New Lanthanide Tag for the Generation of Pseudocontact Shifts in DNA by Site-Specific Ligation to a Phosphorothioate Group.

Pseudocontact shifts (PCS) generated by paramagnetic lanthanides provide a rich source of long-range structural restraints that can readily be measured by nuclear magnetic resonance (NMR) spectroscopy. Many different lanthanide-binding tags have been designed for site-specific tagging of proteins, but established routes for tagging DNA with a single metal ion rely on difficult chemical synthesis. Here we present a simple and practical strategy for site-specific tagging of inexpensive phosphorothioate (PT) oligonucleotides. Commercially available PT oligonucleotides are diastereomers with S and R stereoconfiguration at the backbone PT site. The respective SP and RP diastereomers can readily be separated by HPLC. A new alkylating lanthanide-binding tag, C10, was synthesized that delivered quantitative tagging yields with both diastereomers. PCSs were observed following ligation with the complementary DNA strand to form double-stranded DNA duplexes. The PCSs were larger for the SP than the RP oligonucleotide and good correlation between back-calculated and experimental PCSs was observed. The C10 tag can also be attached to cysteine residues in proteins, where it generates a stable thioether bond. Ligated to the A28C mutant of ubiquitin, the tag produced excellent fits of magnetic susceptibility anisotropy (Δχ) tensors, with larger tensors than for the tagged PT oligonucleotides, indicating that the tag is not completely immobilized after ligation with a PT group.

New α-pyrone and phthalide from the Xylariaceae fungus.

A new α-pyrone xylaripyrone A (1) and a new phthalide xylariphthalide A (2) were isolated from the Xylariaceae fungus (no. 63-19-7-3), along with four related known phthalides (3-6): 4-[(acetyloxy)methyl]-7-methoxy-6-methyl-1(3H)-isobenzofuranone (3), convolvulol (4), 7-methoxy-4,6-dimethyl-3H-isobenzofuran-1-one (5), and convolvulanic acid B (6). Their structures were determined on the basis of IR, MS, and NMR spectroscopic analyses.

Selective Distance Measurements Using Triple Spin Labeling with Gd3+, Mn2+, and a Nitroxide.

Distance measurements by pulse electron paramagnetic resonance techniques, such as double electron-electron resonance (DEER, also called PELDOR), have become an established tool to explore structural properties of biomacromolecules and their assemblies. In such measurements a pair of spin labels provides a single distance constraint. Here we show that by employing three different types of spin labels that differ in their spectroscopic and spin dynamics properties it is possible to extract three independent distances from a single sample. We demonstrate this using the Antennapedia homeodomain orthogonally labeled with Gd3+ and Mn2+ tags in complex with its cognate DNA binding site labeled with a nitroxide.

Nodulisporisteriods A and B, the first 3,4-seco-4-methyl-progesteroids from Nodulisporium sp.

Two new 4-methyl-progesteroids, nodulisporisteriod A (1) and nodulisporisteriod B (2), were isolated from the extract of an endolichenic fungal strain Nodulisporium sp. (No. 65-17-2-1), along with two related metabolites, demethoxyviridin (3) and inoterpene B (4). Their structures were determined by detailed spectroscopic analyses, X-ray crystallographic analysis and comparison of the NMR data with those of the closely related compounds previously reported. Nodulisporisteriod A (1) and nodulisporisteriod B (2) possess new carbon skeletons, which are the first cases of fission at C-3,4 in 4-methyl-progesteroids. A hypothetical biosynthetic pathway for 1 and 2 was proposed. Moreover, the Aß42 aggregation inhibitory activities of 1-4 were evaluated using standard thioflavin T (ThT) fluorescence assay with epigallocatechin gallate (EGCG) as positive control. Demethoxyviridin (3) displayed anti-Aß42 aggregation activity with IC50 value of 13.4µM.