Joaquín Altarejos

Virus‐induced gene silencing identifies Catharanthus roseus 7‐deoxyloganic acid‐7‐hydroxylase, a step in iridoid and monoterpene indole alkaloid biosynthesis

Iridoids are a major group of biologically active molecules that are present in thousands of plant species, and one versatile iridoid, secologanin, is a precursor for the assembly of thousands of monoterpenoid indole alkaloids (MIAs) as well as a number of quinoline alkaloids. This study uses bioinformatics to screen large databases of annotated transcripts from various MIA-producing plant species to select candidate genes that may be involved in iridoid biosynthesis. Virus-induced gene silencing of the selected genes combined with metabolite analyses of silenced plants was then used to identify the 7-deoxyloganic acid 7-hydroxylase (CrDL7H) that is involved in the 3rd to last step in secologanin biosynthesis. Silencing of CrDL7H reduced secologanin levels by at least 70%, and increased the levels of 7-deoxyloganic acid to over 4 mg g(-1) fresh leaf weight compared to control plants in which this iridoid is not detected. Functional expression of this CrDL7H in yeast confirmed its biochemical activity, and substrate specificity studies showed its preference for 7-deoxyloganic acid over other closely related substrates. Together, these results suggest that hydroxylation precedes carboxy-O-methylation in the secologanin pathway in Catharanthus roseus.

Synthesis of biologically active drimanes and homodrimanes from (−)-sclareol

Abstract Three drimanes, polygodial (2), albicanyl acetate (3) and 7-oxo-8,12-drimen-11-al (5), and two homodrimanes, 13,14,15,16-tetranorlabd-7-en-12,17-dial (6) and 7-oxo-13,14,15,16-tetranorlabd-8(17)-en-12-al (7), were synthesized from (−)-sclareol (1), and their antifeedant, antitumor and antimicrobial properties tested. In most cases, 6 and 7 were found to be more active than 2.

Synthesis of Ambrox® from(-)-sclareol and (+)cis-abienol

Abstract Short and efficient syntheses of (−)-Ambrox® (12) from (−)-sclareol (1) and (+)-cis-abienol (11) are described. In constrast to previously described procedures, the transformation of 1 to 12, involving in the key step, an oxidative degradation by catalytic osmium tetroxide, in the presence of sodium periodate, has the advantage of using the more suitable sodium borohydride, as the reducing agent. The isolation and characterization of some reaction intermediates allowed us to confirm the degradation mechanism.

Chemical studies of essential oils of Juniperus oxycedrus ssp. badia.

Leaf and (unripe and ripe) berry essential oils of Juniperus oxycedrus ssp. badia (H. Gay) Debeaux grown wild in Spain have been analysed by capillary GC and GC-MS in combination with retention indices. A seasonal investigation of both leaf and berry oils was also performed. Among the approximately 80 constituents investigated (representing 90-98% of the oils) 60-68 were identified (80-97% of the oil composition). The leaf oils were mainly composed of alpha-pinene (40-57%) and manoyl oxide (5-10%). The (unripe) berry oils were dominated by alpha-pinene (65%) with moderate amounts of myrcene, limonene, germacrene D or gamma-muurolene. Several differences in yields and chemical composition from a qualitative and quantitative point of view were detected when comparing all the oil samples analysed. In addition, two oil samples were examined, but found inactive, against the replication of HIV-1(III(B)) and HIV-2(ROD) in cell culture, whereas the samples were toxic for the cells at a 50% cytotoxic concentration of 106 and 123 microg/ml, respectively.

Stereochemistry of 14-hydroxy-β-caryophyllene and related compounds

The isomerization of β-caryophyllene (3), under treatment with SeO2, is described. Chemical correlations, between 3 14--hydroxy-β-caryophylllene (6z) from Juniperus oxycedrus, are establised. High resolution 1H NMR spectra and analysis by molecular mechanics of 3, 6 and 14-acetoxy-β-caryophyllene (7) indicate the existence of two conformational isomers, βα and ββ, in each compound. At 35°C, the βα conformer predominates in 3 and 7 but the ββ conformer predominates in 6. The higher precentage of 6ββ possibly derives from an intramolecullar hydrogen bond. The treatment of 3, 6 and 7 with m-CPBA generates, in each case, two diasteromeric 4,5-epoxi-derivatives. The epoxides obtained from 6 have been isolated and analysed separately.

Synthesis of Ambrox® from communic acids

Two routes for preparing Ambrox® (1) from the methyl esters of trans-communic acid (2b) and/or cis-communic acid (3b), via selective degradation of their side chains, stereoselective formation of the tetrahydrofurane ring, and reduction of the axial methoxycarbonyl group, are described.

Synthesis of Ambrox® from labdanolic acid

Abstract A synthesis of the valuable amber-type odorant Ambrox® from labdanolic acid (main diterpenoid of the acid fraction of non-polar extracts of Cistus ladaniferus L.) is reported. The conversion is based on (a) the α,β-dehydrogenation of methyl labdanolate using an organoselenium reagent, (b) subsequent oxidative degradations of the side chain, and (c) final acid-promoted cyclization of the resulting tetranorlabdan-8α,12-diol. The influence of the temperature and solvent in the cyclization of the diol with p-toluenesulfonic acid is also described. Thus, Ambrox® has been obtained by a six-step procedure in 33% overall yield from methyl labdanolate.

The Leaf Essential Oils and Taxonomy of Juniperus oxycedrus L. subsp. oxycedrus, subsp. badia(H. Gay) Debeaux, and subsp. macrocarpa (Sibth. & Sm.) Ball.

Abstract The leaf essential oils of Juniperus oxycedrus L. subsp. oxycedrus, subsp. badia (H. Gay) Debeaux and subsp. macrocarpa (Sm.) Ball have been analyzed by GC/MS. One hundred twenty two compounds were found in these three subspecies. The leaf oils of J. oxycedrus were dominated by α-pinene (25–43%), and limonene (4.5–28%), with moderate amounts of β-pinene, myrcene, p-cymene, β-phellandrene and manoyl oxide, whereas the leaf oils of J. oxycedrus subsp. badia were dominated by α-pinene and germacrene D with variable amounts of manoyl oxide and moderate amounts of α-campholenal, β-bourbenene and several unknown sesquiterpenes. The oils of subsp. badia contained nine unknown sesquiterpenes that were only found in this taxon. The oil of subsp. macrocarpa was dominated by sabinene and α-pinene with moderate amounts of p-cymene, γ-terpinene and terpinen-4-ol. Chemically, the three subspecies appear to be distinct and this warrants the continued recognition of these subspecies.

Olive tree wood phenolic compounds with human platelet antiaggregant properties

Oleuropein and (+)-cycloolivil are natural polyphenolic compounds with a significant radical scavenging activity present in olive tree. We have investigated the antiaggregant effects of oleuropein and (+)-cycloolivil isolated from an ethyl acetate extract of olive tree wood. Oleuropein and (+)-cycloolivil reduced the ability of thrombin to stimulate platelet aggregation. Both compounds reduced thrombin-evoked Ca(2+) release and entry to a similar extent to hydroxytyrosol. This effect was greater in platelets from patients with type 2 diabetes mellitus than in controls. Thrombin-, thapsigargin- and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ)-evoked protein tyrosine phosphorylation, which is involved in Ca(2+) signalling and platelet aggregation, is inhibited by oleuropein and (+)-cycloolivil. oleuropein and (+)-cycloolivil are natural oxygen radical scavengers that reduce thrombin-induced protein tyrosine phosphorylation, Ca(2+) signalling and platelet aggregation. These observations suggest that oleuropein and (+)-cycloolivil may prevent thrombotic complications associated to platelet hyperaggregability and be the base for the development of antiaggregant therapeutic strategies.

Chemical Composition and Seasonal Variations of Rosemary Oil from Southern Spain

Abstract The composition of 12 essential oil samples, obtained by steam distillation of twigs of rosemary (Rosmarinus officinalis L.) harvested in four different locations from southern Spain and during three phenological stages, has been analyzed by capillary GC and GC/MS in combination with retention indices. Quantitative data relative to 53 constituents (accounting for 96.7-99.2% of the oils), 12 of which being reported for the first time in Spanish rosemary oil, are described. All samples studied belong to the chemotype α-pinene/l,8-cineole/eamphor, the main and characteristic components being camphor (17.2-34.7%), α-pinene (10.2-21.6%), 1,8-cineole (12.1-14.4%), camphene (5.2-8.6%), borneol (3.2-7.7%), β-pinene (2.3-7.5%), verbenone (2.2-5.8%), β-caryophyllene (1.8-5.1%), limonene (2.0-3.8%), α-terpineol (1.2-2.5%), myrcene (0.9-4.5%), p-cymene (0.2-3.4%), bornyl acetate (0.2-2.3%), linalool (0.3-1.0%) and terpinen-4-ol (0.4-0.9%). The effect of harvest time on the oil production and chemical composition was examined. The highest oil yields (1.6-1.8%) were recorded during the fruiting period (summer). In general, minimum amounts of camphor and maximum amounts of α-pinene were observed in winter. The concentration of 1,8-cineole was almost constant throughout the year, though other oil constituent levels varied randomly with the plant life cycle. Thus, seasonal and geographical variations on the content of representative components allowed to achieve a quality assessment of the rosemary oils and, consequently, to deduce the best period for processing rosemary in the studied area.