The Asymmetric Synthesis of ( + )-Sitophilure , the Natural Form of the Aggregation Pheromone of Sitophilus oryzae L . and Sitophilus zeamais

The weevils of the genus Sitophilus are known to cause serious losses of stored cereal grains throughout the world. Effective, cost-efficient grain weevil management could be accomplished by monitoring pest populations with pheromone-baited insect traps and applying control methods only when pest densities reach economic thresholds. In 1984, Burkholder and coworkers isolated the maleproduced aggregation pheromone of the rice weevil (Sitophilus oryzae L.) and of the maize weevil (Sitophilus zeamais M.), named it sitophilure, and identified it as (4R*,5S*)-5-hydroxy-4-methyl-3-heptanone (1) (Fig. 1). The syntheses of the four possible stereoisomers of sitophilure (1) and bioassays by Burkholder and coworkers revealed the (4S,5R) enantiomer as the active form of the pheromone. Somewhat lower but still significant responses were observed for the (4SR,5RS) mixture while very low responses were elicited by either the (4R,5R)-isomer or the (4RS,5RS) racemic mixture. Since effective and cost-efficient control of both maize and rice weevils populations can be foreseen with the aid of the aggregation pheromone, several total syntheses of the racemic and the natural forms of sitophilure (1) have been published. Since chiral β-hydroxyesters are versatile and convenient building blocks for the syntheses of biologically important compounds, asymmetric reduction of β-ketoesters by baker’s yeast (Saccharomyces cerevisiae) has been widely used due to its simplicity, low cost and no need for cofator regeneration. However, it should be emphasized that there are many substrates that afforded low chemical yields and/or low enantioselectivities, and access to both enantiomeric series of a chiral β-hydroxyester from the same prochiral substrate and microorganism 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 β-ketoesters reduction. The incomplete enantioselectivity observed is generally attributed to the existence of several operating oxidoreductases in the baker’s yeast cells and in many cases the use of a purified reductase leads to high enantioselectivity. Nakamura and coworkers reported a quite useful method for the reduction of β-ketoesters with baker’s yeast. The introduction of a third reagent into the reaction system changes the stereoselectivity of the reduction and allows the Article


Introduction
The weevils of the genus Sitophilus are known to cause serious losses of stored cereal grains throughout the world.Effective, cost-efficient grain weevil management could be accomplished by monitoring pest populations with pheromone-baited insect traps and applying control methods only when pest densities reach economic thresholds.
In 1984, Burkholder and coworkers isolated the maleproduced aggregation pheromone of the rice weevil (Sitophilus oryzae L.) and of the maize weevil (Sitophilus zeamais M.), named it sitophilure, and identified it as (4R*,5S*)-5-hydroxy-4-methyl-3-heptanone (1) (Fig. 1) 1 .The syntheses of the four possible stereoisomers of sitophilure 2 (1) and bioassays by Burkholder and coworkers revealed the (4S,5R) enantiomer as the active form of the pheromone 3 .Somewhat lower but still significant responses were observed for the (4SR,5RS) mixture while very low responses were elicited by either the (4R,5R)-isomer or the (4RS,5RS) racemic mixture.Since effective and cost-efficient control of both maize and rice weevils populations can be foreseen with the aid of the aggregation pheromone, several total syntheses of the racemic and the natural forms of sitophilure (1) have been published 4,5 .
Since chiral β-hydroxyesters are versatile and convenient building blocks for the syntheses of biologically important compounds, asymmetric reduction of β-ketoesters by baker's yeast (Saccharomyces cerevisiae) has been widely used due to its simplicity, low cost and no need for cofator regeneration.However, it should be emphasized that there are many substrates that afforded low chemical yields and/or low enantioselectivities, and access to both enantiomeric series of a chiral β-hydroxyester from the same prochiral substrate and microorganism 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 β-ketoesters reduction 6 .
The incomplete enantioselectivity observed is generally attributed to the existence of several operating oxidoreductases in the baker's yeast cells and in many cases the use of a purified reductase leads to high enantioselectivity 7 .
Nakamura and coworkers reported a quite useful method for the reduction of β-ketoesters with baker's yeast.The introduction of a third reagent into the reaction system changes the stereoselectivity of the reduction and allows the Article e-mail: pilli@iqm.unicamp.brand vbriatto@iqm.unicamp.brdesired configuration to be obtained in good enantiomeric excess.Thus, the introduction of allyl alcohol or an α, β-unsaturated carbonyl compound shifts the stereoselectivity of the reduction toward the D isomer 8 , whereas the introduction of ethyl chloroacetate favors the formation of the L isomer (Scheme 1) 9 .The method is useful because the stereoselectivity can be easily controlled without screening microorganisms or modifying the structure of the subtrate.
We recently took advantage of Nakamura's methodology and developed an efficient preparation of (-)-serricornine, the sex pheromone of the cigarette beetle Lasioderma serricorne F., in 9 steps and 12% overall yield from methyl 3-oxopentanoate (2).The use of allyl alcohol as an enzymatic inhibitor allowed the preparation of (-)-3, in 88% yield and 76% enantiomeric excess 10 which underwent stereoselective Fràter's alkylation 11 .
Here we report an enantioselective synthesis of (+)-sitophilure (1) which features the use of ethyl chloroacetate during the microbiological reduction of methyl 3-oxopentanoate with S. cerevisiae to control the stereochemical course of the baker's yeast reduction.

Experimental
1 H-NMR spectra were recorded in CDCl 3 solution at 300 MHz and 13 C-NMR spectra in CDCl 3 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.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.Oxalyl chloride and dimethyl sulfoxide were distilled 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.
The mixture was stirred 30 min at 0 °C and then cooled to -78 °C.A solution of (+)-3 (1.53 g, 11.6 mmol) in THF (5 mL) was added dropwise and the mixture was stirred 45 min at 0 °C.A solution of MeI (1.08 mL, 17.4 mmol) in DMPU (4.7 mL) was added dropwise to the solution at -40 °C.After stirring 45 min at this temperature, the reaction temperature was allowed to reach room temperature.The mixture was quenched with satd.aq.NH 4 Cl (7 mL) at 0 °C and extracted with Et 2 O (3 X 10 mL).The organic phase was washed with brine (10 mL), dried over MgSO 4 , filtered and concentrated to afford 1.19 g of a mixture of 4 and its C-2 epimer (8:1 ratio) which was used in the next step without further purification. 1

Results and Discussion
The microbiological reduction of methyl and ethyl 3oxopentanoate in the presence of ethyl chloroacetate, as described by Nakamura and coworkers 9 , afforded (+)-3 in good enantiomeric excess (Table 1).The nature of the alkoxy moiety (ethyl or methyl) does not significantly affect the enantiomeric excess of the reduction, whereas the use of only 2 equivalents of ethyl chloroacetate surprisingly leads to lower conversion.Having determined the optimal conditions for the reduction of β-ketoester, we were able to isolate gram quantities of (+)-3 in 70% yield and 82% ee, after purification by Kugelrohr distillation (1mmHg, 70-80 °C) when baker's yeast was previously inactivated at 30 °C for 30 min with 4 equivalents of ethyl chloroacetate.
The stereogenic center at C-4 (sitophilure numbering) was established after alkylation of the lithium dianion derived from (+) 3, according to Fràter conditions. 10A 8:1 mixture of (S,S)-3-hydroxy-2-methylpentanoate 4 and its C-2 epimer was obtained and the configuration of the major isomer was confirmed to be 2S,3S by analysis of its 1 H-NMR spectrum which displayed a coupling constant (JH 2-3 = 7.0 Hz) and a deshielding effect at C-2 and C-3 (27.4 and 74.5 ppm, respectively) in the 13 C-NMR spectrum characteristic of its anti relative configuration 12 (the same carbons in the minor component appeared at 26.7 and 73.2 ppm, respectively).
Attempts to gain direct access to hydroxy ester (+)-4 through the reduction of methyl 2-methyl-3-oxopentanoate with S. cerevisiae in order to simplify the synthetic scheme met with failure: esters of 2-methyl-3-oxopentanoic acid are slowly converted by S. cerevisiae to the corresponding hydroxyesters in low yields and very poor diastereoselection.At this point it seems that a more straightforward route to (+)-4 or even to (+)-1 requires microbial screening.
The conversion of the aldehyde 10 to the diastereoisomeric mixture of alcohols 11 and 12 has been previously reported by Mori and Ebata 2 .The 1 H-and 13 C-NMR spectral data of our less polar isomer were in accord with the values reported by these authors for the syn isomer 11, but the value of the specific optical rotation of our less polar isomer was in accordance with the value reported by Mori and Ebata for the anti isomer 12 (Table 2).Since the data were divergent we decided to carry out an NMR analysis of our less polar isomer.After desilylation, its 1 H-and 13 C-NMR spectra revealed a symmetrical diol (only 5 signals in the 13 C-NMR spectrum), fully confirming that our as well as Mori and Ebata's less polar and more abundant isomer displayed all syn stereochemistry as depicted in 11, and that the specific optical rotation of 11 and 12 were erroneously interchanged in Mori and Ebata's paper 2 .

Table 1 .
Conversions and enantiomeric excesses in the reduction of methyl and ethyl 3-oxopentanonate with baker's yeast in the presence of ethyl chloroacetate.-Conversion determined by capillary CG analysis after 24h at 30 °C; b-Enantiomeric excess determined by CG on capillary column having heptakis-(2,6-methyl-3-pentyl)-β-cyclodextrin as stationary phase; n.d.= not determined.
aOR O O

Table 2 .
Literature and experimental values of optical rotation of 11 and 12.