Full NMR Assignment Research Articles

Phenolic constituents from Rhopalocnemis phalloides with DPPH radical scavenging activity

The 80% aqueous acetone extract of the whole plant of Rhopalocnemis phalloides Jungh. (Balanophoraceae) showed obvious radical scavenging activity (SC50 = 32.1 μg/mL) on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assay. Further chemical investigation of the extract led to the isolation of 12 phenolic compounds (1–12). Among these, bis-6,8’-catechinylmethane (2), a flavan-3-ol dimer, was first isolated as a natural product from Rhopalocnemis phalloides. The full NMR assignments of bis-catechinylmethanes (1 and 2) are reported for the first time, on the basis of detailed spectroscopic analysis. The DPPH assay showed that flavan-3-ol dimers (1–3) had remarkable free radical scavenging activity, while flavonoid aglycones (4 and 9) presented stronger activities than those of their glycosides (5–8 and 10).

Phytochemical and antifungal studies on Terminalia mollis and Terminalia brachystemma

Phytochemical investigation of the stem bark of Terminalia mollis afforded friedelin (1), catechin with epicatechin (2), gallocatechin with epigallocatechin (3) and 3-O-methylellagic acid 4'-O-alpha-rhamnopyranoside (4). Arjunolic acid with 2alpha, 3beta, 23-trihydroxy-urs-12-en-28-oic acid (5), 2alpha-hydroxyursolic acid (6), gallic acid (7), chebulanin (8) and 2''-O-galloylvitexin (9) were isolated from the leaf. Chebulanin (8), betulinic acid (10), ursolic acid (11), catechin (12), isoorientin (13), orientin (14), isovitexin (15) and punicalagin (16) were isolated from Terminalia brachystemma leaf. The first full unambiguous NMR assignments for (4) and (8), and revised assignments for (9), are reported. Compound (16) showed good activity against three Candida species.

Alkaloids from Lindera Aggregata

Eight alkaloids, including a new compound, (+)-norboldine acetate (1), and seven known ones, (+)-norboldine (2), (+)-boldine (3), (+)-laurotetanine (4), (+)-N-methyllaurotetanine (5), (+)-reticuline (6), (-)-pronuciferine (7), and pallidine (8) were isolated from the roots of Lindera aggregata. The structures of these alkaloids were determined by spectroscopic and chemical methods, especially 2D NMR techniques, which also allowed the first full NMR assignments of alkaloids 2, 4 and 5. Among them, the 1D NMR chemical shifts of (+)-norboldine (2) showed a remarkable environmental sensitive behavior. All the alkaloids were tested in cytotoxicity assays against L1210 and K562 tumor cell lines; only (+)-norboldine (2) showed weak activity against the L1210 cell line.

Complete assignments of NMR data of salens and their cobalt (III) complexes

Salens, derived from 1,2-ethylenediamine and salicylaldehydes, have been widely used as ligands for metal complexes which have been showing enormous potential in chemical properties of asymmetric catalysts as well as biological properties such as anticancer agents. Almost all of the salen-metal complexes with their corresponding metal (II)-complexes show the evidences of chelation of two oxygens in salens. However, several metal (II) complexes, especially cobalt (II) complexes, could not show NMR spectra due to their paramagnetism. Recently, it has been reported that one of the cobalt (III) complexes was used for NMR spectroscopy to evaluate its stereoselectivity as a catalyst. Even though many salen ligands are known, their NMR data are not assigned completely. It was possible that modification in northern part of salen with 2-hydroxyphenyl group afforded another oxygen chelation site in salen ligand. Here we report that synthesis and full NMR assignment of new salen ligands, which form meso 1,2-bis(2-hydroxyphenyl)ethylenediamine) and their cobalt (III) complexes. The assignments of (1)H and (13)C NMR data obtained in this experiment can help us to predict the NMR data of other salen ligands.

Isolation, purification, and full NMR assignments of cyclopamine from Veratrum californicum

BackgroundThe Hedgehog signaling pathway is essential for embryogenesis and for tissue homeostasis in the adult. However, it may induce malignancies in a number of tissues when constitutively activated, and it may also have a role in other forms of normal and maladaptive growth. Cyclopamine, a naturally occurring steroidal alkaloid, specifically inhibits the Hedgehog pathway by binding directly to Smoothened, an important Hedgehog response element. To use cyclopamine as a tool to explore and/or inhibit the Hedgehog pathway in vivo, a substantial quantity is required, and as a practical matter cyclopamine has been effectively unavailable for usage in animals larger than mice.ResultsIn this paper, we report a rapid and efficient isolation and purification of large quantities of cyclopamine from the roots and rhizomes of Veratrum californicum Dur. (the Corn Lily or Western false hellebore). We also provide unambiguous assignments of the carbon and proton resonances by using the multinuclear spectra and the spin coupling networks.ConclusionThis method could meet a very real need within diverse scientific communities by allowing cyclopamine to become more readily available.

Local Structural Preferences of Calpastatin, the Intrinsically Unstructured Protein Inhibitor of Calpain

Calpain, the calcium-activated intracellular cysteine protease, is under the tight control of its intrinsically unstructured inhibitor, calpastatin. Understanding how potent inhibition by calpastatin can be reconciled with its unstructured nature provides deeper insight into calpain function and a more general understanding of how proteins devoid of a well-defined structure carry out their function. To this end, we performed a full NMR assignment of hCSD1 to characterize it in its solution state. Secondary chemical shift values and NMR relaxation data, R 1, R 2, and hetero-NOE, as well as spectral density function analysis have shown that conserved regions of calpastatin, subdomains A and C, which are responsible for calcium-dependent anchoring of the inhibitor to the enzyme, preferentially sample partially helical backbone conformations of a reduced flexibility. Moreover, the linker regions between subdomains are more flexible with no structural preference. The primary determinant of calpain inhibition, subdomain B, also has a non-fully random conformational preference, resembling a beta-turn structure also ascertained by prior studies of a 27-residue peptide encompassing the inhibitory region. This local structural preference is also confirmed by a deviation in chemical shift values between full-length calpastatin domain 1 and a truncated construct cut in the middle of subdomain B. At the C-terminal end of the molecule, a nascent helical region was found, which in contrast to the overall structural properties of the molecule may indicate a previously unknown functional region. Overall, these observations provide further evidence that supports previous suggestions that intrinsically unstructured proteins use preformed structural elements in efficient partner recognition.

Neo-clerodane diterpenes from Ajuga turkestanica

The ethyl acetate extract of the aerial parts of Ajuga turkestanica afforded 6 neo-clerodane diterpenes, including two novel compounds, 14,15-dihydroajugachin B ( 1) and 14-hydro-15-methoxyajugachin B ( 2), in addition to the known diterpenoids chamaepitin ( 3), ajugachin B ( 4), ajugapitin ( 5) and lupulin A ( 6). Structures were established through exhaustive NMR spectroscopic analysis and chemical transformation in the case of 1. The full 1H and 13C NMR assignment of the C-15 R and S configurations of 14-hydro-15-methoxyajugachin B and chamaepitin were elucidated.

6-Halogenopyridin-2-olato complexes with the diruthenium(2+) core: Equilibria between head-to-head and head-to-tail structures

The dinuclear bis(6-X-pyridin-2-olato) ruthenium complexes [Ru 2(μ-XpyO) 2(CO) 4(PPh 3) 2] (X = Cl ( 4B) and Br ( 5B)), [Ru 2(μ-XpyO) 2(CO) 4(CH 3CN) 2] (X = Cl ( 6B), Br ( 7B) and F (8B)) and [Ru 2(μ-ClpyO) 2(CO) 4(PhCN) 2] ( 9B) were prepared from the corresponding tetranuclear coordination dimers [Ru 2(μ-XpyO) 2(CO) 4] 2 ( 1: X = Cl; 2: X = Br) and [Ru 2(μ-FpyO) 2(CO) 6] 2 ( 3) by treatment with an excess of triphenylphosphane, acetonitrile and benzonitrile, respectively. In the solid state, complexes 4B– 9B all have a head-to-tail arrangement of the two pyridonate ligands, as evidenced by X-ray crystal structure analyses of 4B, 6B and 9B, in contrast to the head-to-head arrangement in the precursors 1– 3. A temperature- and solvent-dependent equilibrium between the yellow head-to-tail complexes and the red head-to-head complexes 4A– 7A and 9A, bearing an axial ligand only at the O, O-substituted ruthenium atom, exists in solution and was studied by NMR spectroscopy. Full 1H and 13C NMR assignments were made in each case. Treatment of 1 and 2 with the N-heterocyclic carbene (NHC) 1-butyl-3-methylimidazolin-2-ylidene provided the complexes [Ru 2(μ-XpyO) 2(CO) 4(NHC)], X = Cl ( 11A) or Br ( 12A). An XRD analysis revealed the head-to-head arrangement of the pyridonate ligands and axial coordination of the carbene ligand at the O, O-substituted ruthenium atom. The conversion of 11A and 12A into the corresponding head-to-tail complexes was not possible.

Phytochemical and biological studies on Saudi Commiphora opobalsamum L.

The aerial part of Commiphora opobalsamum L. (Burseraceae) growing in Saudi Arabia was subjected to a phytopharmacological investigation in order to identify its major chemical constituents and to evaluate its extracts and isolated compounds in preliminary in vitro assays for antimicrobial, antimalarial, antitumor, anti-inflammatory (COX-2 inhibition), antioxidant and estrogenic activity. Six compounds were isolated and identified as the triterpenes friedelin, canophyllal, and oleanonic acid; the flavonols mearnsetin and quercetin; and syringic acid. The ethyl acetate extract was moderately active against Staphylococcus aureus, Pseudomonas aeruginosa, and Plasmodium falciparum; while the petroleum ether and chloroform extracts inhibited COX-2 at 5 and 10 µg mL−1, respectively. Of the isolated compounds, syringic acid showed moderate antimalarial, anticandidal, and antimycobacterial activity; while mearnsetin and quercetin exhibited antioxidant activity comparable to ascorbic acid and trolox. This is the first detailed phytochemical investigation of C. opobalsamum L. growing in Saudi Arabia and elsewhere. The isolated compounds are reported from this plant for the first time and their full 1H and 13C NMR assignments are included.

Total synthesis and full NMR assignment of ourouparine, a yohimbane-type indole alkaloid isolated from Gelsemium sempervirens

A yohimbane-type indole alkaloid, ourouparine, was found in Gelsemium sempervirens, and its structure was elucidated by spectroscopic analysis and total synthesis.

Multiple Pentafluorophenylation of 2,2,3,3,5,6,6-Heptafluoro-3,6-dihydro-2H-1,4-oxazine with an Organosilicon Reagent: NMR and DFT Structural Analysis of Oligo(perfluoroaryl) Compounds

The reagent Me3Si(C6F5) was used for the preparation of a series of perfluorinated, pentafluorophenyl-substituted 3,6-dihydro-2H-1,4-oxazines (2–8), which, otherwise, would be very difficult to synthesize. Multiple pentafluorophenylation occurred not only on the heterocyclic ring of the starting compound 1 (Scheme), but also in para position of the introduced C6F5 substituent(s) leading to compounds with one to three nonafluorobiphenyl (C12F9) substituents. While the tris(pentafluorophenyl)-substituted compound 3 could be isolated as the sole product by stoichiometric control of the reagent, the higher-substituted compounds 5–8 could only be obtained as mixtures. The structures of the oligo(perfluoroaryl) compounds were confirmed by 19F- and 13C-NMR, MS, and/or X-ray crystallography. DFT simulations of the 19F- and 13C-NMR chemical shifts were performed at the B3LYP-GIAO/6-31++G(d,p) level for geometries optimized by the B3LYP/6-31G(d) level, a technique that proved to be very useful to accomplish full NMR assignment of these complex products.

19F NMR Studies of the Native and Denatured States of Green Fluorescent Protein

Biosynthetic preparation and (19)F NMR experiments on uniformly 3-fluorotyrosine-labeled green fluorescent protein (GFP) are described. The (19)F NMR signals of all 10 fluorotyrosines are resolved in the protein spectrum with signals spread over 10 ppm. Each tyrosine in GFP was mutated in turn to phenylalanine. The spectra of the Tyr --> Phe mutants, in conjunction with relaxation data and results from (19)F photo-CIDNP (chemically induced dynamic nuclear polarization) experiments, yielded a full (19)F NMR assignment. Two (19)F-Tyr residues (Y92 and Y143) were found to yield pairs of signals originating from ring-flip conformers; these two residues must therefore be immobilized in the native structure and have (19)F nuclei in two magnetically distinct positions depending on the orientation of the aromatic ring. Photo-CIDNP experiments were undertaken to probe further the structure of the native and denatured states. The observed NMR signal enhancements were found to be consistent with calculations of the HOMO (highest occupied molecular orbital) accessibilities of the tyrosine residues. The photo-CIDNP spectrum of native GFP shows four peaks corresponding to the four tyrosine residues that have solvent-exposed HOMOs. In contrast, the photo-CIDNP spectra of various denatured states of GFP show only two peaks corresponding to the (19)F-labeled tyrosine side chains and the (19)F-labeled Y66 of the chromophore. These data suggest that the pH-denatured and GdnDCl-denatured states are similar in terms of the chemical environments of the tyrosine residues. Further analysis of the sign and amplitude of the photo-CIDNP effect, however, provided strong evidence that the denatured state at pH 2.9 has significantly different properties and appears to be heterogeneous, containing subensembles with significantly different rotational correlation times.

Larvicidal Diterpenes from Pterodon polygalaeflorus

Ethanolic extract from seeds of Pterodon polygalaeflorus (Benth) has been shown to possess significant larvicidal activity against the mosquito Aedes aegypti. Bioassay-guided fractionation of the extract led to the isolation and characterization of the know diterpenoid furans 6alpha-hydroxyvouacapan-7beta,17beta-lactone (1), 6alpha,7beta-dihydroxyvouacapan- 17beta-oic acid (2) and methyl 6alpha,7beta-dihydroxyvouacapan-17beta-oate (3). The structures were established from infrared (IR), ultraviolet (UV), 1H-nuclear magnetic resonance (NMR), 13C-NMR, and mass spectral data: full NMR assignments are presented for compounds 1-3 and the diacetyl derivative of 3. Compounds 1-3 exhibited LC50 values of 50.08, 14.69, and 21.76 microg/mL against fourth-instar Aedes aegypti larvae.

Pelseneeriol‐1 and ‐2: New Furanosesquiterpene Alcohols from Porostome Nudibranch Doriopsilla pelseneeri

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

PH Dependence of Amide Chemical Shifts in Natively Disordered Polypeptides Detects Medium-Range Interactions with Ionizable Residues

A growing number of natively disordered proteins undergo a folding/binding process that is essential for their biological function. An interesting question is whether these proteins have incompletely solvated regions that drive the folding/binding process. Although the presence of predominantly hydrophobic buried regions can be easily ascertained by high-sensitivity differential scanning calorimetry analysis, the identification of those residues implicated in the burial requires NMR analysis. We have selected a partially solvated natively disordered fragment of Escherichia coli, thioredoxin, C37 (38–108), for full NMR spectral assignment. The secondary chemical shifts, temperature coefficients, and relaxation rates (R1 and R2) of this fragment indicate the presence of a flexible backbone without a stable hydrogen bond network near neutral pH. 1H-15N heteronuclear single quantum coherence analysis of the pH dependence of amide chemical shifts in fragment C37 within pH 2.0 and 7.0 suggests the presence of interactions between nonionizable residues and the carboxylate groups of four Asp and four Glu residues. The pH midpoints (pHm) of the amides in the ionizable residues (Asp or Glu) and, consequently, the shifts in the pHm (ΔpHm) of these residues with respect to model tetrapeptides, are sequence-dependent; and the nonionizable residues that show pH dependence cluster around the ionizable ones. The same pH dependence has been observed in two fragments: M37 (38–73) and C73 (74–108), ruling out the participation of long-range interactions. Our studies indicate the presence of a 15-residue pH-dependent segment with the highest density of ionizable sites in the disordered ensembles of fragments C37 and M37. The observed correlations between ionizable and nonionizable residues in this segment suggest the organization of the backbone and side chains through local and medium-range interactions up to nine residues apart, in contrast to only a few interactions in fragment C73. These results agree qualitatively with the predominantly hydrophobic buried surface detected only in fragments C37 and M37 by highly sensitive differential scanning calorimetry analysis. This work offers a sensitive and rapid new tool to obtain clues about local and nonlocal interactions between ionizable and nonionizable residues in the growing family of natively disordered small proteins with full NMR assignments.

Pelseneeriol-1 and -2: new furanosesquiterpene alcohols from porostome nudibranch Doriopsilla pelseneeri

The paper reports the first chemical study of the porostome nudibranch Doriopsilla pelseneeri collected off the Portuguese coast (Atlantic Ocean). Two new furanosesquiterpene alcohols, pelseneeriol-1 ( 1 ) and pelseneeriol-2 ( 2 ), have been isolated together with known compounds, 15-acetoxy- ent-pallescensin-A ( 5 ), and dendocarbin-A ( 6 ), from the mantle of the nudibranch, whereas euryfuran ( 3 ) and drimane ester mixture 4 were identified in the extract of the internal glands. The structures of 1 and 2 have been determined by extensive spectroscopic studies as well as by comparison with literature model compounds. In order to assess the relative stereochemistry of 1 and 2 , full NMR assignment of related sponge metabolite microcionin-2 ( 8 ) and of co-occurring sesquiterpenes 9– 11 , that have been re-isolated from the Mediterranean sponge Fasciospongia cavernosa, has been also conducted. In particular, the relative stereochemistry of tricyclic sesquiterpene microcionin-1 ( 9 ) has now been rigorously assigned by detailed analysis of NOE difference experiments.

Full NMR assignment and revised structure for the capsular polysaccharide from Streptococcus pneumoniae type 15B

The capsular polysaccharide from Streptococcus pneumoniae Type 15B is a component of the 23-valent polysaccharide vaccine against pneumococcal disease. We report full NMR assignments for the native and de- O-acetylated polysaccharide, and confirm that the phosphorylated substituent is glycerol-2-phosphate rather than phosphocholine, located on O-3 of the side chain β-Gal p residue. The polysaccharide is O-acetylated on the terminal α-Gal residue, distributed between O-2, O-3, O-4 and O-6 in a ratio of 6:12:12:55, with approximately 15% of the repeat units not O-acetylated.

NMR analysis of (1S, 1aR, 6aR)-2’, 3’, 6, 6a-tetrahydrospiro cycloprop a indene-1(1aH), 1’- 1H indene

The aldol condensation product of 1H-indan-1-one, (2E)-2-(2,3-dihydro-1H-in den-1-ylidene)-2,3-dihydro-1H-inden-1-one, subjected to Huang?Minlon reduction conditions was shown, via 1D and 2D NMR analysis, to be a mixture of (1S,1aR,6aR)-2?,3?,6,6a-tetrahydro-spiro cycloprop a indene-1(1aH),1? 1H indene and its 1R,1aS,6aS enantiomer and not 2,3,1?,3?-tetrahydro- 1,2? -biindenylidene as originally expected. The full NMR assignment, the coupling constants in the proton NMR, and the couplings in the HMBC and NOESY of the title compound are summarized in the Table.

Isolation of 41a-Homoyessotoxin and the Identification of 9-Methyl-41a-homoyessotoxin and Nor-ring A-yessotoxin from Protoceratium reticulatum

41a-homoyessotoxin (1), a new analogue of yessotoxin (7), was isolated from extracts of Protoceratium reticulatum grown in culture. In addition, 9-methyl-41a-homoyessotoxin (2) and a nor-ring A-yessotoxin (3) were identified as the major components of a mixed fraction. The structural information was initially obtained from LC-UV and LC-MS3 chromatograms, with subsequent definitive structure determination by NMR, LC-MS/MS, and high-resolution MS. Full 1H and indirectly detected 13C NMR assignments for all but two carbon atoms were obtained from ca. 100 microg of 1. Full 1H NMR assignments for 2 and 3 and identification of three new heptanor-41-oxo-analogues of 3 (4-6) during LC-MS3 analysis of a fraction containing 1-3 and 7 are also reported.

Isolation of pectenotoxin-2 from Dinophysis acuta and its conversion to pectenotoxin-2 seco acid, and preliminary assessment of their acute toxicities

We have developed a simple and effective method for isolating pectenotoxin-2 (PTX-2) from Dinophysis cells collected from a natural bloom. A two-step extraction procedure followed by two column chromatography steps produced PTX-2 in high purity suitable for use as an analytical standard and for toxicological studies. Incubation of purified PTX-2 with the supernatant from ultracentrifuged blue ( Mytilus edulis) or Greenshell ® ( Perna canaliculus) mussel hepatopancreas homogenate caused rapid conversion to pectenotoxin-2 seco acid (PTX-2 SA). Purification of PTX-2 SA was achieved by solvent extraction followed by column chromatography. PTX-2 and PTX-2 SA were fully characterized by LC–MS and NMR, and full 1H and 13C NMR assignments were obtained. Okadaic acid C 8-diol ester was isolated during the purification of PTX-2, and its identity confirmed by NMR and LC–MS analyses. Pectenotoxin-2 seco acid methyl ester, identified by LC–MS, was also produced during the hydrolytic procedure due to the presence of methanol. PTX-2 was acutely toxic to mice by i.p. injection (LD 50=219 μg/kg) but no effects were seen with PTX-2 SA at 5000 μg/kg. Neither PTX-2 nor PTX-2 SA was overtly toxic to mice by the oral route at doses up to 5000 μg/kg. No diarrhea was observed in mice dosed with either compound, suggesting that pectenotoxins do not belong in the diarrhetic shellfish poison group.