ISCE Meeting 2018 Budapest, Hungary (August 14, 2018)

Lepidopteran Sex Pheromones: Wonderland for a Natural Product Chemist

Tetsu Ando Graduate School of BASE, Tokyo University of Agriculture and Technology, Japan Representative lepidopteran sex pheromones Sex pheromones have been identified from 687 . Male attractants have been reported for other 1277 species. Type I rice stem borer silkworm smaller tea tortrix Unsaturated fatty OH O H alcohols, acetates bombykol OAc O and aldehydes with H OA c a C10 –C18 chain Found most commonly (75%)

Type II O O Polyunsaturated hydrocarbons and Z3,Z6,Z9-21:H epo3,Z6,Z9-19:H Z3,epo6,Z9-19:H their epoxides with a C17 –C23 chain Identified from evolved- groups (15%) plum cankerworm moth basalis giant geometrid moth

Photos from http://www.jpmoth.org/ The beginning of pheromone researches in Japan

Kyoto Univ. Prof. S. Ishii

National Inst. of 1971 Almond moth Mediterranean flour moth Agri. Tech. (Z9,E12-14:OAc) Dr. Y. Tamaki 1971 Smaller tea tortrix ＊＊ (Z9-14OAc ＊ + Z11-14:OAc) 1973 Common cutworm (Z9,E11-14:OAc + Z9,E12-14:OAc) 1973-1978 PhD student in a gibberellin laboratory (The Univ. of Tokyo) Trigger for pheromone studies 1973-1978 PhD student in a gibberellin laboratory of Prof. N. Takahashi (The Univ. of Tokyo) Participation in the project for identification of a pheromone of the rice stem borer ⇒ Systematic synthesis of pheromone candidates Known sex pheromones of female 1959 Silkworm moth (E10Z12-16:OH) 1966 Cabbage looper (Z7-12:OAc) 1967 Fall armyworm (Z9-14:OAc) 1968 Soybean looper (Z7-12:OAc) 1968 Redbanded leafroller (Z11-14:OAc) 1969 Oriental fruit moth (Z8-12:OAc) 1971 Codling moth (E8,E10-12:OH) 1971 Smaller tea tortrix (Z9-14OAc + Z11-14:OAc) 1971 Almond moth (Z9,E12:OAc) ⇒ No hit (natural pheromone: new aldehydes) Roelofs & Comeau, 1970. Lepidopterous sex attractants discovered by field screening tests. J. Econ. Entomol., 63: 969-974 ⇒ Systematic screening ⇒ Attraction of 93 species (Homerun !!) Random screening test ① Monoene acetates, Sept. 3 – 10, 1974 Carbon length of chain Double bond C-10 C-12 C-14 C-16 5-en Z ─ , ─ 0 , 1 ─ , ─ ─ , ─ E ─ , ─ 0 , 1 ─ , ─ ─ , ─ 7-en Z 1 , 0 3 + 1 +1 , 2 + 1 2 , 0 1 + 1 , 0 E 1+1 , 1+1 1 , 1 1 , 0 2 + 1 , 0 8-en Z ─ , ─ 23 + 2 , 5 0 , 1 0 , 0 E ─ , ─ 0 , 0 0 , 0 0 , 0 9-en Z ─ , ─ 1 , 1 +1 1 , 1 0 , 1 + 1 E ─ , ─ 0 , 0 1 , 0 0 , 0 11-en Z ─ , ─ 25 + 1 +1 , 26 +1 1 , 2 E ─ , ─ 1 , 1 1 , 7 + 3 control 0 , 0

Nippoptilia issikiiﾞ Cryptophlebia Choristoneura ombrodelta longicellana Dysaethria moza

Insect species were identified by Prof. H. Kuroko of Univ. Osaka Pref. Hedya auricristana Orthogonia sera Random screening test ② Monoene acetates, Sept. 11 – 18, 1974 Carbon length of chain Double bond C-10 C-12 C-14 C-16 5-en Z ─ , ─ 0 , 1 ─ , ─ ─ , ─ E ─ , ─ 0 , 0 ─ , ─ ─ , ─ 7-en Z 1 , 0 2 , 4 + 1 0 , 0 0 , 1 E 1+1 , 0 0 , 0 1 , 0 0 , 0 8-en Z ─ , ─ 7 + 1 , 3 0 , 0 0 , 0 E ─ , ─ 0 , 0 0 , 0 0 , 0 9-en Z ─ , ─ 0 , 0 1+1 , 1 0 , 0 E ─ , ─ 0 , 0 0 , 0 0 , 0 11-en Z ─ , ─ 28 , 32 1 , 0 E ─ , ─ 0 , 0 2 , 31 This is the trigger of my pheromone research. control 0 , 0

Nippoptilia issikiiﾞ Cryptophlebia Choristoneura Namely, the rootombrodelta of my researchlongicellana is synthesis.Dysaethria moza

InsectWhile species I had were no identified special skill of organic synthesis, simple structuresby Prof. H. of Kuroko the pheromones of Univ. Osaka workedPref. Hedya favorably auricristana for myOrthogonia research. sera Utilization of synthetic pheromone candidates 1973-1981 PhD student and post Doc. 1982-1995 Pesticide Chemistry Lab. in TUAT 1996-2015 Chemical Ecology Lab. In TUAT (BASE) (1) Screening tests 1-1) Type I compounds: Monoenes (single-component lures) 1-2) Type I compounds: Monoenes (multi-component lures) 1-3) Type I compounds: Dienyl compounds 1-4) Type II compounds (2) Authentic standards for analysis of natural pheromones 2-1) EAG analysis to determine a position and configuration of the double bond 2-2) Co-chromatography of GC-MS analysis (3) Study on chemical analysis 3-1) Speculation of fragmentations by mass spectrometry 3-2) Selection of an enantioselective HPLC column (4) Study on biosynthesis 4-1) Examination of substrate specificity of enzymes Electroantennogram (EAG) Oscilloscope Amplifier

Sample

Air Recording 1cm electrode 2cm Schneider, 1969 First record Reference electrode Roelofs et al., 1971 First application of EAG as a bioassay tool in a study with the grape berry moth Identification of a 8,10-diene by EAG activities of monoenes in a study with the codling moth Review: “Crop Protection Agnetns – Their Biological Evaluatin” The scope and limitation of the EAG technique in identifying pheromone components. Electroantennogram (EAG) Oscilloscope Amplifier

Sample

Air Recording 1cm electrode 2cm Schneider, 1969 First record Reference electrode Roelofs et al., 1971 First application of EAG as a bioassay tool In Japan, 1976 First equipment: Physical and Chemical Research Institute 1990 Purchase by Ando’s laboratory Arn et al., 1975 GC-EAD 2000 Purchase by Ando’s laboratory GC-EAD GC equipped with an EAG detector (EAD) One of the most important instrument for pheromone researches antenna

electrodes EAD injection FID Noctuidae Tortricoidea Drs. M. Ishitani Structure determination & F. Komai Noctuidae Crambidae Saturniidae Dr. O. Saito Stathmopodidae Dr. K. Kishino ＊ Drs, H. Naka, T. Suzuki & Noctuidae Limacodidae ＊ Sphingidae K. Tsuchida ＊ Drs. H. Honda Geometridae Dr. H. Naka & T. Uehara Yponomeutidae Dr. M. Nomura ＊＊ Noctuidae ＊ Dr. O. Saito ＊ ＊ ＊ ＊ ＊ Saturniidae ＊ Dr. N. Ohbayashi ＊ Lasiocampidae & S. A. Mafi Crambidae ＊ Dr. H. Tatsuta

Drs. T. Koshihara Geometridae Noctuidae S. Yamada Gracillariidae Drs. K. Katagiri, T. Ikeda Dr. M. Nomura & A. Yamane Geometridae Geometridae Dr. T. Horikawa Dr. H. Yasui Erebidae Dr. K. Ohtani Erebidae ＊ Dr. M. Kiso Erebidae Erebidae Tortricoidea Tortricoidea Drs. S Wakamura N. Arakaki ＊ Drs. F. Komai & M. Kinjo Dr. Y. Terashima Photos from http://www.jpmoth.org/ Lepidopteran Sex Pheromones: Wonderland for a Natural Product Chemist 1) Identification by collaboration with many entomologists Systematic synthesis of pheromone analogues Chemical analysis by GC-EAD and GC-MS Diversity of Type I and II pheromones https://lepipheromone.sakura.ne.jp/index_eng.html 2) Application Monitoring and Mating disruption 3) Biosynthesis and its endocrine regulation 4) Methyl-branched pheromones Type III ? Lepidopteran Sex Pheromones: Wonderland for a Natural Product Chemist 1) Identification by collaboration with many entomologist Systematic synthesis of pheromone analogues Chemical analysis by GC-EAD and GC-MS Diversity of Type I and II pheromones https://lepipheromone.sakura.ne.jp/index_eng.html 2) Application Monitoring and Mating disruption 3) Biosynthesis and its endocrine regulation 4) Methyl-branched pheromones Type III ? Commercialized lures for monitoring ①

1) Toleria romanovi ( of vine )

(8:1) OH Z3,Z13-18:OH OAc Z3,Z13-18:OAc

Diurnal moth mimicking a wasp Lure: rubber septum including 1mg of Larvae bore into the trunk, the synthetic pheromone causing the whole vine to death. Sold from Japan Protection Assoc. Recently, the damage suddenly spread. Commercialized lures for monitoring ②

1) Toleria romanovi (pest of vine trees) Sold from Japan Plant Protection Association 2) Rice green

(8:1) (4:1:1) OH Z3,Z13-18:OH OAc Z11-16:OAc OAc Z3,Z13-18:OAc OAc Z9-16:OAc 3) Asian corn borer OAc Z9-14:OAc 4) Diamond back moth OAc

OAc Z12-14:OAc (10:10:1) E12-14:OAc (3:2) O H Z11-16:Ald

5) Tea leafroller OAc Z11-16:OAc Z11-16:OH E11-16:Ald OH Z11-16:Ald (9:1) ６） Soybean pod borer O OAc H O H E8,E10-12:OAc Mating disruption with a Type II pheromone ①

Pheromone components of Ascotis selenaria cretacea, Japanese Giant Looper (Geometridae: Ennominae)

epo3,Z6,Z9-19:H Z3,Z6,Z9-19:H

TIC

GC-MS analysis of the pheromone gland extract

(6Z,9Z)-cis-3,4-epoxy-6,9-nonadecadiene O 1 3 4 69 19 (3Z,6Z,9Z)-3,6,9-nonadecatriene

1 9 Serious damage by the larvae 3 6 in a tea garden 19 Synthesis of Type II pheromones (racemic mixture)

Linolenic acid R

1. EtOH / H+ PPh3-I2 R = CO2H R = CH OH R = CH I 2 / imidazole 2 2. LiAlH4

MeMgBr MCPBA Epoxydiene mixture / Li CuCl 2 4 Z3,Z6,Z9-19:H

epo3,Z6,Z9-19:H MPLC of the mixture O epo9 epo3 epo6 Z3,epo6,Z9-19:H MPLC O

Z3,Z6,epo9-19:H 30 40 50 60 O Rt (min) Lober column n-hexane + THF (1 %) Mating disruption with a Type II pheromone ① Field evaluation of dispenser: polyethylene tube including 80 mg chemical 2 (i) 500 tubes / ha (16 tubes / 180 m2) (ii) 1000 tubes / ha (16 tubes / 90 m )

Tethered 2.78 m female 3.7 m

Dispenser 5.4 m 3.6 m (iii) 3000 tubes / ha (48 tubes / 82 m2) (iv) 5000 tubes / ha (48 tubes / 70 m2)

1.85 m 1.11 m

1.8 m 1.8 m Evaporation of triene: 0.546 mg/tube/day Evaporation of epoxydiene mixture : 0.386 mg/tube/day Mating disruption with a Type II pheromone ② Mating ratios of tethered females in the tea gardens which were permeated with triene or an epoxydiene mixture released from dispensers （polythylene tubes) a (A) Triene (B) Epoxydiene mixture b Tubes No. of females Mating No. of females Mating (N / ha) ratio ratio Tethered Mated (%) Tethered Mated (%)

0 11 11 100 14 14 100 250 - -- 13 3 23 500 10 6 60 14 4 29 1000 9 6 67 14 1 7 3000 10 8 80 12 0 0 5000 10 4 40 12 0 0

a Tested from Sept. 7 to 14, 1999. b Tested from Sept. 7 to 18, 1999. Preparation of optical active epoxy dienes Resolution by enantioselective HPLC

epo3 Chiralpak AD epo6 epo9 Normal phase n-hexane + 2-propanol (0.1 %)

O O O * * R * * R * * R cis-Epoxides (＊ chiral center） Preparation of optical active epoxy dienes Resolution by enantioselective HPLC

epo3 Chiralpak AD epo6 epo9 Normal phase n-hexane + 2-propanol (0.1 %)

Determination of absolute configuration

MeO OH O 1. MeOH / H+ esters of (S)- and 4-ol (R)-MTPA ester 2. MPLC HO OMe epo3,Z6,Z9-19:H esters of (S)- and 3-ol (R)-MTPA ester (fraction 1) Mosher’s method CF 3S,4R-isomer O 3 CF C C O 3 H OMe MeO O OMe H13C6 C6H13 MeO (S)-MTPA ester of 4-ol Diversity of Type II pheromones

Linoleic acid secretion O Z6,Z9-dienes OH E4,Z6,Z9-trienes mono- Z6,Z9,E11-trienes epoxides desaturation Z6,Z9,Z12-trienes Δ12

CnH2n+1 Δ4 Δ11 emerald moth secretion Linolenic acid O Z3,Z6,Z9-trienes OH 1,Z3,Z6,Z9-tetraenes mono- and Z3,Z6,Z9,E11-tetraenes di-epoxides desaturation Z3,Z6,Z9,Z11-tetraenes

CnH2n+1 Δ1 Δ11 The known chemical diversity is still limited. Novel compounds must be found. Z2,E4,Z6,Z9-21H Diagnostic ions of Type II pheromones ①

-H M-98 110 M-43 163 -H M-96 110 -H (A) -H 79 -H R R R R -H 79 79 81 150 -H 121 -H -H M-85

108 -H M-96 -H M-56 106 -H 108 -H -H 91 -2H 79 -H 79 -H R R R -H R 79 79 -H M-82 148 -H -H M-54 M-100 110 (B) O -H O R R -H -H -H M-125 99 M-114 M-111 111 122 97 M-58 108 O -H O -H O

R R -H R -H -H -H M-123 79 M-83 79 M-109 -H M-72 M-69

167 unknown (C) 128 111 M-125 M-125 * +H OOOOO O* RR R* -H M-85 M-69 M-139 -2H M-87 Diagnostic ions of Type II pheromones ②

-H M-98 110 M-43 163 -H M-96 110 -H (A) -H 79 -H R R R R -H 79 79 81 150 -H 121 -H -H M-85

108 -H M-96 -H M-56 106 -H 108 -H -H 91 -2H 79 -H 79 -H R R R -H R 79 79 -H M-82 148 -H -H M-54 136 -H 109 139 79 -H M-151 (B) 111 O O -H unknown R* R* R O * -H 79 97 M-111 M-111 109 113 -H M-114 111 122 97 M-58 108 O -H O -H O

R R -H R -H -H -H M-123 79 M-83 79 M-109 -H M-72 M-69 120 M-41 109 95 106 -H O * -H O * -H O * unknown R R -H R -H -H M-121 79 79 79 149 M-107 -H M-70 Phylogenetic tree of

yellow peach moth E10-16:Ald satin moth Z3,Z6,Z9-23:H plum cankerworm moth Z3,epo6,epo9-21:H Z3,Z6,Z9-21:H

Z3,Z6,Z9-23:H Papillionoidea Hesperioidea Pterophoroidea Tortricoidea Castnioidea Incurvarioidea

Eriocranioidea Zeugloptera Dacnonypha Type I pheromone Type II pheromone Paramecoptera Lepidopteran Sex Pheromones: Wonderland for a Natural Product Chemist 1) Identification by collaboration with many entomologist Systematic synthesis of pheromone analogues Chemical analysis by GC-EAD and GC-MS Diversity of Type I and II pheromones https://lepipheromone.sakura.ne.jp/index_eng.html 2) Application Monitoring and Mating disruption 3) Biosynthesis and its endocrine regulation 4) Methyl-branched pheromones Type III ? Pheromone biosynthesis

(A) How different between Type I and Type II pheromones? Determination of the biosynthetic pathways Type I silkworm moth (Bombycidae) chrysanthemum golden plusia (Noctuidae) Type II giant looper (Geometridae) (Erebidae) Synthesis of 14C- or D-labeled precursors → Topical application to a pheromone gland → Counting of radioactivity or GC-MS analysis

(B) How to apply the biosynthetic study? Block of the biosynthesis O Cyclopropenyl compound: R n X position-specific inhibitor of a desaturase Biosynthetic pathways ＊ Step whose enzyme Type I pheromone was cloned pheromone de novo Acetyl-CoA 16:Acyl gland α,α’-desaturation Desaturation＊ bombykol Z11-16:Acyl ＊ OH Reduction E10,Z12-16:Acyl E10,Z12-16:OH secretion

de novo pheromone Acetyl-CoA 16:Acyl gland

Desaturation α,β-desaturation Z7-12:Acyl Z11-16:Acyl β-Oxidation Desaturation E5,Z7-12:Acyl OAc Reduction E5,Z7-12:OAc Acetylation secretion Biosynthetic pathways ＊ Step whose enzyme Type I pheromone was cloned pheromone de novo Acetyl-CoA 16:Acyl gland α,α’-desaturation Desaturation＊ bombykol Z11-16:Acyl ＊ OH Reduction E10,Z12-16:Acyl E10,Z12-16:OH secretion Type II pheromone Linolenic acid (plant) Z9,Z12,Z15-18:Acid pheromone oenocytes gland Chain elongation O secretion Z11,Z14,Z17-20:Acyl epo3,Z6,Z9-19:H −CO Transportation Epoxydation＊ 2 via hemolymph Z3,Z6,Z9-19:H Z3,Z6,Z9-19:H (lipophorin) secretion PBAN Bioassay decapitation head Pheromone Biosynthesis extract Activating Neuropeptide 4 hr 24 hr eclosion circadian rhythm pheromone injection pheromone titer = 0 gland extraction 3 hr Reproduction of bombykol Endocrine system

Amino acid sequence of PBAN Amino acid Type I identities Silkworm LSEDMPATPADQEMYQPDPEEMESRTRYFSPRL-NH2 76% H. zea LSDDMPATPADQEMYRQDPEQIDSRTKYFSPRL-NH 2 44% Type II Giant looper QLVDDVPQRQQIEEDRLGSRTRFFSPRL-NH2

Shorter by 5 amino acids Essential active motif Similarity is low, but the N-amidated terminal motif of 5 amino acids is conserved. Activation steps by PBAN Type I pheromone pheromone de novo Acetyl-CoA 16:Acyl gland Desaturation bombykol Z11-16:Acyl PBAN OH

E10,Z12-16:Acyl E10,Z12-16:OH Reduction secretion Type II pheromone Linolenic acid (plant) Z9,Z12,Z15-18:Acid pheromone oenocyte gland Chain elongation O secretion Z11,Z14,Z17-20:Acyl epo3,Z6,Z9-19:H PBAN −CO2 Epoxydation Z3,Z6,Z9-19:H Z3,Z6,Z9-19:H (lipophorin) Summary Biosynthetic pathway Endocrine regulation Type I Acetyl-CoA smaller tea tortrix silkworm moth de novo 9-desaturase OAc Saturated acyl 11-desaturase OH 12-desarurase OA c desaturation etc. bombykol Unsaturated acyl Acyl reduction PBAN

Type II Linolenic acid giant geometrid Linolic acid giant looper O O chain elongation −CO2 Polyenyl hydrocarbon uptake by a gland PBAN epoxidation 3,4-epoxidase 6,7-epoxydase etc. 1) The biosyntheses of Type I and II pheromones are quite different, and differently regulated by PBAN. 2) The pheromones in each group are biosynthesize by some common steps. 3) Structural diversity is mainly promoted by reaction selectivity of the desaturases in Type I and the epoxidases in Type II. Summary Biosynthetic pathway Endocrine regulation Type I Acetyl-CoA smaller tea tortrix silkworm moth de novo 9-desaturase OAc Saturated acyl 11-desaturase OH 12-desarurase OA c desaturation etc. bombykol Unsaturated acyl Acyl reduction PBAN

Type II Linolenic acid giant geometrid Linolic acid giant looper O O chain elongation −CO2 Polyenyl hydrocarbon uptake by a gland PBAN epoxidation 3,4-epoxidase 6,7-epoxydase etc. New interesting target to understand the diversity Others Lyclene dharma apple leafminer Methyl-branched hydrocarbons, O ketones, secondary alcohols, O and etc. Lepidopteran Sex Pheromones: Wonderland for a Natural Product Chemist 1) Identification by collaboration with many entomologist Systematic synthesis of pheromone analogues Chemical analysis by GC-EAD and GC-MS Diversity of Type I and II pheromones https://lepipheromone.sakura.ne.jp/index_eng.html 2) Application Monitoring and Mating disruption 3) Biosynthesis and its endocrine regulation 4) Methyl-branched pheromones Type III ? Pheromones of moth ① (Number of the species Noctuoidea Noctuidae (100) whose pheromones were known) Erebidae (69) Lymantriinae (26) Nolidae (1) Arctiinae (27) Arctiini (24) Notodontidae (9) Synthomini (3) Lithosiini (0)

Because of harmless , nobody has been interested in their pheromones. Tokyo ⇒ Novel pheromones are expected.

Lichen moths Many species appear throughout the year. ⇒ Moths collected by a light trap were sent to Tokyo, Okinawa and their pheromones were analyzed. Iriomote Island Pheromones of lichen moth ②

Lyclene dharma dharma O Barsine expressa

(S)-Me6-18:2-one O O O (S)-Me14-18:2-one O O (S)-17:7-OPr O Me6,Me14-18:2-one

Miltochrista calamin (S)-17:8-OPr OH

(5R,7R)-Me5-17:7-OH Each species produces the pheromone with a unique structure. Pheromones of other species？ Methyl-branched 2-ketones acting as a pheromone

Lepidoptera Coleoptera O Diabrotica balteata (S)-Me6-18:2-one (banded cucumber beetle) O O

Lyclene dharma (S)-Me14-18:2-one Me6,Me12-15: 2-one O Chuman et al. , JCE 13: 1601 (1987)

Me6,Me14-18: 2-one Diabrotica undecimpunctata Yamamoto et al. , BBB 71: 2860 (2007) (spotted cucumber beetle) Me10-13: 2-one Blattodea Guss et al. , JCE 9: 1363 (1983) Blattella germanica Arachnida (German cockroach) Chortoglyphus arcuatus O (Acari) C H (storage mite) 18 37 O

Me3,Me11-29: 2-one Me4,Me6,Me8-10: 2-one Nishida et al. , Experientia 30: 978 (1974) chortolure Schulz et al. , Chembiochem 5: 1500 (2004) Methyl-branched 2-ketones acting as a pheromone

Lepidoptera Coleoptera O Diabrotica balteata (S)-Me6-18:2-one (banded cucumber beetle) O O

Lyclene dharma (S)-Me14-18:2-one Me6,Me12-15: 2-one O Chuman et al. , JCE 13: 1601 (1987)

Me6,Me14-18: 2-one Diabrotica undecimpunctata Yamamoto et al. , BBB 71: 2860 (2007) (spotted cucumber beetle) “Propanogenins” Me10-13: 2-one formed by incorporation of propanoate units Blattodea Guss et al. , JCE 9: 1363 (1983) derived from methylmalonyl-CoA Blattella germanica Arachnida (German cockroach) Chortoglyphus arcuatus O (Acari) C H (storage mite) 18 37 O

Me3,Me11-29: 2-one Me4,Me6,Me8-10: 2-one Nishida et al. , Experientia 30: 978 (1974) chortolure Schulz et al. , Chembiochem 5: 1500 (2004) Methyl-branched sex pheromones of Lepidoptera

Lichen moths inhabiting Iriomote Island O OH O

O Lyclene dharma Yamakawa et al., 2011, Miltochrista Tetrahedron Lett., 52, 5808 calamina Adachi et al., 2010, JCE, 36, 814

Leafminer moths () Hemlock looper (Geometridae) Gries et al., 1991, Sugiei et al., 1984, Naturwiss.., 78, 315 AEZ, 36, 814 peach leafminer Gries et al., 1993, JCE, 19, 1501 Gries et al., 1997, JCE, 23, 1119 apple leafminer Bagworm moth (Psychidae ) O Francke et al., 1987, mountain-ash bentwing Naturwiss., 74, 143 O Gries et al., 2006, JCE, 32, 1673 Synthetic strategy for 5,9-dimethyl compounds

Tamagawa et al., 1999 EJOC, 973

O O + C3 + C7 (Grignard) Block A Block B (Grignard) SN2 SN2 OTs X TsO + C Y 1 + C2 Key Points How to accomplish OH MeO C HO the perfect inversion 2 How to confirm it

SN2 reaction between a secondary tosylate and a carbanion associates with stereospecific inversion. Me Me OTs： leaving group Nu OTs Nu H H Nu： nucleophile (carbanion) R R Block A Block B Synthesis of the chiral block A (5S.9R)-1

1) n-C H MgBr NaCH(CO Me) LiCl / DMSO-H O 3 7 2 2 CO Me 2 OTs 2 O 2) TsCl, Et N / DME 170-180ºC 3 CO Me (R)-2 (R)-3 (S)-4 2

LiAlH I , PPh CO Me 4 2 3 2 OH I / THF imidazole / THF (S)-5 (S)-6 (S)-A

Enantioselective HPLC Analysis OH

Chiralpak (R)-3 Chiralpak AS-H AY-H (S)-6 (S)-3 (R)-6 10% IPA 0.4 % IPA in hexane in hexane UV 254 nm RI detector

010203020 25 30 t tR R (min) (min) Application of the SN2 Reaction Synthesis of a 1,11-dimethyl pheromone produced by the bagworm moth SN2

SO2Ph OTs + C O Block A 1 (S)-oxide (p-Me(C6H4)SO2CH2Li) CO H O 2 H N O 2 pheromone (3R,13R,3'S)- (S)-valine

RuCl3, NaIO4 I R R CO2H Block B Carlsen et al. 1981 JOC, 46, 3936

SN2 O HO + C2 MsO Block C or TsO- (R)-oxide A useful chiral building block including a methyl branch between two functional groups Biosynthesis of methyl-branched pheromones

Δ1,Me10,Me14-18:H Me6,Me14-18:2-one O

Propanogenins： formed by incorporation of propanoate units derived from methylmalonyl-CoA The biosynthesis is still an untouched research area. Future expected studies

1) How is the branched skeleton constructed? PKS (polyketide synthase) or FAS (fatty acid synthase)? 2) How is a specific configuration introduced at the branched position? 3) How are the functional groups created? 4) Which organs produce them, pheromone gland or oenocytes? 5) Is there a circadian rhythm? Is PBAN involved in the biosynthesis?

Selection of optimal species, which can be used for these researches Pheromone-dependent speciation Biosynthetic pathways of Plusiinae pheromones 18:Acid chemical diversity Δ11-desaturation 11-18 16:Acid 16:Acid 16:Acid Δ9-desaturation Δ11-desaturation Δ13-desaturation 9-16 11-16 13-16 β-oxidation 14:Acid P. festucae Δ11-desaturation 7-14 9-14 M. confusa 11-14 M. purissima 5-12 7-12 T. ni 9-12 Δ5-desatu- C. agnata ration 5,7-12 3-10 5-10 A. peponis 7-10 T. intermixta D. chrysitis ω7-compounds ω5-compounds ω3-compounds

ManyThe birth pheromones of a new speciesare composed of a 7-12 compound as a key component. ♀: Mutation of a pheromone biosynthesis system The ancestor species acquired an ability to producesynchronized it. ♂: Mutation of a pheromone receptor system The descendants developed the additional ability to produce Actuallya structure happened related in acom longpound. history Acknowledgments The University of Tokyo: Drs. A. Suzuki, H. Nagasawa, S. Tatsuki, Y. Ishikawa, & Y. Rong The Inst. of Chem. & Phys. Res.: Drs. S. Matsumoto & K. Moto University of Osaka Prefecture: Dr. H. Kuroko Osaka University of Arts: Dr. F. Komai Tottori University: Dr. H. Naka University of Ryukyus: Drs. M. Kinjo, Y. Terashima & H. Tatsuta University of Tsukuba: Drs. H. Honda & T. Uehara Nat. Inst. of Sericult. & Entomol. Sci.: Drs. S. Wakamura & H. Yasui Shin-Etsu Chemical Company: Drs. F. Mochizuki & T. Hukumoto Other many collaborators Thank you for your attention!! Chemical Ecology Laboratory of TUAT (1982-2015) PhD students Dr. X.-R. QI Dr. H. Watanabe Dr. K. Matsuoka Dr. Wijaksono Dr. W. Wei Dr. MD A. Islam Dr. M. Yamamoto Dr. T. Mouri Dr. R. Yamakawa Dr. S. Inomata Dr. S.-J. Lee Dr. J. Suzuki Dr. S.-J. Lee Dr. L. V. Vang Dr. N. D. Do Dr. H. Yamazawa Dr. T. Fujii Dr. T. Taguri Dr. A. Ono Dr. T. Kawai Dr . Q. Yan