C andH NMR spectroscopy as a tool in the configurational analysis of iridoid glucosides

The ¹³C NMR data of 51 iridoid glucosides or glucoside acetates are tabulated. The collection includes 20 pairs of C-6, C-7 or C-8 epimers. Three parameters in using the data for the configurational assignment of 6-O-substituents are given. The chemical shift for C-9 in a range of substituted compounds is shown to be numerically related to the stereochemistry at C-8. This allows the determination of the configuration at this centre for most types of substitution patterns by calculation of the C-9 shift using increments for each substituent. Such increments are given for 25 substituents in three different solvents. A method for simulation of spectra of unknown iridoid glucosides is presented. By this method, the structures of five novel iridoid glucosides have been elucidated, and that of tecomoside has been revised. The methods used to assign the configurations to C-6 and C-8 epimeric iridoid glucosides by ¹H NMR spectroscopy are discussed and a table with selected data is presented. It is suggested that the structures in the literature for ajugol and myoporoside should be interchanged. Consequently, Horeau's method has failed in these instances. Finally, the differences in the ¹³C NMR spectra of pairs of C-6 and C-8 epimeric iridoid glucosides have been interpreted as originating from cis/trans-interactions.     

A critical evaluation of the conformational requirements of fusogenic peptides in membranes

It is generally assumed that fusogenic peptides would require a certain conformation, which triggers or participates in the rate-determining step of membrane fusion. Previous structure analyses of the viral fusion peptide from gp41 of HIV-1 have yielded contradictory results, showing either an α-helical or a β-stranded conformation under different conditions. To find out whether either of these conformations is relevant in the actual fusion process, we have placed sterically demanding substitutions into the fusion peptide FP23 to prevent or partially inhibit folding and self-assembly. A single substitution of either D- or L-CF₃-phenylglycine was introduced in different positions of the sequence, and the capability of these peptide analogues to fuse large unilamellar vesicles was monitored by lipid mixing and dynamic light scattering. If fusion proceeds via a β-stranded oligomer, then the D- and L-epimers are expected to differ systematically in their activity, since the D-epimers should be unable to form β-structures due to sterical hindrance. If an α-helical conformation is relevant for fusion, then the D-epimers would be slightly disfavoured compared to the L-forms, hence a small systematic difference in fusion activity should be observed. Interestingly, we find that (1) all D- and L-epimers are fusogenically active, though to different extents compared to the wild type, and – most importantly – (ii) there is no systematic preference for either the D- or L-forms. We therefore suggest that a well-structured α-helical peptide conformation or a β-stranded oligomeric assembly can be excluded as the rate-determining state. Instead, fusion appears to involve conformationally disordered peptides with a pronounced structural plasticity. Dedicated to Prof. K. Arnold on the occasion of this 65th birthday.     

Erythrinaline alkaloids from the flowers and pods of Erythrina lysistemon and their DPPH radical scavenging properties

Fourteen different erythrinaline alkaloids have been isolated from the flowers and pods of Erythrina lysistemon with four being reported for the first time in nature and five for the first time in this species and the rest having been re-isolated. The new compounds are (+)-11beta-hydroxyerysotramidine (1), (+)-11beta-methoxyerysotramidine (2), (+)-11beta-hydroxyerysotrine N-oxide (4) and (+)-11beta-hydroxyerysotrine (8). (+)-11alpha-Hydroxyerysotrine N-oxide (3), earlier misidentified as erythrartine N-oxide (beta-hydroxyerysotrine N-oxide 4), was also re-isolated along with four other alkaloids. Correct identification of compounds 4 and 8 was aided by the fact that the two sets of C-11 epimers 3, 4 and 8, 9 were both isolated in this study thus making it easier to identify and assign the individual epimers. (+)-Erythristemine (14) was found distributed in most of the plant parts investigated. Preliminary work on the crude chloroform/methanol (1:1) showed moderate toxicity to brine shrimp (LC50 23 ppm) and moderate (IC50 86 microg/ml) radical scavenging properties against stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. The DPPH radical scavenging properties of the isolated compounds were assessed using TLC autographic and spectrophotometric assays whereupon only compounds 11 (1 microg; 90 microg/ml) and 12 (0.1 microg; 160 microg/ml) showed any notable activity. It appears the two compounds are slow reacting and do not reach steady state conditions within the standard half an hour time frame but only seemed to have reached steady state conditions after 4 h.     

Successful prediction of the hydrogen bond network of the 3-oxo-12alpha-hydroxy-5beta-cholan-24-oic acid crystal from resolution of the crystal structure of deoxycholic acid and its three 3,12-dihydroxy epimers

Crystal structures of p-xylene-crystallized deoxycholic acid (3alpha,12alpha-dihydroxy-5beta-cholan-24-oic acid) and its three epimers (3beta,12alpha-; 3alpha,12beta-; and 3beta,12beta-) have been solved. Deoxycholic acid forms a crystalline (P21) complex with the solvent with a 2:1 stoichiometry whereas crystals of the three epimers do not form inclusion compounds. Crystals of the 3beta,12beta-epimer are hexagonal, whereas the 3alpha,12beta-and 3beta,12alpha-epimers crystallize in the P2(1)2(1)2(1) orthorhombic space group. The three hydrogen bond sites (two hydroxy groups, i. e. O3-H, and O12-H, and the carboxylic acid group of the side chain, O24bO24a-H) simultaneously act as hydrogen bond donors and acceptors. The hydrogen bond network in the crystals was analyzed and the following sequences have been observed: two chains (abcabc... or acbacb... ) and two rings (abc or acb), which constitute a complete set of all the possible sequences which can be drawn for an intermolecular hydrogen bond network formed by three hydrogen bond donor/acceptor sites forming crossing hydrogen bonds. The orientation of O3-H (alpha or beta) determines the sequence of the acceptor and the donor groups involved in the pattern: O24a --> O12 --> O3 --> O24b when it is alpha and O24a --> O3 --> O12--> O24B when it is beta. These observations were used to predict the hydrogen bond network of p-xylene-crystallized 3-oxo,12alpha-hydroxy-5beta-cholan-24-oic acid. This compound has two hydrogen bond donor and three potential hydrogen bond acceptor sites. According to the previous sequence set, this compound should crystallize in the monoclinic P21 system, should form a complex with the solvent, O24b should not participate in the hydrogen bond network, and the chain sequence O24a --> O12 --> O3 would be followed. All predictions were confirmed experimentally.     

The efficient structure elucidation of minor components in heparin digests using microcoil NMR

The structural complexity and microheterogeneity of the glycosaminoglycans heparin and heparan sulfate make their characterization a daunting task. The methodology described herein utilizes a combination of enzymatic digestion, size-exclusion chromatography, strong anion-exchange HPLC, reverse-phase ion-pair ultrahigh performance liquid chromatography–mass spectrometry, and microcoil NMR for the efficient sequencing of heparin-derived tetrasaccharides. The high mass sensitivity of microcoil NMR makes this technique well suited for the characterization of mass-limited samples removing a bottleneck in the analysis workflow and permitting structural characterization of minor components isolated from a heparin enzymatic digestion. Complete characterization of one tetrasulfonated, five pentasulfonated isomers and two hexasulfonated tetrasaccharide sequences is described. To our knowledge, two of the identified minor tetrasaccharides are unique, and have not been previously reported: IdoA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS(6S) and ΔUA(2S)-GlcNS(6S)-IdoA-GlcNS(6S).     

The 220 MHz NMR spectra of phytosterols

The 220MHz NMR spectra of forty two steroids are reported. Eight pairs of C-24 epimers (24α- and 24β) and two pairs of double bond isomers (cis and trans) can be distinguished by this technique. The influence of substituents, solvents and stereochemistry on methyl group chemical shifts is discussed.     

Epimeric methylsulfinyladenosine derivatives from the marine ascidian Herdmania momus

Investigation of the secondary metabolites of the ascidian Herdmania momus led to the isolation and characterization of four new nucleoside derivatives (1–4). Structural studies showed that these derivatives represent a series of rare methylsulfinyladenosine derivatives of interconvertible transesterification isomers and/or sulfinyl epimers. The antiviral activities of these rare nucleosides were evaluated against a series of human pathogenic viruses.     

New hexalactone derivatives and a pair of new oxaspiro-carbon epimeric glycosides from the fruits of Illicium lanceolatum

Five new compounds (1–5), including three hexalactone derivatives (1–3) and a pair of new oxaspiro-carbon epimeric glycosides (4 and 5), and six known compounds (6–11) were obtained from the fruits of Illicium lanceolatum. The structures of the new compounds were elucidated using extensive spectroscopic data. The absolute configurations of compounds 1–3 were determined by an analysis of their CD spectra. It was determined that compounds 4 and 5, which are epimeric at C-5, possess the same 1-oxaspiro[4,5]decane-7α,8α,9β-triol moiety. Plausible biogenetic pathways for 4 and 5 derived from the key precursor shikimic acid were proposed. Compounds 1–11 were all assayed on monosodium glutamate-induced human neuroblastoma SH-SY5Y cell damage. The results demonstrated that compounds 4, 5, and 8–10 possess potential neuroprotective effects. The anti-inflammatory, antiviral, and cytotoxic activities of 1–11 were also evaluated.     

Accurate and reliable quantification of 25-hydroxy-vitamin D species by liquid chromatography high-resolution tandem mass spectrometry

In general, mass spectrometric quantification of small molecules in routine laboratory testing utilizes liquid chromatography coupled to low mass resolution triple-quadrupole mass spectrometers (QQQs). Here we introduce high-resolution tandem mass spectrometry (quadrupole-Orbitrap) for the quantification of 25-hydroxy-vitamin D [25(OH)D], a marker of the vitamin D status, because the specificity of 25(OH)D immunoassays is still questionable and mass spectrometric quantification is becoming increasingly important. Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/HR-MS) was used to quantify 25-hydroxy-cholecalciferol [25(OH)D₃], 25-hydroxy-ergocalciferol [25(OH)D₂], and their C3-epimers 3-epi-25(OH)D₃ and 3-epi-25(OH)D₂. The method has a run time of 5 min and was validated according to the US Food and Drug Administration and the European Medicines Agency guidelines. High mass resolution was advantageously applied to separate a quasi-isobaric interference of the internal standard D₆-25(OH)D₂ with 3-epi-25(OH)D₃. All analytes showed an imprecision of below 10% coefficient of variation (CV), trueness between 90% and 110%, and limits of quantification below 10 nM. Concentrations measured by LC-MS/HR-MS are in good agreement with those of the National Institute of Standards and Technology reference methods using LC-MS/MS (QQQ). In conclusion, quantification of 25(OH)D by LC-MS/HR-MS is applicable for routine testing and also holds promise for highly specific quantification of other small molecules.     

EI/MS fragmentation of abieta-8,11,13-triene diterpenoid derivatives as a tool to identify swartziarboreols in Swartzia extracts

A systematic molecular fragmentation pathway (SMFP) was designed as an identifier to detect swartziarboreols (abieta-8,11,13-triene diterpenoid derivatives) in Swartzia extracts. The SMFP was rationalised through the interpretation of the EI/MS of swartziarboreols isolated from S. arborescens and S. langasdorffii and by analogy with the fragmentations observed for previously isolated compounds. Two pairs of swartziarboreol epimers were identified by GC-MS in an extract of S. langsdorffii using SMFP. Acetylation and methylation of the dichloromethane extract furnished, respectively, 3 and 4, which suggested the occurrence of a new natural orthodiphenolic swartziarboreol.