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.

Enhanced Antiviral Function of Magnesium Chloride-Modified Heparin on a Broad Spectrum of Viruses.

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.

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.

Structural insights into the calmodulin-Munc13 interaction obtained by cross-linking and mass spectrometry.

Munc13 proteins are essential regulators of synaptic vesicle priming and play a key role in adaptive synaptic plasticity phenomena. We recently identified and characterized the Ca(2+)-dependent interaction of Munc13 and calmodulin (CaM) as the molecular mechanism linking changes in residual Ca(2+) concentrations to presynaptic vesicle priming and short-term plasticity. Here, we used peptidic photoprobes covering the established CaM-binding motif of Munc13 for photoaffinity labeling (PAL) of CaM, followed by structural characterization of the covalent photoadducts. Our innovative analytical workflow based on isotopically labeled CaM and mass spectrometry revealed that, in the bound state, the hydrophobic anchor residue of the CaM-binding motif in Munc13s contacts two distinct methionine residues in the C-terminal domain of CaM. To address the orientation of the peptide during binding, we obtained additional distance constraints from the mass spectrometric analysis of chemically cross-linked CaM-Munc13 peptide adducts. The constraints from both complementary cross-linking approaches were integrated into low-resolution three-dimensional structure models of the CaM-Munc13 peptide complexes. Our experimental data are best compatible with the structure of the complex formed by CaM and a CaM-binding peptide derived from neuronal NO synthase and show that Munc13-1 and ubMunc13-2 bind to CaM in an antiparallel orientation through a 1-5-8 motif. The structural information about the CaM-Munc13 peptide complexes will facilitate the design of Munc13 variants with altered CaM affinity and thereby advance the detailed functional analysis of the role of Munc13 proteins in synaptic transmission and plasticity.

Chromophore/DNA interactions: femto- to nanosecond spectroscopy, NMR structure, and electron transfer theory.

The mechanism of photoinduced hole injection into DNA has been studied using an integrated approach that combines NMR structural analysis, time-resolved spectroscopy, and quantum-chemical calculations. A covalently linked acridinium derivative, the protonated 9-amino-6-chloro-2-methoxyacridine (X+), is replacing a thymine and separated from either guanine (G) or the easier to oxidize 7-deazaguanine (Z) by one adenine.thymine (A.T) base pair. The key features of this donor/acceptor system are the following: (i) In more than 95% of the duplexes, X+ is located in a central, coplanar position between the neighboring A.T base pairs with its long axis in parallel showing minimal twist and tilt angles (<15 degrees). The complementary adenine base is turned out into the extrahelical space. In a minority of less than 5%, X+ is found to be still attached to the duplex. X+ is most probably associated with one of the phosphates, since it is neither intercalated between more remote base pairs nor bound to sugars or grooves. This minority characterized by an excited state lifetime >10 ns gives rise to a small background signal in time-resolved measurements and contributes predominantly to steady-state fluorescence spectra. (ii) Although the intercalation mode of X+ is well defined, the NMR structure reveals that there are two conformations of X+ with respect to the arrangement of its methoxy substituent. In one conformation, the methoxy group is in the plane of the chromophore, while, in the other extraplanar conformation, the methoxy group forms an angle of 70 degrees with the acridinium ring. The fluorescence decay of 5'-ZAX and 5'-GAX tracts can be fitted to a biexponential function with similar amplitudes, reflecting the oxidation dynamics of G and Z, with the slower rate being determined by larger thermal activation energy. The attribution of biexponential electron transfer (ET) dynamics to the bimodal orientation of the methoxy group at the acridinium is supported by quantum-chemical calculations. These predict a larger free energy change for hole transfer in the nonplanar conformation as compared to the planar one, whereas the difference in the electronic couplings is negligible. (iii) Kinetic studies of the directionality of the 1(X+)* induced hole injection reveal similarly fast decay components in both directions of the duplex, that is, in 5'-ZAX and 5'-XAZ, with the amplitude of the fast component being significantly reduced in 5'-XAZ. The NMR structure shows that local structural deviations from B-DNA are much more pronounced in the 3'-5' direction than in the 5'-3' direction. According to quantum-chemical calculations, the directionality of charge injection is not a universal feature of the DNA duplex but depends critically on the rotation angle of the aromatic plane of the acridinium within the pi stack. The arrangement of X+ in 5'-ZAX and 5'-XAZ corresponds to a conformation with weak directionality of the electronic couplings. The increased disorder in the 3'-5'direction favors slow hole transfer components at the expense of the fast ones. (iv) A comparison of the hole transfer in 5'-GAX and 5'-ZAG shows that classical Marcus theory can explain the ratio of the charge shift rates of more than 2 orders of magnitude on the basis of a free energy difference between G and Z of 0.3 eV. Both NMR structures and quantum-chemical calculations justify the appreciable neglect of differences of electronic couplings as well as in the reorganization energy in 5'-GAX and 5'-ZAG. Despite the attractive concept for the behavior of floppy DNA oligonucleotides, in this acridinium/DNA system, there is no evidence for conformational gating, that is, for fluctuations in the electronic couplings that permit the ET to occur.