A Chemoenzymatic Synthesis of the Sex Pheromone of Lasioderma serricorne

A síntese quimioenzimática da forma enantiomericamente enriquecida (80% ee) da (-)-serricornina (1), feromônio sexual da praga do fumo Lasioderma serricorne F., foi alcançada em 8 etapas e 13% de rendimento total a partir de (R)-3-hidroxipentanoato de metila, obtido da redução de 3-oxopentanoato de metila com fermento de padaria (S. cerevisiae) na presença de álcool alílico como inibidor enzimático.


Introduction
The two-electron reduction of carbonyls by NAD(P)H is a highly valued process for the preparation of chiral building blocks in the total synthesis of enantiomerically pure natural products due to its ability to differentiate the enantiotopic faces of carbonylic substrates 1,2 .
However, the nicotinamide cofactors are rather expensive to be used in stoichiometric amounts and the preparative use of nicotinamide cofactor dependant oxidoredutases usually requires the recycling of the cofator or the use of whole cell preparations.The later may show a different reactivity profile from the purified enzyme due to the action of other oxidoreductases, and may be even superior for many applications.
In both cases, the stereoselectivity and yield may be generally high but access to both enantiomeric series of a chiral alcohol from the same prochiral substrate is generally not possible.Much effort has been directed towards screening different microorganisms, modifying the substrate and the reaction conditions in order to improve the scope of biocatalyzed carbonyl reductions 3,4,5 .
The reduction of methyl 3-oxopentanoate with baker's yeast (Saccharomyces cerevisiae) can be directed to afford either methyl (R)-3-hydroxypentanoate (86% ee) when baker's yeast immobilized in polyurethane is employed 6 or its antipode (89% ee) when the reduction is carried out with added MgCl2 and baker's yeast immobilized in magnesium alginate 7 .
The scenario is worse for the baker's yeast reduction of 2-methyl-3-oxopentanoates where up to four stereoisomers can conceivably be formed.So far a preparatively useful procedure employing baker's yeast has not been developed for such substrates, although the preparation of (2S,3S) isomer has been achieved from the reduction of ethyl 2-methyl-3-oxopentanoate with some fungal strains 8 .
Nakamura and coworkers have devised a practical method for the baker's yeast reduction of 3-oxopentanoates either to the corresponding (R)-or (S)-3-hydroxypentanoates 9,10 taking advantage of the selective inhibition of either the L-or D-enzymes present in S. cerevisiae 11 .
This protocol not only provides an enantiodivergent approach to these highly useful building blocks but also allows the preparation of (R,R)-and (S,S)-2-methyl-3-hydroxypentanoates in good diastereoisomeric and enantiomeric excesses when the reduction is followed by stereoselective Fràter alkylation 12 .In fact, Mori and Watanabe have used Fràter's alkylation of methyl (R)-2hydroxypentanoate, prepared by oxidation of pentanoic acid with Candida rugosa, to synthesize (-)-serricornine (1), the sex pheromone of the cigarette beetle Lasioderma serricorne F., in 13 steps and 7.6% overall yield 13 .Experimental 1 H-NMR spectra were recorded in CDCl3 solution at 300 MHz and 13 C-NMR spectra in CDCl3 solution at 75.5 MHz (unless otherwise noted) with a Varian Gemini 2000 or a Bruker AC-300P instrument.Chemical shifts are expressed in ppm relative to tetramethylsilane followed by multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; qt, quintet; m, multiplet), coupling constant (Hz) and number of protons.Infrared spectra were recorded on a Perkin-Elmer 399B or 1600 series spectrophotometer.Mass spectra were obtained via electron impact (30 eV) on a Varian MAT 311A spectrometer.Optical rotations were measured at 25 °C in a Polamat A (Carl Zeiss) at 546 nm (mercury line) and corrected to 589 nm (sodium D line).
Tetrahydrofuran was treated with sodium/benzophenone and distilled immediately prior to use.Dichloromethane, triethylamine, diisopropylamine and benzene were treated with calcium hydride and distilled immediately prior to use.Acetic and propionic anhydride, and allyl alcohol were distilled prior to use.Potassium tert-butoxide was sublimed immediately prior to use.The remaining reagents employed were purchased from commercial suppliers and used without further purification.The reactions involving anhydrous solvents were carried out under argon atmosphere.

Results and Discussion
Baker's yeast is an inexpensive and versatile microorganism which does not require any special growth conditions to be used on a preparative scale.On the other hand, major limitations are generallly associated with non-repro-ducible results due to the different strains of S. cerevisiae available from industrial sources.
As the methodology which employs an enzymatic inhibitor to control the stereochemical course of the reduction seemed appropriate for the preparation of some insect pheromones 9 , we initially evaluated two commercially available baker's yeast brands (Fleischmann, Brazil and Sigma, USA) in the reduction of methyl 3-oxopentanoate aiming to reach a preparatively useful access to enantiomerically enriched methyl (R)-3-hydroxypentanoate.
While both strains tested were able to efficiently reduce ethyl acetoacetate to ethyl (S)-3-hydroxybutanoate (70-75% isolated yield, 90-92% ee) only the yeast suplied by Sigma, USA efficiently converted methyl 3-oxopentanoate to the corresponding hydroxyester, albeit in low enantiomeric excess (Table 1, entry 1).At this point it is rather difficult to rationalize the lack of reactivity towards methyl 3-oxopentanoate observed for the baker's yeast supplied by Fleischmann, Brazil but morfological differences were evident when samples of each brand were examined by atomic force microscopy as the sample from Sigma, USA displayed extensive cell wall preservation when compared to the one from Fleischmann, Brazil 14 .
On a preparative scale, we were able to isolate gram quantities of (-)-2 in 88% yield and 76-80% ee, after purification by Kugelrohr distillation (1 mm Hg, 70-80 °C) when baker's yeast was previously incubated at 30 °C for 30 min with 0.5 equivalents of allyl alcohol.
The stereogenic center at C-6 (serricornine numbering) was established after Fràter's alkylation 12 of (-)-2 which provided an 8:1 mixture of methyl (R,R)-3-hydroxy-2methylpentanoate (-)-3 and its epimer at C-2.The configuration of the major isomer was confirmed to be 2R,3R by analysis of its 1 H-NMR spectrum which displayed a coupling constant (JH2-H3 = 7.0 Hz) characteristic of its anti relative configuration and a deshielding effect at C-2 and C-3 (27.4 and 74.5 ppm, respectively) in the 13 C-NMR spectrum 17 of the major isomer when compared to the same carbons in the minor component (26.7 and 73.2 ppm, respectively).
In order to reduce the number of purification steps, epimer separation was deferred until the preparation of (-)-4b which was obtained after lithium aluminum hydride reduction and selective monotosylation of the diol (+)-4a.After column chromatography on silica gel, alcohol (-)-4b was isolated in 56% overall yield from (-)-2.
The intramolecular alkylation, the key step in our approach to (-)-serricornine (1), was accomplished after Opropionylation of (-)-6a (82% yield) and treatment of a THF solution of the corresponding propionate (-)-6b with freshly sublimed potassium tert-butoxide at 0 °C followed by acidification of the reaction mixture with conc.HCl and stirring at room temperature 15,16 .An 8:1 mixture of (-)-7 and its epimer at C-4 (serricornine numbering) was formed and the configuration of the major isomer was established as 4S,6S,7S (serricornine numbering) by comparison of the 1 H-NMR spectrum of the mixture with literature data for (-)-7 19 .

Table 1 .
Yields and enantiomeric excesses in the reduction of methyl 3-oxopentanonate with baker's yeast in the presence of allyl alcohol a .Mean value for 3 experiments; b Conversion determined by capillary GC analysis after 24 h at 30 °C; c Enantiomeric excess determined by chiral GC analysis (see Experimental section). a