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Supplementary Data

Proposed Mechanism for the Diterpene Synthases in the Formation of Phomactatriene andTaxadiene: Experimental Results on Biosynthetic Study, Biomimetic Reactions andExamination of the Cyclization Pathway by ab initio Calculations

Tetsuo Tokiwano, Taeko Endo, Tae Tsukagoshi, Hitoshi Goto, Eri Fukushi, and Hideaki Oikawa*

Table of Contents:

1. Table S1. MMFF94 and ab Initio HF/6-31G* Energies of Top Five Conformers Found by CONFLEX

Search

--------------------S2-S3

2. Figure S1. Optimized Structures of Top Five Conformers with ab Initio HF/6-31G*Calculation

--------------------S4-S8

3. 1H and 13C NMR spectra for compounds 9a isolated from Phoma sp.

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4. 1H and 13C NMR spectra for compounds 9b, 9c, 13a, 13b and 14 obtained from the acid-catalyzed

rearrangements of verticillol (5)

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Table S1. MMFF94 and ab Initio HF/6-31G* Energies of Top Five Conformers Found by CONFLEX Search

A+

MMFF94 HF/6-31G(d)No. ∆E a E b ∆E a

1 0.00 -776.2916 0.002 2.29 -776.2872 2.763 2.65 -776.2873 2.664 3.40 -776.2862 3.395 4.94 -776.2817 6.19

a kcal/mol. b au.

G+

MMFF94 HF/6-31G(d)No. ∆E a E b ∆E a

1 0.00 -776.2906 0.002 0.32 -776.2893 0.833 1.36 -776.2915 -0.544 1.85 -776.2895 0.705 1.94 -776.2860 2.93

a kcal/mol. b au.

F+

MMFF94 HF/6-31G(d)No. ∆E a E b ∆E a

1 0.00 -776.2965 0.002 3.05 -776.2931 2.123 4.07 -776.2891 4.694 4.21 -776.2883 5.155 4.45 -776.2890 4.74

a kcal/mol. b au.

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D+

MMFF94 HF/6-31G(d)No. ∆E a E b ∆E a

1 0.00 -776.2877 0.002 0.02 -776.2844 2.093 0.40 -776.2850 1.704 0.84 -776.2813 4.075 2.05 -776.2818 3.74

a kcal/mol. b au.

E+

MMFF94 HF/6-31G(d)No. ∆E a E b ∆E a

1 0.00 -776.2856 0.002 0.58 -776.2851 0.303 0.95 -776.2862 -0.404 0.98 -776.2846 0.595 1.40 -776.2829 1.67

a kcal/mol. b au.

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A+ No. 1 A+ No. 2

A+ No. 3 A+ No. 4

A+ No. 5

Figure S1. Optimized Structures of Top Five Conformers with ab Initio HF/6-31G* Calculation

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G+ No. 1 G+ No. 2

G+ No. 3 G+ No. 4

G+ No. 5

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5 4 3 2 1 0δ / ppm

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180 160 140 120 100 80 60 40 20δ / ppm

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Exp 1.2

CommentFile piccpmt/3COSYOikawa PMT 1mg /90uL C6D6dH(Solv)=7.15ppm, dC(solv)=128.0pBruker AMX-500/ GC-MS & NMR Lab.Grad. Sch. Agric., Hokkaido Univ.H=piccpmt/1 C=piccpmt/2

$ F2-Acquisition Parameters Date Mon Jun 17 16:48:33 2002PULPROG cosygsTD 1024NS 1DS 2D[0] 0.000003 secD[1] 1.999999 secHL1 5 dBIN[0] 0.000218 secP[1] 12.000000 usecSW 9.171891 ppmSWH 4587.155963 HzFIDRES 0.223232 HzAQ 0.111616 secRG 4096NUCLEUS 1HSFO1 500.131982 MHzBF1 500.130000 MHzO1 1982.147592 MHzSFO2 0.000000 MHzBF2 500.130000 MHzO2 1982.147592 MHzLOKNUC 2HSOLVENT C6D6PROBHD 2.5 mm DUL 13C-1H-D Z-GRTE 300 KGRDPROG 2sineCNST[21] 10 %CNST[22] -10 %D[16] 0.000100 secP[16] 1000.000000 usecL[21] 100 $ F1-Acquisition Parameters TD 256ND0 1SW 9.171891 ppmSWH 4587.155963 HzFIDRES 0.055808 Hz $ F2-Processing Parameters SI 1024SF 500.130062538213OFFSET 8.424184HZpPT 4.479648 HzMC2 qfWDW SINESSB 0.000000 $ F1-Processing Parameters SI 512SF 500.130062538213OFFSET 8.424184REVERSE yesHZpPT 8.959297 HzMC2 qfWDW SINESSB 0.000000expt = ca. 9 min

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Exp 1.2

CommentFile piccpmt/4noesyOikawa PMT 1mg /90uL C6D6dH(Solv)=7.15ppm, dC(solv)=128.0pBruker AMX-500/ GC-MS & NMR Lab.Grad. Sch. Agric., Hokkaido Univ.H=piccpmt/1 C=piccpmt/2

$ F2-Acquisition Parameters Date Mon Jun 17 16:59:41 2002PULPROG noesygs.jpTD 1024NS 16DS 4D[0] 0.000003 secD[1] 2.002048 secD[9] 1.000000 secHL1 5 dBIN[0] 0.000109 secP[1] 12.000000 usecV9 2.000000 %SW 9.171891 ppmSWH 4587.155963 HzFIDRES 0.223232 HzAQ 0.111616 secRG 2048NUCLEUS 1HSFO1 500.131982 MHzBF1 500.130000 MHzO1 1982.147592 MHzSFO2 0.000000 MHzBF2 500.130000 MHzO2 1982.147592 MHzLOKNUC 2HSOLVENT C6D6PROBHD 2.5 mm DUL 13C-1H-D Z-GRTE 300 KGRDPROG 1sineCNST[21] 30 %D[16] 0.000100 secP[16] 1000.000000 usecL[21] 100 $ F1-Acquisition Parameters TD 256ND0 2SW 9.171891 ppmSWH 4587.155963 HzFIDRES 0.055808 Hz $ F2-Processing Parameters SI 512SF 500.130062538213OFFSET 8.424184HZpPT 8.959297 HzMC2 qfWDW QSINESSB 2.000000PH_mod pkPHC0 35.422300 degreePHC1 25.800010 degree $ F1-Processing Parameters SI 512SF 500.130062538213OFFSET 8.424184REVERSE noHZpPT 8.959297 HzMC2 TPPIWDW QSINESSB 2.000000PH_mod pkPHC0 15.443750 degreePHC1 -29.200000 degreeexpt = ca. 4 hr

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Exp 1.2Comment

File piccpmt/7hsqcOikawa PMT 1mg /90uL C6D6dH(Solv)=7.15ppm, dC(solv)=128.0pBruker AMX-500/ GC-MS & NMR Lab.Grad. Sch. Agric., Hokkaido Univ.H=piccpmt/1 C=piccpmt/2

$ F2-Acquisition Parameters Date Thu Jun 20 15:38:53 2002PULPROG invigstp.mTD 1024NS 96DS 2D[0] 0.000003 secD[1] 2.181247 secD[4] 0.001800 secDL[0] 19 dBDL[5] 41 dBHL1 5 dBIN[0] 0.000013 secP[1] 12.000000 usecP[3] 5.800000 usecP[31] 0.000000 usecSW 9.171891 ppmSWH 4587.155963 HzFIDRES 0.223232 HzAQ 0.111616 secRG 32768NUCLEUS 1HSFO1 500.131982 MHzBF1 500.130000 MHzO1 1982.147592 MHzSFO2 125.768146 MHzBF2 125.757739 MHzO2 10407.375084 MHzLOKNUC 2HSOLVENT C6D6PROBHD 2.5 mm DUL 13C-1H-D Z-GRTE 300 KGRDPROG 3sineCNST[21] 40 %CNST[22] 15 %CNST[23] 10 %D[16] 0.000000 secP[16] 1000.000000 usecL[21] 100 $ F1-Acquisition Parameters TD 256ND0 4SW 159.022778 ppmSWH 20000.000000 HzFIDRES 0.012800 Hz $ F2-Processing Parameters SI 1024SF 500.130062538213OFFSET 8.424184HZpPT 4.479648 HzMC2 qfWDW QSINESSB 2.000000PH_mod pkPHC0 -100.325000 degreePHC1 -25.600000 degree $ F1-Processing Parameters SI 512SF 125.757762832763OFFSET 162.0858REVERSE noHZpPT 39.062500 HzMC2 TPPIWDW QSINESSB 2.000000PH_mod pkPHC0 289.530900 degreePHC1 -253.800000 degreeexpt = ca. 16 hr

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Exp 1.2

Comment File piccpmt/6

HMBCOikawa PMT 1mg /90uL C6D6dH(Solv)=7.15ppm, dC(solv)=128.0pBruker AMX-500/ GC-MS & NMR Lab.Grad. Sch. Agric., Hokkaido Univ.H=piccpmt/1 C=piccpmt/2

$ F2-Acquisition Parameters Date Tue Jun 18 02:02:20 2002PULPROG inv4gslplrnd.jpTD 1024NS 192DS 2D[0] 0.000003 secD[1] 2.000000 secD[2] 0.003450 secD[6] 0.070000 secDL[0] 19 dBHL1 5 dBIN[0] 0.000025 secP[1] 12.000000 usecP[3] 5.800000 usecSW 9.171891 ppmSWH 4587.155963 HzFIDRES 0.223232 HzAQ 0.111616 secRG 32768NUCLEUS 1HSFO1 500.131982 MHzBF1 500.130000 MHzO1 1982.147592 MHzSFO2 125.768146 MHzBF2 125.757739 MHzO2 10407.375084 MHzLOKNUC 2HSOLVENT C6D6PROBHD 2.5 mm DUL 13C-1H-D Z-GRTE 300 KGRDPROG 3sineCNST[21] 10 %CNST[22] 10 %CNST[23] 5 %D[16] 0.000000 secP[16] 1000.000000 usecL[21] 100 $ F1-Acquisition Parameters TD 256ND0 2SW 159.022778 ppmSWH 20000.000000 HzFIDRES 0.012800 Hz $ F2-Processing Parameters SI 1024SF 500.130062538213OFFSET 8.424184HZpPT 4.479648 HzMC2 qfWDW GMGB 0.300000LB -10.000000 $ F1-Processing Parameters SI 512SF 125.757762832763OFFSET 162.0858REVERSE noHZpPT 39.062500 HzMC2 qfWDW QSINESSB 6.000000expt = ca. 29 hr

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CommentFile piccpmt/104noesyOikawa verticillol-(BF3)-(-78)-fr3 ? mg /60uL CDCl3dH(Solv)=7.24ppm, dC(solv)=77.0ppmBruker AMX-500/ GC-MS & NMR Lab.Grad. Sch. Agric., Hokkaido Univ.H=piccpmt/101 C=piccpmt/102

$ F2-Acquisition Parameters Date Sat Nov 15 15:07:52 2003PULPROG noesygs.jpTD 1024NS 32DS 4D[0] 0.000003 secD[1] 1.943680 secD[9] 1.000000 secHL1 5 dBIN[0] 0.000166 secP[1] 12.000000 usecV9 2.000000 %SW 6.022513 ppmSWH 3012.048193 HzFIDRES 0.339968 HzAQ 0.169984 secRG 1024NUCLEUS 1HSFO1 500.131493 MHzBF1 500.130000 MHzO1 1492.510573 MHzSFO2 0.000000 MHzBF2 500.130000 MHzO2 1492.510573 MHzLOKNUC 2HSOLVENT CDCl3PROBHD 2.5 mm DUL 13C-1H-D Z-GRD Z5541/008TE 300 KGRDPROG 1sineCNST[21] 30 %D[16] 0.000100 secP[16] 1000.000000 usecL[21] 100 $ F1-Acquisition Parameters TD 256ND0 2SW 6.022513 ppmSWH 3012.048193 HzFIDRES 0.084992 Hz $ F2-Processing Parameters SI 512SF 500.130023435046OFFSET 5.948652HZpPT 5.882910 HzMC2 qfWDW QSINESSB 2.000000PH_mod pkPHC0 -11.780910 degreePHC1 104.200000 degree $ F1-Processing Parameters SI 512SF 500.130023435046OFFSET 5.948652REVERSE noHZpPT 5.882910 HzMC2 TPPIWDW QSINESSB 2.000000PH_mod pkPHC0 11.223440 degreePHC1 -23.200000 degreeexpt = ca. 7 hr

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1δ / ppm

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Exp 1.5 CommentFile piccpmt/206noesyOikawa No.2 (verticillol-27.5-L(OTf)3-10min-m/z=121,135) 0.3mg/90uL C6D6dH(Solv)=7.15ppm, dC(solv)=128.ppmBruker AMX-500/ GC-MS & NMR LabGrad. Sch. Agric., Hokkaido Uni.H=piccpmt/201 C=piccpmt/202

$ F2-Acquisition Parameters Date Wed Feb 11 10:36:27 204PULPROG noesy.jpTD 1024NS 256DS 4D[0] 0.000003 secD[1] 1.750000 secD[9] 1.000000 secHL1 5 dBIN[0] 0.000123 secP[1] 12.000000 usecV9 2.000000 %SW 8.127936 ppmSWH 4065.040650 HzFIDRES 0.251904 HzAQ 0.125952 secRG 2048NUCLEUS 1HSFO1 500.131973 MHzBF1 500.130000 MHzO1 1972.771511 MHzSFO2 0.000000 MHzBF2 500.130000 MHzO2 1972.771511 MHzLOKNUC 2HSOLVENT C6D6PROBHD 2.5 mm DUL 13C-1H-D Z-RD Z5541/008TE 300 K $ F1-Acquisition Parameters TD 128ND0 2SW 8.127936 ppmSWH 4065.040650 HzFIDRES 0.031488 Hz $ F2-Processing Parameters SI 512SF 500.130062049003OFFSET 7.884435HZpPT 7.939531 HzMC2 qfWDW QSINESSB 2.000000PH_mod pkPHC0 23.159380 degreePHC1 47.200000 degree $ F1-Processing Parameters SI 512SF 500.130062049003OFFSET 7.884435REVERSE noHZpPT 7.939531 HzMC2 TPPIWDW QSINESSB 2.000000PH_mod pkPHC0 11.223440 degreePHC1 -23.200000 degreeME_mod LPfrNCOEF 16LPBIN 256expt = ca. 27 hr

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CommentFile piccpmt/506noesyOikawa Ver2-7 TiCl4-95 mg /90uL C6D6dH(Solv)=7.15ppm, dC(solv)=128.0ppmBruker AMX-500/ GC-MS & NMR Lab.Grad. Sch. Agric., Hokkaido Univ.H=piccpmt/501 C=piccpmt/502

$ F2-Acquisition Parameters Date Mon Aug 30 18:28:05 2004PULPROG noesy.jpTD 1024NS 256DS 4D[0] 0.000003 secD[1] 1.750000 secD[9] 1.000000 secHL1 5 dBIN[0] 0.000123 secP[1] 12.000000 usecV9 2.000000 %SW 8.127936 ppmSWH 4065.040650 HzFIDRES 0.251904 HzAQ 0.125952 secRG 2048NUCLEUS 1HSFO1 500.131973 MHzBF1 500.130000 MHzO1 1972.771511 MHzSFO2 0.000000 MHzBF2 500.130000 MHzO2 1972.771511 MHzLOKNUC 2HSOLVENT C6D6PROBHD 2.5 mm DUL 13C-1H-D Z-GRD Z5541/008TE 300 K $ F1-Acquisition Parameters TD 128ND0 2SW 8.127936 ppmSWH 4065.040650 HzFIDRES 0.031488 Hz $ F2-Processing Parameters SI 512SF 500.130062049003OFFSET 7.884435HZpPT 7.939531 HzMC2 qfWDW QSINESSB 2.000000PH_mod pkPHC0 45.822670 degreePHC1 55.400020 degree $ F1-Processing Parameters SI 512SF 500.130062049003OFFSET 7.884435REVERSE noHZpPT 7.939531 HzMC2 TPPIWDW QSINESSB 2.000000PH_mod pkPHC0 11.223440 degreePHC1 -23.200000 degreeME_mod LPfrNCOEF 16LPBIN 256expt = ca. 27 hr

14

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