Expanding the Limits of Structural Characterization of Marine Dissolved Organic Matter Using Nonuniform Sampling Frequency-Reversed Edited HSQC NMR.

The multiplicity-edited heteronuclear single quantum correlation (ME-HSQC) NMR method is widely used for the structural characterization of marine dissolved organic matter (DOM), which is a complex molecular mixture comprising millions of individual compounds. However, the standard ME-HSQC suffers from significant signal cancellation and subsequent loss of crucial structural information due to the overlap between CH3/CH (positive) and CH2 (negative) cross-peaks in overcrowded regions. This study introduces nonuniform sampling in frequency-reversed ME-HSQC (NUS FR-ME-HSQC), highlighting its remarkable potential for the comprehensive structural characterization of marine DOM. By reversing the frequency of CH2 cross-peaks into an empty region, the FR-ME-HSQC method effectively simplifies the spectra and eliminates signal cancellation. We demonstrate that nonuniform sampling enables the acquisition of comparable spectra in half the time or significantly enhances the sensitivity in time-equivalent spectra. Comparative analysis also identifies vulnerable CH2 cross-peaks in the standard ME-HSQC that coincide with CH3 and CH cross-peaks, resulting in the loss of critical structural details. In contrast, the NUS FR-ME-HSQC retains these missing correlations, enabling in-depth characterization of marine DOM. These findings highlight the potential of NUS FR-ME-HSQC as an advanced NMR technique that effectively addresses challenges such as signal overcrowding and prolonged experimental times, enabling the thorough investigation of complex mixtures with implications in several fields, including chemistry, metabolomics, and environmental sciences. The advantages of NUS FR-ME-HSQC are experimentally demonstrated on two solid-phase-extracted DOM (SPE-DOM) samples from the surface and deep ocean. With this new technology, differences in the composition of DOM from various aquatic environments can be assigned to individual molecules.

Challenging Structure Elucidation of Lumnitzeralactone, an Ellagic Acid Derivative from the Mangrove Lumnitzera racemosa.

The previously undescribed natural product lumnitzeralactone (1), which represents a derivative of ellagic acid, was isolated from the anti-bacterial extract of the Indonesian mangrove species Lumnitzera racemosa Willd. The structure of lumnitzeralactone (1), a proton-deficient and highly challenging condensed aromatic ring system, was unambiguously elucidated by extensive spectroscopic analyses involving high-resolution mass spectrometry (HRMS), 1D 1H and 13C nuclear magnetic resonance spectroscopy (NMR), and 2D NMR (including 1,1-ADEQUATE and 1,n-ADEQUATE). Determination of the structure was supported by computer-assisted structure elucidation (CASE system applying ACD-SE), density functional theory (DFT) calculations, and a two-step chemical synthesis. Possible biosynthetic pathways involving mangrove-associated fungi have been suggested.

Cu2+-Induced self-assembly and amyloid formation of a cyclic D,L-α-peptide: structure and function.

In a wide spectrum of neurodegenerative diseases, self-assembly of pathogenic proteins to cytotoxic intermediates is accelerated by the presence of metal ions such as Cu2+. Only low concentrations of these early transient oligomeric intermediates are present in a mixture of species during fibril formation, and hence information on the extent of structuring of these oligomers is still largely unknown. Here, we investigate dimers as the first intermediates in the Cu2+-driven aggregation of a cyclic D,L-α-peptide architecture. The unique structural and functional properties of this model system recapitulate the self-assembling properties of amyloidogenic proteins including ß-sheet conformation and cross-interaction with pathogenic amyloids. We show that a histidine-rich cyclic D,L-α-octapeptide binds Cu2+ with high affinity and selectivity to generate amyloid-like cross-ß-sheet structures. By taking advantage of backbone amide methylation to arrest the self-assembly at the dimeric stage, we obtain structural information and characterize the degree of local order for the dimer. We found that, while catalytic amounts of Cu2+ promote aggregation of the peptide to fibrillar structures, higher concentrations dose-dependently reduce fibrillization and lead to formation of spherical particles, showing self-assembly to different polymorphs. For the initial self-assembly step to the dimers, we found that Cu2+ is coordinated on average by two histidines, similar to self-assembled peptides, indicating that a similar binding interface is perpetuated during Cu2+-driven oligomerization. The dimer itself is found in heterogeneous conformations that undergo dynamic exchange, leading to the formation of different polymorphs at the initial stage of the aggregation process.

Marine Dissolved Organic Matter Shares Thousands of Molecular Formulae Yet Differs Structurally across Major Water Masses.

Most oceanic dissolved organic matter (DOM) is still not fully molecularly characterized. We combined high-field nuclear magnetic resonance (NMR) and ultrahigh-resolution mass spectrometry (Fourier-transform ion cyclotron resonance mass spectrometry, FT-ICR-MS) for the structural and molecular formula-level characterization of solid-phase extracted (SPE) DOM from surface, mesopelagic, and bathypelagic Atlantic and Pacific Ocean samples. Using a MicroCryoProbe, unprecedented low amounts of SPE-DOM (∼1 mg carbon) were sufficient for two-dimensional NMR analysis. Low proportions of olefinic and aromatic relative to aliphatic and carboxylated structures (NMR) at the sea surface were likely related to photochemical transformations. This was consistent with lower molecular masses and higher degrees of saturation and oxygenation (FT-ICR-MS) compared to those of the deep sea. Carbohydrate structures in the mesopelagic North Pacific Ocean suggest export and release from sinking particles. In our sample set, the universal molecular DOM composition, as captured by FT-ICR-MS, appears to be structurally more diverse when analyzed by NMR, suggesting DOM variability across oceanic provinces to be more pronounced than previously assumed. As a proof of concept, our study takes advantage of new complementary approaches resolving thousands of structural and molecular DOM features while applying reasonable instrument times, allowing for the analysis of large oceanic data sets to increase our understanding of marine DOM biogeochemistry.

Combined High-Pressure and Multiquantum NMR and Molecular Simulation Propose a Role for N-Terminal Salt Bridges in Amyloid-Beta.

Several lines of evidence point to the important role of the N-terminal region of amyloid-beta (Aß) peptide in its toxic aggregation in Alzheimer's disease (AD). It is known that charge-altering modifications such as Ser8 phosphorylation promote Aß fibrillar aggregation. In this Letter, we combine high-pressure NMR, multiquantum chemical exchange saturation transfer (MQ-CEST) NMR, and microseconds-long molecular dynamics simulation and provide evidence of the presence of several salt bridges between Arg5 and its nearby negatively charged residues, in particular, Asp7 and Glu3. The presence of these salt bridges is correlated with less extended structures in the N-terminal region of Aß. Through density functional theory calculations, we demonstrate how the introduction of negatively charged phosphoserine 8 influences the network of adjacent salt bridges in Aß and favors more extended N-terminal structures. Our data propose a structural mechanism for the Ser8-phosphorylation-promoted Aß aggregation and define the N-terminal salt bridges as potential targets for anti-AD drug design.

Structure and Absolute Configuration of Phenanthro-perylene Quinone Pigments from the Deep-Sea Crinoid Hypalocrinus naresianus.

Two new water-soluble phenanthroperylene quinones, gymnochrome H (2) and monosulfated gymnochrome A (3), as well as the known compounds gymnochrome A (4) and monosulfated gymnochrome D (5) were isolated from the deep-sea crinoid Hypalocrinus naresianus, which had been collected in the deep sea of Japan. The structures of the compounds were elucidated by spectroscopic analysis including HRMS, 1D 1H and 13C NMR, and 2D NMR. The absolute configuration was determined by ECD spectroscopy, analysis of J-couplings and ROE contacts, and DFT calculations. The configuration of the axial chirality of all isolated phenanthroperylene quinones (2-5) was determined to be (P). For gymnochrome H (2) and monosulfated gymnochrome A (3), a (2'S,2″R) configuration was determined, whereas for monosulfated gymnochrome D (5) a (2'R,2″R), configuration was determined. Acetylated quinones are unusual among natural products from an echinoderm and gymnochrome H (2) together with the recently reported gymnochrome G (1) represent the first isolated acetylated phenanthroperylene quinones.

Probing the Accuracy of Explicit Solvent Constant pH Molecular Dynamics Simulations for Peptides.

Protonation states of titratable amino acids play a key role in many biomolecular processes. Knowledge of protonatable residue charges at a given pH is essential for a correct understanding of protein catalysis, inter- and intramolecular interactions, substrate binding, and protein dynamics for instance. However, acquiring experimental values for individual amino acid protonation states of complex systems is not straightforward; therefore, several in silico approaches have been developed to tackle this issue. In this work, we assess the accuracy of our previously developed constant pH MD approach by comparing our theoretically obtained pKa values for titratable residues with experimental values from an equivalent NMR study. We selected a set of four pentapeptides, of adequately small size to ensure comprehensive sampling, but concurrently, due to their charge composition, posing a challenge for protonation state calculation. The comparison of the pKa values shows good agreement of the experimental and the theoretical approach with a largest difference of 0.25 pKa units. Further, the corresponding titration curves are in fair agreement, although the shift of the Hill coefficient from a value of 1 was not always reproduced in simulations. The phase space overlap in Cartesian space between trajectories generated in constant pH and standard MD simulations is fair and suggests that our constant pH MD approach reasonably well preserves the dynamics of the system, allowing dynamic protonation MD simulations without introducing structural artifacts.

Secophragmalin-Type Limonoids from Trichilia connaroides: Isolation, Semisynthesis, and Their Cytotoxic Activities.

Two new secophragmalin-type limonoids, secotrichagmalins B (1) and C (2) along with two known compounds, were isolated from the fruits of Trichilia connaroides. The structures of the new compounds were elucidated by analysis of spectroscopic (IR, MS, and 2D NMR) data and single crystal X-ray diffraction studies. In addition, semisynthetic derivatives (2a-2l) were efficiently synthesized and evaluated for their in vitro cytotoxicity along with the isolated limonoids against a panel of human cancer cell lines. The results indicated that new analogues 2a, 2d, and 2e showed cytotoxicity on the DU145 cell line with IC50 values of 3.6, 4.2, and 5.2 µM, respectively. Flow cytometric analysis revealed that these analogues arrested the cell cycle in the G0/G1 phase and markedly induced apoptosis.

Selective measurement of 1 H-1 H scalar couplings from crowded chemical shift regions: Combined pure shift and spin-echo modulation approach.

JHH scalar couplings carry rich structural information and their measurements are fundamental in the 1 H NMR based elucidation of small and medium molecules, which, however, are hampered in the presence of large J-coupling network. Further, enhanced spectral resolution is often essential for precise determination of a specific set of 1 H-1 H J-couplings among the complex J-multiplets. In the light of the recent advancements in homodecoupling pure shift strategies, here, we report absorption mode, band-selective refocused pure shift spin-echo method, which helps in determining 1 H-1 H J-couplings from crowded spectral regions. The importance of the present band-selective refocused pure shift spin-echo experiment is exemplified for 2 steroid molecules, estradiol and testosterone.

Band-selective excited ultrahigh resolution PSYCHE-TOCSY: fast screening of organic molecules and complex mixtures.

Precise assignments of (1) H atomic sites and establishment of their through-bond COSY or TOCSY connectivity are crucial for molecular structural characterization by using (1) H NMR spectroscopy. However, this exercise is often hampered by signal overlap, primarily because of (1) H-(1) H scalar coupling multiplets, even at typical high magnetic fields. The recent developments in homodecoupling strategies for effectively suppressing the coupling multiplets into nice singlets (pure-shift), particularly, Morris's advanced broadband pure-shift yielded by chirp excitation (PSYCHE) decoupling and ultrahigh resolution PSYCHE-TOCSY schemes, have shown new possibilities for unambiguous structural elucidation of complex organic molecules. The superior broadband PSYCHE-TOCSY exhibits enhanced performance over the earlier TOCSY methods, which however warrants prolonged experimental times due to the requirement of large number of dwell increments along the indirect dimension. Herein, we present fast and band-selective analog of the broadband PSYCHE-TOCSY, which is useful for analyzing complex organic molecules that exhibit characteristic yet crowded spectral regions. The simple pulse scheme relies on band-selective excitation (BSE) followed by PSYCHE homodecoupling in the indirect dimension. The BSE-PSYCHE-TOCSY has been exemplified for Estradiol and a complex carbohydrate mixture comprised of six constituents of closely comparable molecular weights. The experimental times are greatly reduced viz., ~20 fold for Estradiol and ~10 fold for carbohydrate mixture, with respect to the broadband PSYCHE-TOCSY. Furthermore, unlike the earlier homonuclear band-selective decoupling, the BSE-PSYCHE-decoupling provides fully decoupled pure-shift spectra for all the individual chemical sites within the excited band. The BSE-PSYCHE-TOCSY is expected to have significant potential for quick screening of complex organic molecules and mixtures at ultrahigh resolution. Copyright © 2015 John Wiley & Sons, Ltd.

Precise Determination of Enantiomeric Excess by a Sensitivity Enhanced Two-Dimensional Band-Selective Pure-Shift NMR.

Unambiguous identification and precise quantification of enantiomers in chiral mixtures is crucial for enantiomer specific synthesis as well as chemical analysis. The task is often challenging for mixtures with high enantiomeric excess and for complex molecules with strong (1)H-(1)H scalar (J) coupling network. The recent advancements in (1)H-(1)H decoupling strategies to suppress the J-interactions offered new possibilities for NMR based unambiguous discrimination and quantification enantiomers. Herein, we discuss a high resolution two-dimensional pure-shift zCOSY NMR method with homonuclear band-selective decoupling in both the F1 and F2 dimensions (F1F2-HOBS-zCOSY). This advanced method shows a sharp improvement in resolution over the other COSY methods and also eliminates the problems associated with the overlapping decoupling sidebands. The efficacy of this method has been exploited for precise quantification of enantiomeric excess (ee) ratio (R/S) up to 99:1 in the presence of very low concentrations of chiral lanthanide shift reagents (CLSR) or chiral solvating agents (CSA). The F1F2-HOBS-zCOSY is simple and can be easily implemented on any modern NMR spectrometers, as a routine analytical tool.

A concise diastereoselective approach to enantioenriched substituted piperidines and their in vitro cytotoxicity evaluation.

A library of diversely stereo-oriented, highly substituted 2,6-cis piperidine derivatives were synthesized, and evaluated for their anticancer activity in cancer cells that included A549 (lung cancer, CCL-185), MCF7 (breast cancer (HTB-22), DU145 (prostate cancer (HTB-81), and HeLa (cervical cancer, CCL-2). One stereo-variant emerged as a promising candidate for further design based structure-activity studies.

Enantioselective synthesis of δ-/γ-alkoxy-ß-hydroxy-α-alkyl-substituted Weinreb amides via DKR-ATH: application to the synthesis of advanced intermediate of (-)-brevisamide.

A method of preparing stereodefined δ-/γ-alkoxy-ß-hydroxy-α-alkyl-substituted Weinreb amides containing two successive hydroxyl-alkyl stereocenters has been developed. Further, this strategy coupled with organo-catalyzed asymmetric epoxidation culminates in the synthesis of a critical intermediate of (-)-brevisamide and its diastereomers.