Bastien Nay

Natural products from Cephalotaxus sp.: chemical diversity and synthetic aspects

The Cephalotaxus genus belongs to the Cephalotaxaceae family of conifers. Over the past decades it has proved to be a fruitful source of interesting natural products, especially alkaloids (cephalotaxine esters) and terpenoids (abietanes, troponoids), which often display medicinal properties, especially in the anticancer area. Homoharringtonine is active against some orphan leukaemia and is nowadays approaching marketability. A phytochemical update will be provided and the total synthesis of alkaloids and terpenoids will be discussed in detail.

Chemical communication between the endophytic fungus Paraconiothyrium variabile and the phytopathogen Fusarium oxysporum.

Paraconiothyrium variabile, one of the specific endophytic fungi isolated from the host plant Cephalotaxus harringtonia, possesses the faculty to inhibit the growth of common phytopathogens, thus suggesting a role in its host protection. A strong antagonism between the endophyte P. variabile and Fusarium oxysporum was observed and studied using optic and electronic microscopies. A disorganization of the mycelium of F. oxysporum was thus noticed. Interestingly, the biological effect of the main secondary metabolites isolated from P. variabile against F. oxysporum did not account for this strong antagonism. However, a metabolomic approach of pure fungal strains and confrontation zones using the data analysis tool XCMS were analyzed and pointed out a competition-induced metabolite production by the endophyte in the presence of the phytopathogen. Subsequent MS/MS fragmentations permitted to identify one of the induced metabolites as 13-oxo-9,11-octadecadienoic acid and highlighted a negative modulation of the biosynthesis of beauvericin, one of the most potent mycotoxin of F. oxysporum, during the competition with the endophyte.

Total synthesis of isotopically labelled flavonoids. Part 5: Gram-scale production of 13C-labelled (−)-procyanidin B3☆

Gram amounts of 13C-labelled dimer (−)-procyanidin B3 5 were prepared from 1-[13C]acetic acid by coupling the optically labelled flavan-3,4-diol (+)-7 with protected natural catechin (+)-3 in acidic medium.

Mechanism of an insect glutathione S-transferase: kinetic analysis supporting a rapid equilibrium random sequential mechanism with housefly I1 isoform

The steady-state kinetics of glutathione S-transferase I1 (GST I1) from housefly Musca domestica expressed in Escherichia coli were investigated with glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Concentrations of the varied substrates were from 0.03 to 1 mM for GSH and 0.05 to 1 mM for CDNB. Within this range, Michaelis-Menten behaviour was observed and convergent straight lines in double reciprocal plots excluded a ping-pong kinetic mechanism. Instead, data were consistent either with rapid-equilibrium random or with steady-state ordered sequential mechanisms because of abscissa convergence. Discrimination was achieved by studying the reaction with another electrophilic partner, p-nitrophenyl-acetate (PNPA). Concentrations of PNPA and GSH varied within the ranges 0.5 to 10 mM and 0.03 to 0.6 mM, respectively. The complete set of data supports the proposal of a rapid-equilibrium random-sequential model with strictly independent sites for GSH and CDNB or PNPA. Kinetic parameters are thus true dissociation equilibrium constants with values of 0.15 mM for GSH, 0.15 mM for CDNB, and 7 mM for PNPA. Analysis of the inhibition by the product (S-(2,4-dinitrophenyl)-glutathione, 10 to 100 microM), on the coupling reaction between GSH and CDNB with either GSH (0.05 to 0.5 mM, CDNB 0.2 mM) or CDNB (0.05 to 0.5 mM, GSH 0.2 mM) varied, consistent with the proposed mechanism. Binding of product to the free enzyme excludes GSH (competitive inhibition pattern with Kp = 12 microM) but only slightly hinders binding of CDNB. Binding free energies, together with the inhibition pattern, suggest that the non-peptidic moiety of product interacts with an alternative sub-site within the large open pocket accommodating the various electrophilic substrates. These results lead us to propose a model for intra-pocket shifting of the non-peptidic moiety upon product formation which contributes to the product release.

Further studies of the norditerpene (+)-harringtonolide isolated from Cephalotaxus harringtonia var. drupacea: absolute configuration, cytotoxic and antifungal activities.

Harringtonolide (= hainanolide) is a complex polycyclic fused norditerpene isolated from CEPHALOTAXUS HARRINGTONIA var. DRUPACEA. In spite of its appealing biological properties - we measured an IC (50) of 43 nM on KB cells and a significant antifungal activity - its absolute configuration has not yet been firmly established. This was done herein using X-ray anomalous scattering after bromination of the tropone ring, unambiguously giving the stereochemistry 5 R,6 R,7 S,13 S,14 S,15 R,16 R. Detailed IN VITRO biological measurements are provided.

Total synthesis of isotopically labelled flavonoids. Part 3:†13C-labelled (−)-procyanidin B3 from 1-[13C]-acetic acid

Abstract The regioselectively 13C-labelled (−)-procyanidin B3 was prepared in eight steps from 1-[13C]-acetic acid.

Gram-Scale Production and Applications of Optically Pure13C-Labelled (+)-Catechin and (−)-Epicatechin

In this paper gram-scale asymmetric total syntheses of pure (+)-4-[13C]catechin (1) and (−)-4-[13C]epicatechin (2) are described. Labelling was introduced through acylation of a phloroglucinol derivative 4 by 1-[13C]acetic acid, after activation by TFAA. Condensation of the resulting C6−C2 acetophenone building block 6 with a C1−C6 benzaldehyde unit 9 provided the C6−C3−C6 flavonoid skeleton, with benzyl ethers as phenol protecting groups. Asymmetry was introduced by an inexpensive and very efficient resolution process, performed at the penultimate step towards naturally occurring flavan-3-ols, by using tartaric acid derivatives. Both enantiomers of 4-[13C]catechin were obtained with a high level of enantiomeric purity, especially (+)-1 of the natural series. The other enantiomer (−)-1 was a valuable precursor of natural (−)-4-[13C]epicatechin by epimerisation at C-2. These two natural flavanols are thus obtained for the first time in large quantities in optically pure form as labelled compounds and will be useful tools for biological studies using NMR or isotopic mass spectrometry in forthcoming experiments.

Geographic locality greatly influences fungal endophyte communities in Cephalotaxus harringtonia

Although endophytes of conifers have been extensively studied, few data are available on Cephalotaxaceae. We examined foliar and stem endophytes of Cephalotaxus harringtonia, within its natural range in Japan and outside its natural range in France to study the effect of geography on endophyte community composition. In Japan, rapidly growing endophytes were dominant and may have masked the real diversity, in comparison to France where most endophytes were growing slowly. Analyses of ITS rDNA revealed 104 different Blast Groups among 554 isolates. Almost no overlap between endophyte assemblages of C. harringtonia from the two countries was observed. It seems that Japanese C. harringtonia trees, which should be well adapted to their native site, would host a specific, endemic endophyte community, while trees that have been introduced recently to a foreign site, in France, should have captured existing cosmopolitan and more generalist taxa. In Japan the majority of xylariaceous taxa, which dominated the communities, were unknown and, although closely related to Asian taxa, may be new to science. Dothideomycetes were more prevalent in France. Locally, urban environment, particularly in Japan, may have introduced some perturbations in the native endophyte community of C. harringtonia, with an abundance of generalist fungi such as Nigrospora and Colletotrichum.

Asymmetric Synthesis of the Oxygenated Polycyclic System of (+)-Harringtonolide

A straightforward asymmetric synthesis of the cage oxygenated structure of (+)-harringtonolide has been accomplished for the first time. The key steps involved (i) a templated stereoselective IMDA reaction to build a highly functionalized cyclohexene ring D, (ii) functionalization of the cycloadduct, (iii) ring-closing metathesis providing the five-membered ring C, and finally (iv) a challenging one-step cascade cyclization of an epoxy-alcohol toward the target structure, whose mechanism was investigated.

Methods in synthesis of flavonoids.: Part 2:1 High yield access to both enantiomers of catechin

Abstract Resolution of racemic synthetic tetra- O -benzylcatechin 2 is described, through the formation of esters 5 and 6 derived from dibenzoyl- l -tartaric acid. The diastereoisomer of the natural series 6 was separated by crystallization, the other one being an oil. This process allowed us to prepare enantiomerically pure (+)-catechin 8 in high yield. The pure isomer in the ent -series 9 could be obtained, following the same scheme of reactions.