Mid Sweden University

Resolving complexes of closely related and cryptic insect species can be challenging, especially when dealing with rare and protected taxa that are difficult to collect for genetic and morphological analyses. Until recently, populations of the genus Osmoderma (Scarabaeidae), widespread in Europe, were treated as a single species O.eremita (Scopoli, 1763) in spite of observed geographic variation in morphology. A previous survey using sequence data from the mtDNA cytochrome C oxidase I gene (COI) revealed the occurrence of at least two distinct lineages within this species complex: O.eremita in the west and O.barnabita Motschulsky, 1845, in the east. Interestingly, beetles confined to Sicily have been described as a distinct species, O.cristinae Sparacio, 1994, based on morphological traits. Only few Sicilian specimens were included in the former genetic analysis, and the results led to a still questionable taxonomic rank for these populations. To explore the robustness of the previous taxonomic arrangement for O.cristinae, a combination of genetic, morphological and pheromonal analyses was used. A 617-bp fragment of the COI gene, aligned with O.cristinae and O.eremita sequences already available in GenBank, showed a clear genetic divergence between the two species (interspecific mean distance=6.6%). Moreover, results from AFLP markers sustained the distinction of the two species. In addition, geometric morphometric analyses of the shape of male genitalia revealed a clear differentiation between the two species. Via scent analysis and field trapping, we demonstrated the production of the sex pheromone (R)-(+)--decalactone by males of O.cristinae, the attraction by conspecific individuals (mostly females) to this compound, and a lack of antagonistic effect of (S)-(-)--decalactone. The fact that O.eremita and O.eremita use the same compound for mate finding suggests that this sex pheromone has not undergone a differentiation and probably the allopatry of these two species compensates for the absence of a mechanism to avoid cross-attraction. Our genetic and morphological data support the divergence of the two species and confirm the species status for O.cristinae, while sex pheromones are confirmed to be invariant among different species of the genus Osmoderma.

Candida rugosa lipase (CRL) catalysed enantioselective esterification of racemic 2-methylhexanoic acid was performed with 1-decanol in organic solvent at constant water activity (aw). The aw was maintained either by a salt hydrate pair added in the reaction mixture or by performing the reaction in an air tight desiccator over an aqueous saturated salt solution. It was found that the enatiomeric ratio and average reaction rate profiles were similar when controlling the water activity for the esterification reaction with these two different methods. But, in some cases the average rate of the reaction and the enantioselectivity of CRL was affected when ions from the salt hydrate pairs were present in the reaction mixture.

A series of antimalarial chiral 1,2,4-trioxanes (1–8) were synthesised in high enantiomeric purities. Enantioselective addition of R2Zn reagent to 3-methyl-2-butenal catalysed by (+)-MIB or (−)-MIB yielded both the enantiomers of the chiral allylic alcohols 9–11 (90–98% ee), which were subjected to diastereoselective photooxygenation in the presence of tetraphenylporphine (TPP) to obtain (R,R)-threo- or (S,S)-threo-β-hydroperoxy alcohols (12–14). Reaction of β-hydroperoxy alcohols (12–14) with different cyclic ketones produced optically active trioxanes 1–8.

Dispiro N-Boc-protected 1,2,4-trioxane 2 was synthesised via Mo(acac)(2)

catalysed perhydrolysis of N-Boc spirooxirane followed by condensation of

the resulting beta-hydroperoxy alcohol 10 with 2-adamantanone. N-Boc

1,2,4-trioxane 2 was converted to the amine 1,2,4-trioxane hydrochloride

salt 3 which was subsequently used to prepare derivatives (4-7). Several of

these novel 1,2,4-trioxanes had nanomolar antimalarial activity versus the

3D7 strain of Plasmodium falciparum. Amine intermediate 3 represents a

versatile derivative for the preparation of achiral arrays of trioxane analogues

with antimalarial activity.

A rapid, two step synthesis of a range of dispiro-1,2,4,5-tetraoxanes with potent antimalarial activity both in vitro and in vivo has been achieved. These 1,2,4,5-tetraoxanes have been proven to be superior to 1,2,4-trioxolanes in terms of stability and to be superior to trioxane analogues in terms of both stability and activity. Selected analogues have in vitro nanomolar antimalarial activity, good oral activity and are non-toxic in screens for both cytotoxicity and genotoxicity. The synthesis of a fluorescent NBD-tagged tetraoxane probe has allowed investigation into the mechanism of accumulation of these drugs using laser scanning confocal microscopy techniques.

Enantioselective acylation of some (±)-3-alkyl-3-phenyl-l-propanols was performed with enzymes as catalysts. Moderate enantiomeric ratios (E), ranging up to E = 11.6, were obtained. In the resolution, some of the lipases selectively acylated the (+)-enantiomer while others acylated the (-)-enantiomer of the γ-substituted primary alcohols 1-4. Thus, it is possible to obtain both enantiomers of the alcohols as remaining substrate with high enantiomeric purity. The resolution of (±)-4,4-dimethyl-3-phenyl-1-pentanol 4 was extensively studied and screening experiments were conducted to select suitable lipase(s), reaction medium, acyl donor and appropriate temperature combinations to increase the enantiomeric ratio. Chirazyme® L-6/chloroform/vinyl propionate/38 °C and Chirazyme® L-7/di-iso-propyl ether/vinyl propionate/0 °C were chosen to obtain both enantiomers, (R)-(+)-4 and (S)-(-)-4, respectively, via sequential resolutions in excellent enantiomeric excess (>98%) and in 25% and 22% yield, respectively.

When Candida rugosa lipase (CRL) was immobilised on the porous polypropylene carrier Accurel it was found that differences in the samples of the carrier influenced the enantiomeric ratio (E) when racemic 2-methylhexanoic acid was esterified with 1-decanol in organic solvent at a constant water activity (aw) of 0.76. CRL immobilised on Accurel with smaller particles and smaller pore diameters gave higher enantiomeric ratio in the esterification. The reaction rate was influenced by the different Accurel materials and also by the amount of CRL exposed to the carrier. Characterisation of the different Accurel grades, with and without adsorbed CRL, was performed by light scattering analysis, SEM and FT-IR.