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Males of the closely related species Bombus terrestris and Bombus lucorum attract conspecific females by completely different marking pheromones. MP of B. terrestris and B. lucorum pheromones contain mainly isoprenoid (ISP) compounds and fatty acid derivatives, respectively. Here, we studied the regulation of ISP biosynthesis in both bumblebees. RNA-seq and qRT-PCR analyses indicated that acetoacetyl-CoA thiolase (AACT), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), and farnesyl diphosphate synthase (FPPS) transcripts are abundant in the B. terrestris labial gland. Maximal abundance of these transcripts correlated well with AACT enzymatic activity detected in the LG extracts. In contrast, transcript abundances of AACT, HMGR, and FPPS in B. lucorum were low, and AACT activity was not detected in LGs. These results suggest that transcriptional regulation plays a key role in the control of ISP biosynthetic gene expression and ISP pheromone biosynthesis in bumblebee males.

Buff-tailed bumblebees, Bombus terrestris, use a male sex pheromone for premating communication. Its main component is a sesquiterpene, 2,3-dihydrofarnesol. This paper reports the isolation of a thiolase (acetyl-CoA thiolase, AACT_BT), the first enzyme involved in the biosynthetic pathway leading to formation of isoprenoids in the B. terrestris male sex pheromone. Characterisation of AACT_BT might contribute to a better understanding of pheromonogenesis in the labial gland of B. terrestris males. The protein was purified to apparent homogeneity by column chromatography with subsequent stepwise treatment. AACT_BT showed optimum acetyltransferase activity at pH 7.1 and was strongly inhibited by iodoacetamide. The enzyme migrated as a band with an apparent mass of 42.9 kDa on SDS-PAGE. MS analysis of an AACT_BT tryptic digest revealed high homology to representatives of the thiolase family. AACT_BT has 96 % amino acid sequence identity with the previously reported Bombus impatiens thiolase.

The enantioselective allylation of an aromatic aldehyde to give a chiral homoallylic alcohol was employed as the key step in the syntheses of a flobufen metabolite and dapoxetine. In the former case, the homoallylic alcohol moiety (99 % ee) was converted into a five-membered lactone ring with good preservation of the optical purity, and the target compound, a flobufen metabolite, was obtained in 95 % ee. In the latter case, the homoallylic alcohol moiety (97 % ee) was transformed over several steps into a 3-aminopropanol moiety. During the course of the synthesis, the gradual loss of optical purity was observed, and the target compound, dapoxetine, was obtained in 85 % ee.

Triacylglycerols (TGs) stored in the fat bodies of bumblebee males have a species-specific composition. The striking structural similarities between TG fatty acids (FAs) and components of the male marking pheromone in certain species led to the hypothesis that FAs may serve as precursors in pheromone biosynthesis. Here, we analysed TGs from B. ruderatus, B. bohemicus, and B. campestris. Nonadec-9-ene and icos-15-en-1-ol are the main components of B. ruderatus labial gland secretion, forming up to 92% of the gland extract. The corresponding icos-11-enic and icos-15-enic acids were found in TGs at levels higher than usual for bumblebee species. We found similar relationships in B. campestris and B. bohemicus. These results suggest that FAs might be precursors of aliphatic compounds in the male pheromones. Furthermore, we report for the first time the pheromone structure of B. ruderatus males.

The enantioselective allylation of thio- phene-2-carbaldehyde with allyltrichlorosilane under Lewis base catalysis has been studied. The use of cat- alytic amount (1 mol%) of chiral bipyridine N,N'-di- oxides provided the corresponding 1-(thiophen-2-yl)- but-3-en-1-ol with asymmetric induction reaching 97% ee. The prepared chiral (S)-1-(thiophen-2-yl)- but-3-en-1-ol was used as the crucial chiral building block in a formal total synthesis of duloxetine.

Instrumental human scent analysis is undoubtedly desirable for many forensic as well medical applications. Most of the previous human scent studies were focused on volatile organic compounds (VOCs) which were analysed by head space solid phase micro-extraction gas chromatography/mass spectrometry (HS-SPME-GC/MS). This method is, however, significantly less sensitive to "heavier" less volatile compounds emitted from the human skin. These less volatile organic scent molecules probably create the basis of the individual human scent signature, and therefore, our attention is focused mainly on these "heavier" compounds. The human scent was adsorbed onto purified glass beads and samples were prepared as hexane solutions obtained by extraction from the sampled glass beads. To resolve a lot of very similar molecules, the comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometer (GCxGC-TOFMS) was used to analyse the hexane scent solutions. Using this technique, more than 137 less volatile molecules including organic fatty acids, ketones, aldehydes, simple esters, alcohols, and especially various fatty acid esters with different carbon chains were identified. A considerable number of these molecules were identified in the scent samples for the first time.

Biotransformation of (1S)-2-carene and (1S)-3-carene by Picea abies suspension culture led to the formation of oxygenated products. (1S)-2-Carene was transformed slowly and the final product was identified as (1S)-2-caren-4-one. On the other hand, the transformation of (1S)-3-carene was rapid and finally led to the formation of (1S)-3-caren-5-one and (1S)-2-caren-4-one as equally abundant major products. The time-course of the reaction indicates that some products abundant at the beginning of the reaction (e.g. (1S, 3S, 4R)-3,4-epoxycarane and (1R)-p-mentha-1(7),2-dien-8-ol) were consumed by a subsequent transformations. Thus, a precise selection of the biotransformation time may be used for a production of specific compounds.

Three kinds of turpentine (a waste by-product of industrial wood processing) of various monoterpene compositions were transformed by Picea abies cells and the product yields monitored in relation to the initial turpentine concentration. The major products obtained were trans-verbenol, trans-pinocarveol, myrtenol, α -terpineol, and p-cymen-8-ol depending on the substrate composition. The absolute quantitative values of the major products were evaluated for a substrate concentration of 0.86 g L 1 . The concentration of trans-verbenol and trans-pinocarveol after twelve days of biotransformation was 768 and 388 mg L 1 , respectively. The substrate was uptaken by Picea abies cells within the fi rst three days and the majority of products released in fi ve days. Although not all the starting material was consumed, the Picea abies suspension culture was able to convert concentrations of turpentine as high as 4.3 g L 1 into valuable products. By precise selection of the substrate concentration and time course, favourable conversion to products could be achieved.

Human cerumen was separated by column chromatography into the following groups of compounds: hydrocarbons, squalene, wax esters and cholesterol esters, triacylglycerols, free fatty acids, free fatty alcohols, monoacylglycerols, free cholesterol, free sterols, and free hydroxy acids. The groups of compounds obtained were examined in detail by gas chromatography and gas chromatography-mass spectrometry. In total, about one thousand compounds have been identified.

A detailed analysis of Rhaponticum carthamoides (Willd.) Iljin root essential oil was carried out by GC, GC–MS and GC–FTIR techniques. In total, 30 components were identified, accounting for 98.0% of total volatiles. A norsesquiterpene 13-norcypera-1(5),11(12)-diene (22.6%), followed by aplotaxene (21.2%) and cyperene (17.9%), were isolated and their structures confirmed by 1D and 2D-NMR spectra (COSY, ROESY, HSQC, HMBC and INADEQUATE). Selinene type sesquiterpenes and aliphatic hydrocarbons were among minor constituents of the essential oil. The oil exhibited antimicrobial activity against 5 of 9 strains of bacteria and yeast, when tested using broth micro-dilution method. Minimum inhibitory concentrations ranged between 32 and 256 μg/ml.

A new class of axially chiral bipyridine N,N¢-oxides with bis(tetrahydroisoquinoline) framework were tested as catalysts in the reaction of aliphatic aldehydes with allyl(trichloro)silane to af- ford homoallylic alcohols. The course of the reaction, that is, the catalytic activity as well as enantioselectivity, is strongly dependent on the solvent used. The products were obtained in good yields and up to 68% ee in chloroform.

The knowledge of the molecular basis of communication in bumblebee communities is limited. None of the enzymes that participate in pheromone production have been characterized. Here, we cloned the gene encoding the Delta(9) desaturase from cDNA prepared from the total RNA of the pheromone gland and fat bodies of Bombus lucorum male. Functional expression of BlucNPVE desaturase in Saccharomyces cerevisiae and GC-MS analyses revealed its preference for C(18) saturated fatty acids. This suggests that Delta(9) desaturase is involved in the desaturation of metabolic fatty acids stored in triacylglyceroles (TAGs), because oleic acid is the most abundant fatty acid bound in TAG in B. lucorum and it is present in low concentration in the pheromone blend. The incubation of pheromone precursors with a dissected labial gland as well as direct injection of labelled pheromone substrates into B. lucorum males revealed that esterification of pheromone products occurs in the labial gland. These results support both the biosynthesis of pheromones from common lipids and the de novo synthesis of unsaturated pheromones in the labial gland.

The [2+2+2] cyclotrimerization of 1,7,9,15-hexadecatetrayne with nitriles catalyzed by dicarbonylcyclopentadienylcobalt(I) opened a new pathway for the synthesis of C 2 -symmetrical bis(tetrahydroisoquinolines) that were used as starting material for the preparation of axially chiral bipyridine N,N'-dioxides. The N,N'-dioxides (1 mol%) were found to be highly catalytically active and enantioselective (up to 83% ee) for the asymmetric allylation of aldehydes with allyl(trichloro)silane in various solvents. In addition, a dramatic solvent effect was observed where the use of different solvents induced opposite chiralities of the product with the same enantiomer of the catalyst, e.g., 65% ee (S) in acetonitrile (MeCN) vs. 82% ee (R) in chlorobenzene.

Unusual fatty acids with 24, 26, and 28 carbon atoms were found in triacylglycerols (TAGs) isolated from fat body tissue of bumblebee Bombus pratorum. The most abundant one was (Z,Z)-9,19-hexacosadienoic acid. Its structure was determined by mass spectrometry after derivatization with dimethyl disulfide and by infrared spectroscopy. ECL (equivalent chain length) values of its methyl ester were determined on both DB-1 and DB-WAX capillary columns. (Z,Z)-9,19-Hexacosadienoic acid is quite rare in nature. So far it has been identified only in marine sponges, and this work is the first evidence of its occurrence in a terrestrial organism. HPLC/MS analysis of the bumblebee TAGs showed that (Z,Z)-9,19-hexacosadienoic acid is present in one third of all TAG molecular species. As it was found in all sn-TAG positions, it is likely that (Z,Z)-9,19-hexacosadienoic acid is transported to tissues. Interestingly, labial gland secretion of B. pratorum was found to contain (Z,Z)-7,17-pentacosadiene, a hydrocarbon with markedly similar double bond positions and geometry. Possible biosynthetic relationships between these two compounds are discussed.

The (2 + 2 + 2) cyclotrimerization of 1-iso- quinolinyl-1,7-octadiyne with benzonitrile catalyzed by CpCo(CO)2 opened a new pathway for a synthe- sis of unsymmetrical axially chiral bipyridine N,N'- dioxides. The N,N'-dioxide 3a was found to be highly catalytically active and enantioselective for the asymmetric allylation of aldehydes with allyltri- chlorosilane. The allylation took place with even 1 % of the catalyst with an enantioselectivity up to 87 % ee.

Axially chiral bipyridine N , N ′-dioxides (Lewis basic organocatalysts) are easily accessible in three steps from commercially available material. The key step of this reaction sequence is cobalt-catalyzed heterocyclotrimerization of 1-pyridyl-1,7-octadiynes with nitriles. Our effort was focused on the synthesis of unsymmetrically substituted bipyridines. The scope of the cyclotrimerization reaction was tested under thermal and microwave conditions. Two of the synthesized bipyridine N , N ′-dioxides were successfully resolved into enantiomers and tested in enantioselective allylation of aldehydes.

The absolute configurations of citronellol, 2,3-dihydrofarnesol, and 2,3-dihydrofarnesal in male marking pheromones of seven species of bumblebees and cuckoo bumblebees were determined by enantioselective gas chromatography on a capillary column coated with 60% heptakis(2,3-di-O-acetyl-6-O-TBDMS)-beta-cyclodextrin in polysiloxane PS 268. Pure (-)-S-enantiomers of all three terpenes were found in the labial glands of all investigated specimens of the following species: Bombus (Bombus) terrestris, B. (Bombus) lucorum, B. (Pyrobombus) pratorum, B. (Pyrobombus) pyrenaeus, B. (Pyrobombus) jonellus, B. (Pyrobombus) impatiens, and the cuckoo bumblebee B. (Ashtonipsithyrus) bohemicus. Within species, specimens were collected at different localities and in different years. Except for 2,3-dihydrofarnesol in B. terrestris, this is the first report on the absolute configuration of terpenes in marking pheromones of bumblebees.

When subjected to a Picea abies suspension cell culture, beta-pinene, either one of the pure enantiomers or the racemate, was transformed mainly to trans-pinocarveol along with the minor products myrtenol, alpha-terpineol, pinocarvone, myrtenal and cis-pinocarveol. The absolute configuration of the major products corresponded to that of the starting beta-pinene enantiomer. Some of the primary transformation products, i.e. (1S)-cis- and (1S)-trans-pinocarveol, (1R)-myrtenol and (4S)-alpha-terpineol, were also tested as substrates of the P. abies suspension culture. They reacted more slowly than beta-pinene but, except for (4S)-alpha-terpineol, they were all transformed. Thus, (1R)-myrtenol was converted into both (1R)-myrtenal and (1R)-myrtanol, whereas (1S)-trans-pinocarveol was converted into (1S)-pinocarvone. (4R)-Limonene was slowly transformed by the suspension culture into limonene-(1,2)-epoxide as the major product, with carveol, perillyl alcohol and 1,8-cineole as minor products. Autoxidation of terpenes in cell-free nutrient medium was investigated in detail. Alpha-pinene and beta-pinene were both autoxidized to a certain extent, while limonene remained unaffected. The rate of the autoxidation was more than one order of magnitude slower than that of the biotransformation. Moreover, different products were formed by autoxidation than by biotransformation.

The enantioselective allylation of aldehydes (I) and (VIII) with pinacolborane (II) in the presence of a chiral BINOL-phosphoric-acid-derived catalyst is the key step in the synthesis of the flubofen metabolite (VII) and dapoxetine (X).

Optimized reaction conditions are developed for the enantioselective allylation of thiophen-2-carbaldehyde.

The stereoselectivity of the reduction of rac-3-methyl-1-(phenylsulfonyl)hexan-2-one (1) to 3-methyl-1-(phenylsulfonyl)hexan-2-ol (2) diastereomers by more than 20 yeasts was studied. Reduction of carbonyl group in 1 proceeds with a high Re-face enantioselectivity: Candida guillermondii (989% e.e.), C. zeylanoides (>99.9%) and Kloeckera apiculata (99.6%), respectively and the (R)- 1 enantiomer usually reacted faster. The enantioselectivity was determined by GC on chiral cyclodextrine phases and absolute configurations of products were assigned by NMR spectroscopy and a chemical correlation.

De novo biosynthesis of male sex pheromone from two bumblebee species (Bombus terrestris and Bombus lucorum) was studied by using in vitro incubations of labial glands (LGs) with radioactive [1,2-(14)C]acetate and deuterated [D(3)]acetate. The labeled substrate was incorporated into several types of compounds, such as terpenic alcohols, fatty acids, esters, and hydrocarbons. A similar incubation of [1,2-(14)C]acetate with fat bodies (FB) led to the formation of fatty acids, triacylglycerols (TAG), and hydrocarbons. To support the results from in vitro incubations, PCR analysis of fatty acid synthase (FAS) transcripts in LG and FB was performed. Relative quantification of FAS transcription levels revealed that the abundance of mRNA from the FAS gene is a function of the age of B. terrestris males. A comparison of the relative FAS mRNA gene transcription level in FB and LGs of B. terrestris and B. lucorum males proved that high biosynthetic activity takes place in the LGs of both species. Together, these results indicate that pheromone components are synthesized de novo in the LG.

The age-dependent changes in the composition of triacylglycerols (TAG) in the fat bodies of bumblebee males were studied using HPLC/MS. Two related species (Bombus terrestris and B. lucorum) were compared, with the age of the males being 0–30 days. The total amount of TAG in B. lucorum was about 2.7 times higher than that in B. terrestris for all of the ages studied. One to three-day-old males had the highest content of TAG in their fat bodies (1.6–2.3 mg/individual in B. terrestris and 3.8–4.2 mg/individual in B. lucorum). The analytical data show different patterns in both species. The qualitative composition of fatty acids in TAG was similar, but the mean relative abundance between B. terrestris and B. lucorum differed: 14:0, 7 and 14%; 16:0, 20 and 44%; 18:3, 62 and 23%; 18:1, 3 and 8%, respectively (the data is based on a GC/MS integration). A statistical evaluation of the dynamic changes in the TAG composition revealed that in B. terrestris different age classes were well separated according to their TAG composition while in B. lucorum the TAG did not change substantially during the male’s life. The TAG analyses provide more precise information on the differences between the classes studied than the FA composition alone.

Unsymmetrically 3,3'-substituted axially chiral bis(tetrahydroisoquinoline) N,N'-dioxides can be prepared in just three steps. They exhibit unique catalytic activity (turnover frequency, enantioselectivity, substrate scope) in the asymmetric allylation of aromatic aldehydes (up to 96% ee). The product of the enantioselective allylation of benzaldehyde served as a building block for the preparation of an intermediate useful in the enantioselective synthesis of diospongines.

Male wing gland secretion and volatiles emanating from calling males were investigated in the bumble bee wax moth, Aphomia. sociella, using comprehensive two-dimensional gas chromatography-mass spectrometry (GCxGC-TOF-MS), gas chromatography-infrared spectroscopy (GC-FTIR), enantioselective gas chromatography, electroantennography (EAG), gas chromatography with electroantennographic detection (GC-EAD), and NMR. GC-EAD analysis of the male wing gland secretion revealed seven active areas, corresponding to 1-hexanol (1), 2-phenylethanol (2), [(R),(Z)]-nona-2,6-dien-4-olide (3), [(S),(Z)]-nona-6-en-4-olide (4), mellein (5), phytone (6), and a mixture of C(18) fatty acids (7). Solid-phase microextraction (SPME) confirmed the presence of 2-phenylethanol, nona-2,6-dien-4-olide, nona-6-en-4-olide, mellein, and phytone in volatiles emanating from a calling male. Though the abundance of these compounds slightly differed in SPME and gland secretion analysis, nona-2,6-dien-4-olide and mellein dominated in both samples, followed by 2-phenylethanol, nona-6-en-4-olide, and phytone. The strong antennal responses elicited by components of the secretion suggest that one or more of these compounds constitute the sex pheromone. Both sexes perceived male wing gland secretion, with females being significantly more sensitive compared to males.

Volatiles released by seedlings of Norway spruce infested with the aphid Cinara pilicornis were analyzed using SPME-GC-MS. Among the stress-induced compounds released by the host plant, citronellol, cis-trans-nepetalactone and cis-trans-nepetalactol was found. These compounds originated from the aphids and they were assumed to be pheromone components for this aphid species. To determine the relative stereochemistry of the nepetalactone, a diagnostic method was developed. The method was based on multivariate analysis of tabulated relative intensities of mass fragments of the four nepetalactone diastereomers. In the practical method described, a few pairs of fragments in the mass spectra were compared and, in combination with the Kovat's index, were used to unambiguously identify the relative stereochemistry of the nepetalactone.

Current lipidomics approaches require simple and rapid algorithms enabling the interpretation of mass spectra of lipids. Most lipids are complex mixtures of related components in which the composition of the aliphatic fatty acid chains varies from one molecule to the next. Triacylglycerols (TAGs) are an example of such a lipid class. Fatty acid chains are the only parts of the molecule to change from one species to another. Fatty acids, and consequently also TAGs, can be characterized by two parameters; the number of carbon atoms and the number of double bonds. All calculations reflecting relations among ions in the spectra can be easily made using these parameters. An algorithm for the automated interpretation of TAGs from atmospheric pressure chemical ionization mass spectra (TriglyAPCI) is presented in this paper. The algorithm first identifies diacylglycerol fragments and molecular adducts. In the next step, relations among the ions are searched and possible TAG structures are suggested. Individual features of the algorithm are described in detail and the software performance is demonstrated for the liquid chromatography/mass spectrometric (LC/MS) analysis of TAGs isolated from the termite Prorhinotermes canalifrons.

Higher wax esters within the range of C24 to C44 (205 standards) were analyzed by means of gas chromatography and Kovats indexes (I) and reduced Kovats indexes (RKI) were calculated. The dependences of these retention data on number of carbon atoms and on number and position of double bonds in acid and in alcohol moieties of esters were plotted.

Triacylglycerols (TAGs) from the fat body of several bumblebee species (Bombus lucorum, B. terrestris, B. lapidarius, B. hypnorum, B. hortorum, and B. confusus) were studied using chromatographic techniques. Semi-preparative thin-layer chromatography was used to isolate the TAGs from the tissue extract. Gas chromatography (GC) enabled us to identify the fatty acids (FAs) that form bumblebee TAGs and to quantify their relative proportions. The TAGs were subsequently analysed by high-performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry. Two chromatographic systems, including non-aqueous reversed-phase chromatography and silver ion chromatography on cation exchange resin in silver (I) ionic form, were optimised and their performance compared. The most abundant fatty acids in bumblebees TAGs contained 18 or 16 carbon atoms; oleic acid predominated in most samples. TAGs were found to be a complex mixture of isomers; some of them, e.g. OLnO, PLnO, PoPoO, PoPoP, POO, or OOO (where Po is palmitoleic, P is palmitic, Ln is linolenic, and O is oleic acid) were abundant in particular species. The composition of both FAs and TAGs was found to be species-specific. Only minor differences were found among specimens of the same species.

Both enantiomers and the racemate of alpha-pinene were transformed by Picea abies cells immobilised on alginate. The main products were cis- and trans-verbenol, the later being further transformed to verbenone. The enantiomeric purity of each product more or less corresponded to that of the substrate. Transformation by free cells was faster than that by the immobilised cells. The ratio of products differed to some extent between the transformation by free and immobilised cells.

Graphical Abstract Full-size image; International audience; Female alates of Nasutitermes ephratae termites from Guadeloupe and Nasutitermes sp. from Brazil produce a diterpene hydrocarbon of the molecular formula C20H30 as the main component of their tergal gland secretion. Analysis of NMR, IR, and mass spectra of the diterpene led to a structure of (11E)-trinervita-1(14),2,11-triene. Based on a comparison with the published oxygenated trinervitane skeleton from termites we prefer the enantiomer with absolute configurations (4R,7S,8R,15S,16S). The suggested structure is supported by ab initio quantum chemical calculation of 1H and 13C chemical shifts for the optimized geometry of the molecule.

Citronellal was transformed by Solanum aviculare suspension cultures to menthane-3,8-diols. cis-Menthane-3,8-diol dominated over the trans-isomer (39% and 15%, respectively). Absolute configurations of menthane-3,8-diols were assigned by critical analysis of 1H and 19F NMR spectra of prepared esters with 2-methoxy-2-phenyl-3,3,3-trifluoropropanoic acid. Citronellol and isopulegol were other products of the transformation (23% and 17%, respectively). The reaction course was identical for both citronellal enantiomers.

Pollen of the elder (Sambucus nigra L.) was extracted with chloroform–methanol. The extract was separated by column chromatography into the following groups of compounds: hydrocarbons (8.7%), polycyclic aromatic hydrocarbons (0.2%), complex esters (5.2%), triglycerides (18.7%), hydroxy esters (27.9%), free fatty acids and alcohols (16.8%), free sterols (6.8%), and triterpenic alcohols (4.0%). The nonsaponifiable components (hydrocarbons, fatty acids, alcohols, and sterols) were examined in detail using spectroscopic and chromatographic methods (IR spectroscopy, GC, and GC–MS). The identified compounds were characterized by their mass spectra and Kovats retention indices. The double bond positions and their configurations in unsaturated compounds are also reported.

Volatiles from the essential oil of the liverwort Conocephalum conicum were analysed. The chirality of the monoterpene hydrocarbons was studied by two-dimensional ps chromatography. All compounds e ...

Diacids XXIII and XXIV, triacid XXVII and tetraacid XVI were prepared from 3,4-secodiacid V and from nitrile XVII by modifying the isopropenyl groups in position 5 and 19. Procedures for selective esterification of the acids and the hydrolysis of their esters in position 3, 23 and 28 were elaborated and the methyl esters VII, XI, XII, XXVI, XXVIII, XXX and XXXI prepared. The structures of the derivatives prepared were confirmed by mass spectra. The antibacterial activity of the series of 3,4-secoacids derived from lupane and 19β,28-epoxy-18α-oleanane was also determined.

Release rate and degree of isomerization of pheromones with conjugated double bonds were studied in dispensers prepared from several rubber substrates. The substrates compared were made of rubber, cured with elemental sulfur or accelerators based on organic sulfur compounds or organic peroxides. Isomerization of the double bonds occurs immediately after impregnation of the substrate, and the degree of isomerization increases during field use and/or storage. The propensity of the isomers to isomerize corresponds to their proportion in the equilibrium mixture. AnE,Z isomer is isomerized faster than theE,E isomer, and finally a near-equilibrium mixture of the four isomers is present. Minimal isomerization was found in non-sulfur-cured substrates which are the material of choice.

The direct esterification of enantiomeric 2-methoxy-2-phenyl-3,3,3-trifiuoropropionic acids (MTPA) was reinvestigated. Preferential formation of one of the diastereoisomeric esters was avoided by using three equivalents of MTPA. Chromatographic behaviour of the obtained MTPA esters is discussed. Our recent method! for direct esterification of enantiomeric 2-methoxy-2-phenyl­ -3,3,3-triftuoropropionic acids Ia and Ib (Mosher's acid, MTPA) represents a signifi­ cant advance in the preparation of MTPA esters. The method consists in activation of MTPA by reaction with 2-chloro-l-methylpyridinium iodide (CMPI) in the presence of 4-dimethylaminopyridine (DMPA), followed by reaction with the alcohol. In contrast with the original method 2 , our procedure does not require the not easily available chloride of MTPA and the isolation of products is very simple. Using our new method we found that the ratio of the corresponding diastereoisomeric MTPA esters (amides) did not correspond to the actual proportion of enantiomers in the studied mixture. For racemic alcohols the ratio of the diastereoisomers differed from the theoretical value 50: 50. Naturally, this fact markedly distorted our results of enantiomeric purity determination which differed from the results obtained by other methods3 (such as optical rotation or CD spectra). The same behaviour has also been found for the reacfon of (R)-( + )-l-(t-naphthyl)ethylamine with racemic 2-phenylbutanoic acid 4 (using the same reagents for activation of the acid). The mentioned discrepancy is caused by incomplete reaction of the studied alcohol (amirie) as observed by TLC of the reaction mixture. Under these conditions the reaction mixture is kinetically enriched in the faster arising diastereoisomer. Naturally, no enrichment can occur with pure enantiomers. In order to preserve the advantages of the described method 1 (short reaction time, mild reaction conditions) we tried to solve the problem by using an excess of MTP A. As model compounds we chose various structural types of secondary alcohols: I-phenylethanol (11) (benzylic type), cis-verbenol (Ill, IV) (allylic type), trans-2-(4-methoxybenzyl)-1-cycJohexanoi (V) and its cis-isomer VI (stericallY hindered