Most compounds that support our living have chirality. As the living system can recognize and discriminate chiral isomers, the biological activities depend on their chiral configurations. So, it is very important to make optically pure form of drugs, pheromones, or odorants. Although, the olfactory system of humans and animals can distinguish between optical isomers, the synthetic part of these optically active odorants is underdeveloped.

The methods for preparing an optically pure products can divide into two parts. One is bioconversion and the other is chemical synthesis. In this study, I tried both of these methods for preparing optically active odorant compounds.

Chapter I-1 : Chemo-biological preparation of (R)-4-acetoxy-2-methyl-1-butanol

By the bioconversion method, I tried enantioselective reductions of carbon-carbon double bond. 4-Acetoxy-2-methylene-1-butanol (1) was used as a substrate of bioconversion, and 1 was able to be synthesized from cheap natural compound itaconic acid in 4 steps. The whole growing cells of microbes were used for the bioconversion, because of their efficiency for no need of co-substrates or co-enzymes.

I screened 6 kinds of microbes, and Pseudomonas putida showed the best reduction activity against the double bond of 1. So I proceeded to the optimization using P. putida.

The optimized condition of bioconversion with P. putida was investigated. After adding 1 and the seed cultured P. putida to a new liquid medium, transformation was checked regularly by G.C.

The desired reduction of the double bond of 1 was occurred gradually. But after exponential phase of P. putida, it started making undesired byproducts 3 and 4 from 1 and 2 by simultaneous hydrolysis. So, I stopped the process when the peaks of 3 and/or 4 are observed by G.C. The best bioconversion was achieved when 18 h-cultured seed culture medium 1.50 ml (3% of medium) and 0.10 g (0.70 mmol) of substrate in 50 ml (in 500 ml Erlenmeyer flask) of YPD media was cultured at 30℃, 180 rpm. With this method, I could get the optically active asymmetric alcohol 2, with high conversion rate (>80%) and high optical purity (≧98%).

Chapter I-2 : Synthesis of methyl (R)- and (S )-3-methyloctanoate

Methyl 3-methyloctanoate has been reported as an odor component of African orchids, Aerangis sp. and the odor of this compound is different according to its (R)- and (S)-configuration. I thought the produced alcohol 2 could be used as chiral building block for the synthesis of these methyl (R)- and (S)-3-methyloctanoate (Fig. 3).

The synthesis was carried out by simple organic chemical process. I could synthesize the flowery methyl (S)-3-methyloctanoate 5 in 4 steps with 60% overall yield, and the fruity (R)-form 6 was obtained in 9 steps with 11% overall yield.

Chapter II : Sythesis of δ-nonalactone by asymmetric SN2' reaction

In chemical conversion, I tried to synthesize optically active δ-nonalactone. The δ-lactones are important ingredients for flavor in many foods. They have different flavor according to their absolute configuration, i.e. the (R)-form have milky or creamy, while (S)-form have peach like flavor.

There are many possible approaches to prepare chiral δ-nonalactone, I tried to use the direct enantioselective alkylation reaction, concretely, SN2' reaction which is less developed yet.

Compound 7 was designed as an intermediate. It can be prepared from cheap natural compound limonene. It has many efficient points for the SN2' reaction. At first, it has non-epimerizable ketone, and the direct formation of cyclic carbonate is convenient as a good leaving group. Most of all, the steric effect of neighboring methyl group is expected to enhance the stereoselectivity of SN2' reaction.

(R)-(+)-Limonene was converted to carbonate 7 in 4 steps, which was subjected to addition of cyclopentenyl nucleophile. Unexpectedly, I could get two chiral intermediate, epoxide 8 and cyclic carbonate compound 9. Both of them were possible substrates for the SN2' reaction, so the reaction was performed with both of them after separation.

The epoxide 9 was treated with lithium dibutylcuprate to give (Z)-product 12 with 65% yield. On the other hand, cyclic carbonate 8 was treated with mono-butylcyanocuprate to give (E)-product 10 with 75% yield, while the cyclic carbonate was reductively decarboxylated by the lithium dibutylcuprate. Both SN2' products were converted to δ-nonalactones by ozonoysis and Baeyer-Villiger reaction to afford (R)- and (S)-δ-nonalactone, respectively. The e.e. of these products was checked by chiral G.C. The results showed 90% e.e. of (R)-δ-nonalactone 11 and 89% of e.e. of (S)-form 13.

Conclusion

In this study, I have synthesized optically active odorants, methyl 3-methyloctanoate and δ-nonalactone, by chemobiological and chemical synthetic method.

At first, by chemobiological method, I could synthesize the optically active alcohol (R)-4-acetoxy-2-methyl-1-butanol 2. The cultured cells of P. putida were used for bioconversion with high enantioselectivity. As synthetic application of this chiral alcohol, I synthesized methyl (R)-and x(S)-3-methyloctanoate, the odor compound of African orchids. The methyl (S)- 3-methyloctanoate 5 was synthesized from 2 in 4 steps with 60% overall yield, and the methyl (R)-3-methyloctanoate 6 was obtained in 9 steps with 11% overall yield.

By chemical synthetic method, I have synthesized both (R)-and (S)-δ-nonalactone by asymmetric SN2' reaction with good enantioselectivity. The optically active cyclic carbonate and epoxide, have been developed as SN2' intermediate. And they produced δ-nonalactone with different manner. I expect they could be applied for the synthesis of other optically active compounds by changing vinyl group and alkyl nucleophile.

Fig. 1. Chemo-biological preparation of asymmetric chiral alcohol 2.

Fig. 2. Bioconversion tendency of 1 by P. putida.

Fig. 3. Synthesis of methyl (R)- and (S)-3-methyloctanoates.

Fig. 4. Synthetic strategy of d-nonalactone by stereoselective SN2' reaction.

Fig. 5. Synthesis of (R)- and (S)-δ-nonalactones.

多くの天然有機化合物は不斉点を有しているが、各鏡像体は生体内で異なる作用を示すことから、これらを純粋に合成し利用することが必要である。本論文は微生物変換を用いたエナンチオ選択的還元と、SN2'反応を用いた不斉点導入という二つの手法による光学活性香気成分合成法に関して論じたものであり、二章より構成されている。

第一章では微生物による立体選択的な還元反応を用いた光学活性アセテート2の調製と、これを用いた3-メチルオクタン酸メチルの両鏡像体(3,4)の合成について述べている。安価な原料であるイタコン酸より容易に合成できるアセテート1を基質として、光学活性な2へと選択的に還元する微生物を探索した結果、Pseudomonas putidaを見出した。培養条件の最適化の結果、65%の収率と98%以上の鏡像体過剰率で1から2へ変換することが出来た。アセテート2は種々の光学活性香気成分の合成へ汎用性の高い原料になり得ると考えられる。そこで微生物変換により得られた2を出発原料として、ランの香気成分として知られる3-メチルオクタン酸メチルの両鏡像体(3,4)の合成を短工程で達成し、アセテート2が光学活性原料として有用であることを示すことが出来た。

第二章では新規な立体選択的反応として、(R)-リモネンを不斉補助基として用いたSN2'反応による不斉点の導入法を開発し、これを応用してココナッツやパイナップル様香気を有するδ-ノナラクトンの両鏡像体の合成を行なっている。(沼)-リモネンから4工程で合成が可能なケトン5に対しシク`ペンテニル基を導入し、環状カルボネート6およびエポキシド7を得た。両者を分離後、環状カルボネート6に対する立体選択的SN2'反応によりシクロペンタン環上に不斉点を導入した8とし、オゾン分解とBaeyer-Villiger酸化を経て(R)-δ-ノナラクトン(10)を合成することに成功している。一方、エポキシド7に対し同様の反応を適用することにより、鏡像体である(S)-δ-ノナラクトン(11)の合成にも成功している。本合成経路は共通の中間体から両鏡像体を合成することが可能であるという点で興味深く、立体選択的SN2'反応を利用した香料成分の新規合成経路を確立した。

以上本論文は、微生物を用いた不斉還元反応および新規不斉SN2'反応による光学活性香気成分の両鏡像体の合成に関する研究をまとめたものであり、学術上ならびに応用上貢献するところが少なくない。よって審査委員一同は本論文が博士(農学)の学位論文として価値があると認めた。