoresponsivity observed for these polymers offers the possibility to measure different orientations of analytes before and after helix reversal of the alignment medium at 373â K. Herein we present a synthesized copolymer of phenethyl- and benzylaspartate as a new alignment medium undergoing this helix reversal at 303-313â K. Thus, the measurement of residual dipolar couplings (RDC) before and after the helix reversal is allowed for at ambient temperatures. A complete sign change of all 1 H-13 C RDCs was observed, which is close to the highest possible difference in NMR spectra.
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Ácido Aspártico/análogos & derivados , Ácido Aspártico/química , Espectroscopia de Ressonância Magnética/métodos , Poliésteres/química , Temperatura , Anisotropia , Conformação Molecular , Solventes , TermodinâmicaRESUMO
Lyotropic liquid crystalline (LLC) phases of polyglutamic acid derivatives, such as poly-γ-benzyl-l-glutamate, are suitable alignment media for organic structure elucidation by NMR spectroscopy. Their helical structure is responsible for enantiodiscrimination. As part of our ongoing investigations concerning the alignment mechanism(s) of these systems, we considered whether an additional chiral center in the side chain could improve enantiodiscrimination relative to the helical polymer with an achiral side chain. Therefore, the diastereoisomers poly-γ-S-2-methylbutyl-l-glutamate (PSMBLG) and poly-γ-S-2-methylbutyl-d-glutamate (PSMBDG) were synthesized. These two polymers were tested for their liquid crystallinity and suitability as alignment media. The spatial structure of the solutes (-)-curcumol and isopinocampheol (IPC) were validated by the residual dipolar coupling data obtained. Additionally, enantiodiscrimination of IPC was observed in the new alignment media and compared with the enantiodiscrimination of IPC in other homopolypeptides.
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Espectroscopia de Ressonância Magnética/métodos , Ácido Poliglutâmico/análogos & derivados , Ácido Poliglutâmico/síntese química , Cristais Líquidos/química , Estrutura Molecular , Ácido Poliglutâmico/química , Sesquiterpenos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
This perspective article aims to review the general methodology in the application of residual dipolar couplings (RDCs) in the structure elucidation of small molecules and give the author's view on challenges for future applications. Recent improvements in the availability of alignment media, new pulse sequences for the measurement of couplings and improvements in the analysis software have garnered widespread interest in the technique. However, further generalization is needed in order to make RDC analysis into a truly "routine" method. Copyright © 2016 John Wiley & Sons, Ltd.
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We report a comprehensive study on novel, highly efficient, and biodegradable hybrid molecular transporters. To this end, we designed a series of cell-penetrating, cube-octameric silsesquioxanes (COSS), and investigated cellular uptake by confocal microscopy and flow cytometry. A COSS with dense spatial arrangement of guanidinium groups displayed fast uptake kinetics and cell permeation at nanomolar concentrations in living HeLa cells. Efficient uptake was also observed in bacteria, yeasts, and archaea. The COSS-based carrier was significantly more potent than cell-penetrating peptides (CPPs) and displayed low toxicity. It efficiently delivered a covalently attached cytotoxic drug, doxorubicin, to living tumor cells. As the uptake of fluorescently labeled carrier remained in the presence of serum, the system could be considered particularly attractive for the inâ vivo delivery of therapeutics.
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Antineoplásicos/farmacologia , Peptídeos Penetradores de Células/farmacologia , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Compostos de Organossilício/farmacologia , Antineoplásicos/química , Antineoplásicos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Doxorrubicina/química , Doxorrubicina/metabolismo , Citometria de Fluxo , Células HeLa , Humanos , Microscopia Confocal , Estrutura Molecular , Compostos de Organossilício/química , Compostos de Organossilício/metabolismoRESUMO
We report on a detailed NMR spectroscopic study of the catalyst-substrate interaction of a highly enantioselective oligopeptide catalyst that is used for the kinetic resolution of trans-cycloalkane-1,2-diols via monoacylation. The extraordinary selectivity has been rationalized by molecular dynamics as well as density functional theory (DFT) computations. Herein we describe the conformational analysis of the organocatalyst studied by a combination of nuclear Overhauser effect (NOE) and residual dipolar coupling (RDC)-based methods that resulted in an ensemble of four final conformers. To corroborate the proposed mechanism, we also investigated the catalyst in mixtures with both trans-cyclohexane-1,2-diol enantiomers separately, using advanced NMR methods such as T1 relaxation time and diffusion-ordered spectroscopy (DOSY) measurements to probe molecular aggregation. We determined intramolecular distance changes within the catalyst after diol addition from quantitative NOE data. Finally, we developed a pure shift EASY ROESY experiment using PSYCHE homodecoupling to directly observe intermolecular NOE contacts between the trans-1,2-diol and the cyclohexyl moiety of the catalyst hidden by spectral overlap in conventional spectra. All experimental NMR data support the results proposed by earlier computations including the proposed key role of dispersion interaction.
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Stimuli responsive compounds and materials are of high interest in synthetic chemistry and materials science, with light being the most intriguing stimulus due to the possibility to remote control the physicochemical properties of a molecule or a material. There is a constant quest to design photoswitches with improved switching efficiency and especially diarylethene-type switches promise photo cyclization quantum yields up to unity. However, only limited attention has been paid towards the influence of the solution conformation on the switching efficiency. Here, we describe a detailed NMR spectroscopic investigation on the conformational distribution of bridge-substituted dithienylcyclopentenes in solution. We could discriminate between several photoactive and photoinactive as well as two diastereomorphous conformations and show that the trends observed in the switching efficiency match the conformer populations obtained from state of the art NMR parameters in solution.
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The hygrophorones, a class of cyclopentenones isolated from fruiting bodies of the genus Hygrophorus (basidiomycetes), show promising antifungal activity. While the constitution of 4,6-diacetylhygrophorone A(12) (3) and the relative configuration of the stereogenic centers in the cyclopentenone ring were elucidated using standard NMR and MS techniques, the relative configuration of the exocyclic stereogenic center could not be assigned. By introducing a sample of 3 into an alignment medium and measuring anisotropic NMR parameters, namely, residual dipolar couplings, we were able to unambiguously determine the relative configuration of all three stereogenic centers in 4,6-diacetylhygrophorone A(12) simultaneously by fitting several structure proposals to the experimental data.
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Basidiomycota/química , Ciclopentanos/isolamento & purificação , Ciclopentanos/farmacologia , Fungicidas Industriais/isolamento & purificação , Fungicidas Industriais/farmacologia , Ciclopentanos/química , Ciclopentanos/classificação , Fungicidas Industriais/química , Fungicidas Industriais/classificação , Estrutura Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
There has been much debate about the σ-donor and π-acceptor properties of N-heterocyclic carbenes (NHCs). While a lot of synthetic modifications have been performed with the goal of optimizing properties of the catalyst to tune reactivity in various transformations (e.g. metathesis), direct methods to characterize σ-donor and π-acceptor properties are still few. We believe that dynamic NMR spectroscopy can improve understanding of this aspect. Thus, we investigated the intramolecular dynamics of metathesis precatalysts bearing two NHCs. We chose four systems with one identical NHC ligand (N,N'-Bis(2,4,6-trimethylphenyl)-imidazolinylidene (SIMes) in all four cases) and NHC(ewg) ligands bearing four different electron-withdrawing groups (ewg). Both rotational barriers of the respective Ru-NHC-bonds change significantly when the electron density of one of the NHCs (NHC(ewg)) is modified. Although it is certainly not possible to fully dissect σ-donor and π-acceptor portions of the bonding situations in the respective Ru-NHC-bond via dynamic NMR spectroscopy, our studies nevertheless show that the analysis of the rotation around the Ru-SIMes-bond can be used as a spectroscopic parameter complementary to cyclic voltammetry. Surprisingly, we observed that the rotation around the Ru-NHC(ewg)-bond shows the same trend as the initiation rate of a ring-closing metathesis of the four investigated bis-NHC-complexes.
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For elucidating molecular structure and dynamics in solution, NMR experiments such as NOESY, ROESY and EXSY have been used excessively over the past decades, to provide interatomic distance restraints or rates for chemical exchange. The extraction of such information, however, is often prohibited by signal overlap in these spectra. To reduce this problem, pure shift methods for improving the spectral resolution have become popular. We report on pure shift EASY-ROESY experiments and their application to extract cross-relaxation rates, proton-proton distances and exchange rates. Homonuclear decoupling (pure shift) is applied in the indirect dimension using the PSYCHE or the perfectBASH technique, to enhance the spectral resolution of severely overcrowded spectral regions. The spectral quality is further improved by using a gradient selected F1-PSYCHE-EASY-ROESY, which produces significantly less t1-noise than the experiment used previously, as also demonstrated by employing the recently introduced SAN (signal-artefact-noise) plots. Applications include the quantification of distance restraints in a peptide organocatalyst and the extraction of a number of distance restraints in cyclosporine A, which were previously not available for analysis, because they were either located in overcrowded spectral regions or hidden under t1-noise. Distances extracted and exchange rates obtained are accurate. Also, the 2D gradient-selected F1-perfectBASH-EASY-ROESY with the additional gradient selection proposed herein, which is superior in terms of sensitivity, can be used to accurately quantify cross-relaxation.
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Ressonância Magnética Nuclear Biomolecular/métodos , Peptídeos/química , Algoritmos , Estrutura Molecular , Prótons , Sensibilidade e EspecificidadeRESUMO
The orientational properties of the two enantiomers of an example compound, namely isopinocampheol [(+)- and (-)-IPC] in the two enantiomers of a liquid crystalline phase, namely Poly-gamma-benzyl-L/D-glutamate (PBLG/PBDG) with the organic cosolvent CDCl(3), were investigated. The interactions can be either enantiomorphous, leading to equal orientations and residual dipolar couplings (RDCs), or diastereomorphous, leading to different orientations and RDCs. The difference between the two diastereomorphous orientations was determined to be rather small (5 degrees in the Euler angle beta). Furthermore, we investigated whether one of the two diastereomorphous interactions is favored.
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Cristais Líquidos/química , Ácido Poliglutâmico/análogos & derivados , Espectroscopia de Ressonância Magnética , Ácido Poliglutâmico/química , Solventes , EstereoisomerismoRESUMO
NMR techniques incorporating pure shift methods to improve signal resolution have recently attracted much attention, owing to their potential use in studies of increasingly complex molecular systems. Extraction of frequencies from these simplified spectra enables easier structure determination, but only a few of the methods presented provide structural parameters derived from signal integral measurements. In particular, for quantification of the nuclear Overhauser effect (NOE) it is highly desirable to utilize pure shift techniques where signal overlap normally prevents accurate signal integration, to enable measurement of a larger number of interatomic distances. However, robust methods for the measurement of interatomic distances using the recently developed pure shift techniques have not been reported to date. In this work we discuss some of the factors determining the accuracy of measurements of signal integrals in interferogram-based Zangger-Sterk (ZS) pure shift NMR experiments. The ZS broadband homodecoupling technique is used in different experiments designed for quantitative NOE determination from pure shift spectra. It is shown that the techniques studied can be used for quantitative extraction of NOE-derived distance restraints, as exemplified for the test case of strychnine.
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Contemporary catalytic procedures involving alkylpalladium(ii) have enriched the arsenal of synthetic organic chemistry. Those transformations usually rely on internal coordination through "directing groups", carefully designed to maximize catalytic efficiency and regioselectivity. Herein, we report structural and reactivity studies of a series of internally coordinated monohaptoallylpalladium complexes. These species enable the direct spectroscopic observation and theoretical study of π-σ-π interconversion processes. They further display unusual dynamic behavior which should be of direct relevance to chemistries beyond catalytic allylic alkylation.