Nucleotidylation of Unsaturated Carbasugar in Validamycin ... · RG 1625.5 AQ 1.4418420 sec FIDRES...
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1 Nucleotidylation of Unsaturated Carbasugar in Validamycin Biosynthesis Jongtae Yang, 1 Hui Xu, 1,2 Yirong Zhang, 2 Linquan Bai, 2 Zixin Deng, 2 Taifo Mahmud 1, * 1 Department of Pharmaceutical Sciences, Oregon State University, Corvallis, OR 97331; 2 Laboratory of Microbial Metabolism and School of Life Sciences & Biotechnology, Shanghai Jiaotong University, Shanghai 200030, China Table S1. Strains and plasmids used in this study Strain/plasmid Relevant genotype/comments Source/ref. Escherichia coli DH10B F - mcrA Δ(mrr-hsdRMS-mcrBC)φ80lacZ ΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ - rspL nupG GibcoBRL ET12567(pUZ8002) dam dcm hsdS, pUZ8002 1 BW25113 K12 derivative: ΔaraBAD, ΔrhaBAD 2 S. hygroscopicus 5008 wild-type producer of validamycin 3 ZYR-2 5008 derivative generated by replacement of a 936-bp DNA fragment internal to valB with the 1.40-kb oriT-aac(3)IV cassette This work ZYR-2/pJTU918 ZYR-2 complemented with pJTU918 harboring full-length valB This work Plasmids pHZ1358 tsr, bla, oriT, ori (pIJ101) 4 # Supplementary Material (ESI) for Organic & Biomolecular Chemistry # This journal is (c) The Royal Society of Chemistry 2010
Transcript of Nucleotidylation of Unsaturated Carbasugar in Validamycin ... · RG 1625.5 AQ 1.4418420 sec FIDRES...
1
Nucleotidylation of Unsaturated Carbasugar in Validamycin Biosynthesis
Jongtae Yang,1 Hui Xu,1,2 Yirong Zhang,2 Linquan Bai,2 Zixin Deng,2 Taifo Mahmud1,*
1Department of Pharmaceutical Sciences, Oregon State University, Corvallis, OR 97331; 2Laboratory of
Microbial Metabolism and School of Life Sciences & Biotechnology, Shanghai Jiaotong University, Shanghai
200030, China
Table S1. Strains and plasmids used in this study
Strain/plasmid Relevant genotype/comments Source/ref.
Escherichia coli
DH10B F- mcrA Δ(mrr-hsdRMS-mcrBC)φ80lacZ
ΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara,
leu)7697 galU galK λ- rspL nupG
GibcoBRL
ET12567(pUZ8002) dam dcm hsdS, pUZ8002 1
BW25113 K12 derivative: ΔaraBAD, ΔrhaBAD 2
S. hygroscopicus
5008 wild-type producer of validamycin 3
ZYR-2 5008 derivative generated by replacement of a
936-bp DNA fragment internal to valB with
the 1.40-kb oriT-aac(3)IV cassette
This work
ZYR-2/pJTU918 ZYR-2 complemented with pJTU918
harboring full-length valB
This work
Plasmids
pHZ1358 tsr, bla, oriT, ori (pIJ101) 4
# Supplementary Material (ESI) for Organic & Biomolecular Chemistry# This journal is (c) The Royal Society of Chemistry 2010
2
pHZ2229 pBluescript II SK(+) with the 6.0 kb BamHI
fragment from cosmid 20E1
3
pIJ790 λ-RED (gam, bet, exo), cat, araC, rep101ts 2
pIJ773 aac(3)IV, oriT 2
pRSET-B PT7 RBS 6×His XpressTM Epitope EK, bla Invitrogen
pMD18 T-vector pUC18 derivative TaKaRa
pPM927 tsr, oriT, int, attP 5
pJTU968 pRSET-B derivative bla, PermE* 6
pJTU700 5.80-kb BamHI fragment from pHZ2229
cloned in BamHI-digested pHZ1358
This work
pJTU702 pJTU700 recombinant with 1384-bp oriT-
aac(3)IV cassette through Redirect
Technology
This work
pJTU707 1.10-kb valB fragment cloned in pRSET-B This work
pJTU915 1.10-kb NdeI/EcoRI fragment from pJTU707
cloned in pJTU968
This work
pJTU918 pPM927 carrying PermE* and valB This work
Figures S1 – S24: NMR Spectra
# Supplementary Material (ESI) for Organic & Biomolecular Chemistry# This journal is (c) The Royal Society of Chemistry 2010
0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5 ppm
2.05
93.
607
3.61
13.
619
3.63
33.
640
3.64
43.
651
3.89
93.
944
4.08
74.
111
4.11
94.
144
4.16
04.
197
4.45
64.
465
4.64
14.
649
4.68
04.
687
4.72
54.
812
4.84
94.
860
4.89
84.
943
4.98
05.
018
5.02
85.
045
5.05
15.
070
5.19
65.
202
5.20
75.
885
5.88
85.
903
5.90
77.
227
7.25
07.
260
7.26
37.
275
7.28
57.
298
7.30
97.
324
7.33
07.
337
7.34
87.
358
7.38
07.
387
7.39
97.
412
0.99
0
1.02
31.
064
2.02
3
2.07
83.
051
1.10
91.
051
1.07
23.
134
1.07
41.
008
1.00
0
2.43
925
.861
2.06
1
PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 300.1300063 MHzSI 32768F2 − Processing parameters
SFO1 300.1319508 MHzPL1 0.00 dBP1 11.00 usecNUC1 1H======== CHANNEL f1 ========
TD0 1D1 1.60000002 secTE 296.4 KDE 158.64 usecDW 111.200 usecRG 287.4AQ 7.2876530 secFIDRES 0.068610 HzSWH 4496.403 HzDS 4NS 9SOLVENT CDCl3TD 65536PULPROG zg30PROBHD 5 mm BBO BB/BBINSTRUM DRX300Time 12.30Date_ 20070507F2 − Acquisition Parameters
PROCNO 1EXPNO 1NAME JTY−1−37−04Current Data Parameters
# Supplementary Material (ESI) for Organic & Biomolecular Chemistry# This journal is (c) The Royal Society of Chemistry 2010
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Figure S1. 1H NMR spectrum of compound 9a
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
69.2
0469
.268
69.3
3969
.978
71.1
6471
.242
72.4
1672
.868
74.6
0175
.182
76.7
4077
.164
77.3
6577
.588
78.3
0978
.377
79.7
4980
.274
121.
319
121.
356
127.
736
127.
763
127.
792
127.
818
127.
853
127.
972
128.
118
128.
305
128.
342
128.
462
128.
492
128.
549
128.
627
136.
035
136.
133
136.
162
136.
265
138.
013
138.
051
138.
542
138.
725
143.
049
PC 1.40GB 0LB 1.00 HzSSB 0WDW EMSF 75.4677404 MHzSI 32768F2 − Processing parameters
SFO2 300.1312005 MHzPL13 19.00 dBPL12 19.00 dBPL2 0.00 dBPCPD2 85.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========
SFO1 75.4783145 MHzPL1 0.00 dBP1 8.95 usecNUC1 13C======== CHANNEL f1 ========
TD0 1DELTA 0.20000002 secd11 0.03000000 secD1 0.30000001 secTE 296.7 KDE 6.00 usecDW 22.000 usecRG 3251AQ 1.4418420 secFIDRES 0.346791 HzSWH 22727.273 HzDS 8NS 1360SOLVENT CDCl3TD 65536PULPROG zgpg30PROBHD 5 mm BBO BB/BBINSTRUM DRX300Time 13.20Date_ 20070507F2 − Acquisition Parameters
PROCNO 1EXPNO 1NAME JTY−1−37−04cCurrent Data Parameters
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Figure S2. 13C NMR spectrum of compound 9a
2.02.53.03.54.04.55.05.56.06.57.07.5 ppm
3.77
63.
788
3.79
93.
867
3.90
94.
177
4.21
84.
290
4.30
84.
430
4.47
04.
485
4.52
54.
680
4.71
64.
763
4.80
04.
816
4.83
74.
854
4.86
24.
923
4.94
24.
960
4.98
15.
007
5.03
35.
066
5.07
65.
824
7.23
37.
240
7.25
47.
277
7.28
97.
302
7.31
0
2.02
40.
999
1.01
00.
987
2.04
9
1.03
4
9.20
3
1.10
3
1.00
2
31.0
84
PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 300.1300061 MHzSI 32768F2 − Processing parameters
SFO1 300.1319508 MHzPL1 0.00 dBP1 11.00 usecNUC1 1H======== CHANNEL f1 ========
TD0 1D1 1.60000002 secTE 303.0 KDE 158.64 usecDW 111.200 usecRG 322.5AQ 7.2876530 secFIDRES 0.068610 HzSWH 4496.403 HzDS 4NS 6SOLVENT CDCl3TD 65536PULPROG zg30PROBHD 5 mm BBO BB−1HINSTRUM DRX300Time 16.19Date_ 20100209F2 − Acquisition Parameters
PROCNO 1EXPNO 2NAME JTY−1−41−02Current Data Parameters
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Figure S3. 1H NMR spectrum of compound 9b
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
69.3
5469
.432
69.5
1469
.883
72.6
7175
.016
75.3
9075
.613
76.7
4077
.164
77.3
6577
.587
78.9
1278
.998
79.7
4482
.709
82.7
9883
.944
124.
180
127.
649
127.
780
127.
807
127.
830
127.
880
127.
949
127.
981
127.
999
128.
015
128.
411
128.
522
128.
558
128.
641
128.
671
136.
012
136.
086
138.
229
138.
486
138.
540
PC 1.40GB 0LB 1.00 HzSSB 0WDW EMSF 75.4677364 MHzSI 32768F2 − Processing parameters
SFO2 300.1312005 MHzPL13 19.00 dBPL12 19.00 dBPL2 0.00 dBPCPD2 85.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========
SFO1 75.4783145 MHzPL1 0.00 dBP1 8.95 usecNUC1 13C======== CHANNEL f1 ========
TD0 1DELTA 0.20000002 secd11 0.03000000 secD1 0.30000001 secTE 303.0 KDE 6.00 usecDW 22.000 usecRG 32768AQ 1.4418420 secFIDRES 0.346791 HzSWH 22727.273 HzDS 8NS 4000SOLVENT CDCl3TD 65536PULPROG zgpg30PROBHD 5 mm BBO BB−1HINSTRUM DRX300Time 18.23Date_ 20100209F2 − Acquisition Parameters
PROCNO 1EXPNO 2NAME JTY−1−41−02cCurrent Data Parameters
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Figure S4. 13C NMR spectrum of compound 9b
3.03.54.04.55.05.56.0 ppm
3.54
33.
556
3.57
73.
589
3.77
33.
799
3.80
83.
834
4.01
34.
040
4.10
04.
148
4.18
44.
230
4.63
74.
656
4.67
54.
692
4.72
64.
790
4.85
54.
858
5.90
55.
920
1.10
2
1.02
0
1.00
9
2.13
8
1.26
9
1.00
0
PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 300.1299715 MHzSI 32768F2 − Processing parameters
SFO1 300.1319508 MHzPL1 0.00 dBP1 11.00 usecNUC1 1H======== CHANNEL f1 ========
TD0 1D1 1.79999995 secTE 293.7 KDE 158.64 usecDW 111.200 usecRG 1024AQ 7.2876530 secFIDRES 0.068610 HzSWH 4496.403 HzDS 2NS 17SOLVENT CDCl3TD 65536PULPROG zg30PROBHD 5 mm BBO BB−1HINSTRUM DRX300Time 17.59Date_ 20091109F2 − Acquisition Parameters
PROCNO 1EXPNO 1NAME JTY−1−V−1PCurrent Data Parameters
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Figure S5. 1H NMR spectrum of compound 1a
PC 1.20GB 0LB 1.00 HzSSB 0WDW EMSF 75.4675366 MHzSI 32768F2 − Processing parameters
SFO2 300.1312005 MHzPL13 19.00 dBPL12 19.00 dBPL2 0.00 dBPCPD2 85.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========
SFO1 75.4783145 MHzPL1 0.00 dBP1 8.95 usecNUC1 13C======== CHANNEL f1 ========
TD0 1DELTA 0.20000002 secd11 0.03000000 secD1 0.30000001 secTE 293.0 KDE 6.00 usecDW 22.000 usecRG 1625.5AQ 1.4418420 secFIDRES 0.346791 HzSWH 22727.273 HzDS 8NS 23769SOLVENT CDCl3TD 65536PULPROG zgpg30PROBHD 5 mm BBO BB−1HINSTRUM DRX300Time 9.16Date_ 20091111F2 − Acquisition Parameters
PROCNO 2EXPNO 1NAME JTY−1−V−1P−03cCurrent Data Parameters
405060708090100110120130140150160170180190 ppm
64.0
2472
.165
72.2
3073
.334
73.3
8874
.841
75.8
25
124.
586
124.
619
144.
693
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Figure S6. 13C NMR spectrum of compound 1a
−30−25−20−15−10−555 50 45 40 35 30 25 20 15 10 5 0 ppm
0.10
PC 1.40GB 0LB 3.00 HzSSB 0WDW EMSF 121.4948510 MHzSI 32768F2 − Processing parameters
SFO2 300.1312005 MHzPL13 17.55 dBPL12 17.55 dBPL2 −3.00 dBPCPD2 80.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========
SFO1 121.4960659 MHzPL1 −3.00 dBP1 7.65 usecNUC1 31P======== CHANNEL f1 ========
TD0 1DELTA 1.89999998 secd11 0.03000000 secD1 2.00000000 secTE 298.2 KDE 6.00 usecDW 43.200 usecRG 20642.5AQ 0.7078388 secFIDRES 0.706425 HzSWH 11574.074 HzDS 2NS 109SOLVENT D2OTD 16384PULPROG zgpg30PROBHD 5 mm QNP 1H/1INSTRUM DPX300Time 13.45Date_ 20070510F2 − Acquisition Parameters
PROCNO 1EXPNO 1NAME JTY−1−40−03Current Data Parameters
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Figure S7. 31P NMR spectrum of compound 1a
3.03.54.04.55.05.56.0 ppm
3.55
03.
560
3.56
63.
576
3.58
53.
590
3.62
5
4.05
84.
105
4.16
74.
187
4.20
9
4.58
74.
614
4.72
24.
790
5.70
1
2.01
1
1.02
7
2.03
5
1.00
2
1.00
0
PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 300.1299716 MHzSI 32768F2 − Processing parameters
SFO1 300.1319508 MHzPL1 0.00 dBP1 11.00 usecNUC1 1H======== CHANNEL f1 ========
TD0 1D1 1.60000002 secTE 294.1 KDE 158.64 usecDW 111.200 usecRG 1824.6AQ 7.2876530 secFIDRES 0.068610 HzSWH 4496.403 HzDS 2NS 21SOLVENT D2OTD 65536PULPROG zg30PROBHD 5 mm BBO BB−1HINSTRUM DRX300Time 16.26Date_ 20080131F2 − Acquisition Parameters
PROCNO 2EXPNO 1NAME JTY−1−98−16Current Data Parameters# Supplementary Material (ESI) for Organic & Biomolecular Chemistry
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Figure S8. 1H NMR spectrum of compound 1b
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
0.00
5
63.8
00
74.4
0277
.111
77.1
6478
.058
78.2
5278
.317
126.
557
126.
605
141.
521
PC 1.20GB 0LB 1.00 HzSSB 0WDW EMSF 75.4675345 MHzSI 32768F2 − Processing parameters
SFO2 300.1312005 MHzPL13 19.00 dBPL12 19.00 dBPL2 0.00 dBPCPD2 85.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========
SFO1 75.4783145 MHzPL1 0.00 dBP1 8.95 usecNUC1 13C======== CHANNEL f1 ========
TD0 1DELTA 0.20000002 secd11 0.03000000 secD1 0.30000001 secTE 292.8 KDE 6.00 usecDW 22.000 usecRG 4096AQ 1.4418420 secFIDRES 0.346791 HzSWH 22727.273 HzDS 8NS 33744SOLVENT CDCl3TD 65536PULPROG zgpg30PROBHD 5 mm BBO BB−1HINSTRUM DRX300Time 7.55Date_ 20080202F2 − Acquisition Parameters
PROCNO 2EXPNO 1NAME JTY−1−98−16cCurrent Data Parameters
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Figure S9. 13C NMR spectrum of compound 1b
−30−25−20−15−10−555 50 45 40 35 30 25 20 15 10 5 0 ppm
2.85
4
PC 1.40GB 0LB 3.00 HzSSB 0WDW EMSF 121.4947551 MHzSI 32768F2 − Processing parameters
SFO2 300.1312005 MHzPL13 17.55 dBPL12 17.55 dBPL2 −3.00 dBPCPD2 80.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========
SFO1 121.4960659 MHzPL1 −3.00 dBP1 7.65 usecNUC1 31P======== CHANNEL f1 ========
TD0 1DELTA 1.89999998 secd11 0.03000000 secD1 2.00000000 secTE 298.2 KDE 6.00 usecDW 43.200 usecRG 20642.5AQ 0.7078388 secFIDRES 0.706425 HzSWH 11574.074 HzDS 2NS 128SOLVENT D2OTD 16384PULPROG zgpg30PROBHD 5 mm QNP 1H/1INSTRUM DPX300Time 15.16Date_ 20080131F2 − Acquisition Parameters
PROCNO 1EXPNO 1NAME JTY−1−98−1Current Data Parameters
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Figure S10. 31P NMR spectrum of compound 1b
2.02.53.03.54.04.55.05.56.06.57.07.5 ppm
1.54
21.
712
1.73
21.
753
2.57
72.
588
3.57
73.
591
3.60
83.
621
4.05
34.
076
4.08
44.
107
4.11
34.
133
4.14
84.
171
4.28
34.
296
4.31
04.
324
4.64
74.
672
4.68
54.
711
4.73
64.
773
4.77
84.
816
4.85
34.
905
4.94
25.
840
5.84
55.
856
5.86
1
7.26
17.
291
7.31
47.
322
7.33
47.
344
7.35
0
0.93
8
0.98
3
1.05
2
3.09
71.
058
1.03
6
2.02
41.
095
2.02
71.
034
1.00
0
15.4
07
PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 300.1300060 MHzSI 32768F2 − Processing parameters
SFO1 300.1319508 MHzPL1 0.00 dBP1 11.00 usecNUC1 1H======== CHANNEL f1 ========
TD0 1D1 1.60000002 secTE 298.0 KDE 158.64 usecDW 111.200 usecRG 2580.3AQ 7.2876530 secFIDRES 0.068610 HzSWH 4496.403 HzDS 2NS 17SOLVENT CDCl3TD 65536PULPROG zg30PROBHD 5 mm BBO BB−1HINSTRUM DRX300Time 17.00Date_ 20080219F2 − Acquisition Parameters
PROCNO 1EXPNO 1NAME JTY−1−109−06Current Data Parameters
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Figure S11. 1H NMR spectrum of compound 10a
190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
63.9
6365
.185
73.0
8074
.147
74.8
8976
.740
77.1
6477
.367
77.5
8779
.056
79.2
7979
.327
124.
129
127.
895
128.
072
128.
132
128.
155
128.
615
128.
700
128.
715
138.
059
138.
293
138.
632
141.
711
PC 1.40GB 0LB 1.00 HzSSB 0WDW EMSF 75.4677363 MHzSI 32768F2 − Processing parameters
SFO2 300.1312005 MHzPL13 19.00 dBPL12 19.00 dBPL2 0.00 dBPCPD2 85.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========
SFO1 75.4783145 MHzPL1 0.00 dBP1 8.95 usecNUC1 13C======== CHANNEL f1 ========
TD0 1DELTA 0.20000002 secd11 0.03000000 secD1 0.30000001 secTE 298.0 KDE 6.00 usecDW 22.000 usecRG 10321.3AQ 1.4418420 secFIDRES 0.346791 HzSWH 22727.273 HzDS 8NS 28490SOLVENT CDCl3TD 65536PULPROG zgpg30PROBHD 5 mm BBO BB−1HINSTRUM DRX300Time 22.00Date_ 20080219F2 − Acquisition Parameters
PROCNO 1EXPNO 1NAME JTY−1−109−06cCurrent Data Parameters
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Figure S12. 13C NMR spectrum of compound 10a
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm
−0.
008
0.00
30.
014
1.58
41.
878
1.89
81.
919
2.13
22.
146
3.52
63.
551
3.56
03.
584
3.83
43.
858
3.86
73.
891
4.07
44.
093
4.32
84.
348
4.69
74.
716
4.73
44.
754
4.80
94.
846
4.86
04.
897
4.96
24.
999
5.66
9
7.26
07.
272
7.28
17.
299
7.30
67.
322
7.33
17.
347
7.35
87.
362
0.99
5
1.00
3
1.04
4
1.03
0
2.04
3
2.12
5
2.05
42.
103
2.06
5
1.00
0
15.4
58
PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 300.1300062 MHzSI 32768F2 − Processing parameters
SFO1 300.1319508 MHzPL1 0.00 dBP1 11.00 usecNUC1 1H======== CHANNEL f1 ========
TD0 1D1 1.60000002 secTE 300.0 KDE 158.64 usecDW 111.200 usecRG 1149.4AQ 7.2876530 secFIDRES 0.068610 HzSWH 4496.403 HzDS 2NS 14SOLVENT CDCl3TD 65536PULPROG zg30PROBHD 5 mm MultinuclINSTRUM DRX300Time 10.06Date_ 20070718F2 − Acquisition Parameters
PROCNO 1EXPNO 2NAME JTY−1−59−05Current Data Parameters
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Figure S13. 1H NMR spectrum of compound 10b
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
0.14
3
63.9
9771
.543
74.8
3475
.213
75.3
1276
.740
77.1
6477
.367
77.5
8780
.488
83.8
4084
.186
126.
738
127.
974
128.
037
128.
129
128.
157
128.
190
128.
266
128.
673
128.
753
128.
828
138.
094
138.
285
138.
338
138.
525
PC 1.40GB 0LB 1.00 HzSSB 0WDW EMSF 75.4677384 MHzSI 32768F2 − Processing parameters
SFO2 300.1312005 MHzPL13 19.00 dBPL12 19.00 dBPL2 0.00 dBPCPD2 85.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========
SFO1 75.4783145 MHzPL1 0.00 dBP1 8.95 usecNUC1 13C======== CHANNEL f1 ========
TD0 1DELTA 0.20000002 secd11 0.03000000 secD1 0.30000001 secTE 295.0 KDE 6.00 usecDW 22.000 usecRG 10321.3AQ 1.4418420 secFIDRES 0.346791 HzSWH 22727.273 HzDS 8NS 4000SOLVENT CDCl3TD 65536PULPROG zgpg30PROBHD 5 mm MultinuclINSTRUM DRX300Time 12.12Date_ 20070718F2 − Acquisition Parameters
PROCNO 1EXPNO 2NAME JTY−1−59−05cCurrent Data Parameters
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Figure S14. 13C NMR spectrum of compound 10b
2.02.53.03.54.04.55.05.56.06.57.07.5 ppm
1.63
5
3.47
93.
507
3.51
24.
021
4.04
94.
415
4.43
64.
532
4.57
24.
608
4.62
24.
659
4.75
24.
788
4.80
14.
840
4.89
24.
929
4.94
94.
954
4.96
54.
973
4.98
14.
989
4.99
25.
002
5.00
85.
029
5.04
15.
045
5.07
05.
078
5.08
85.
096
5.73
05.
748
7.19
97.
211
7.21
87.
232
7.24
47.
253
7.26
07.
271
7.28
87.
295
7.32
5
1.00
0
2.03
6
1.14
20.
933
2.28
71.
172
1.11
91.
037
1.23
78.
351
1.06
8
1.00
5
6.83
128
.623
PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 300.1300061 MHzSI 32768F2 − Processing parameters
SFO1 300.1319508 MHzPL1 0.00 dBP1 11.00 usecNUC1 1H======== CHANNEL f1 ========
TD0 1D1 1.79999995 secTE 298.0 KDE 158.64 usecDW 111.200 usecRG 574.7AQ 7.2876530 secFIDRES 0.068610 HzSWH 4496.403 HzDS 2NS 9SOLVENT D2OTD 65536PULPROG zg30PROBHD 5 mm BBO BB−1HINSTRUM DRX300Time 14.38Date_ 20080306F2 − Acquisition Parameters
PROCNO 1EXPNO 2NAME JTY−1−112−10Current Data Parameters
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Figure S15. 1H NMR spectrum of compound 11a
190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm66
.951
67.0
2169
.297
69.3
6269
.475
69.5
1969
.549
69.5
9270
.743
70.8
2572
.500
74.6
6175
.076
76.7
4377
.166
77.3
6777
.589
78.0
5578
.119
78.8
2480
.036
122.
484
127.
754
127.
785
127.
886
128.
006
128.
062
128.
110
128.
311
128.
393
128.
493
128.
528
128.
583
128.
678
128.
752
135.
792
135.
880
135.
942
136.
095
136.
197
137.
971
138.
218
138.
585
140.
533
140.
628
PC 1.20GB 0LB 1.00 HzSSB 0WDW EMSF 75.4677385 MHzSI 32768F2 − Processing parameters
SFO2 300.1312005 MHzPL13 19.00 dBPL12 19.00 dBPL2 0.00 dBPCPD2 85.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========
SFO1 75.4783145 MHzPL1 0.00 dBP1 8.95 usecNUC1 13C======== CHANNEL f1 ========
TD0 1DELTA 0.20000002 secd11 0.03000000 secD1 0.30000001 secTE 298.0 KDE 6.00 usecDW 22.000 usecRG 10321.3AQ 1.4418420 secFIDRES 0.346791 HzSWH 22727.273 HzDS 8NS 2840SOLVENT CDCl3TD 65536PULPROG zgpg30PROBHD 5 mm BBO BB−1HINSTRUM DRX300Time 17.25Date_ 20080306F2 − Acquisition Parameters
PROCNO 1EXPNO 2NAME JTY−1−112−10cCurrent Data Parameters
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Figure S16. 13C NMR spectrum of compound 11a
2.02.53.03.54.04.55.05.56.06.57.07.5 ppm
1.56
1
3.64
73.
671
3.68
13.
705
3.71
73.
741
3.75
13.
776
4.19
84.
222
4.38
04.
400
4.42
34.
442
4.55
14.
579
4.59
34.
606
4.62
14.
642
4.71
74.
754
4.75
84.
771
4.79
54.
809
4.81
44.
852
4.90
44.
944
4.96
64.
972
4.98
74.
992
4.99
85.
014
5.03
15.
771
7.19
17.
204
7.20
87.
215
7.22
37.
233
7.23
87.
246
7.25
47.
260
7.26
8
2.04
4
1.00
5
1.05
80.
875
1.19
42.
249
1.90
91.
806
8.15
1
1.00
0
12.1
4722
.658
PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 300.1300066 MHzSI 32768F2 − Processing parameters
SFO1 300.1319508 MHzPL1 0.00 dBP1 11.00 usecNUC1 1H======== CHANNEL f1 ========
TD0 1D1 1.79999995 secTE 298.0 KDE 158.64 usecDW 111.200 usecRG 1824.6AQ 7.2876530 secFIDRES 0.068610 HzSWH 4496.403 HzDS 2NS 23SOLVENT CDCl3TD 65536PULPROG zg30PROBHD 5 mm BBO BB−1HINSTRUM DRX300Time 16.21Date_ 20080317F2 − Acquisition Parameters
PROCNO 1EXPNO 1NAME JTY−1−111−04Current Data Parameters
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Figure S17. 1H NMR spectrum of compound 11b
190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm66
.780
66.8
4869
.448
69.4
9469
.527
69.5
6669
.606
75.1
4675
.460
75.6
0376
.747
77.1
7077
.373
77.5
9478
.325
78.4
0978
.823
82.3
6982
.454
83.8
8212
5.28
012
7.73
212
7.86
112
7.93
912
7.96
512
8.04
612
8.06
312
8.10
812
8.44
912
8.56
312
8.58
812
8.67
112
8.71
912
8.76
913
5.81
013
5.90
313
6.39
113
6.49
613
8.07
513
8.26
813
8.31
1
PC 1.40GB 0LB 1.00 HzSSB 0WDW EMSF 75.4677378 MHzSI 32768F2 − Processing parameters
SFO2 300.1312005 MHzPL13 19.00 dBPL12 19.00 dBPL2 0.00 dBPCPD2 85.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========
SFO1 75.4783145 MHzPL1 0.00 dBP1 8.95 usecNUC1 13C======== CHANNEL f1 ========
TD0 1DELTA 0.20000002 secd11 0.03000000 secD1 0.30000001 secTE 297.8 KDE 6.00 usecDW 22.000 usecRG 5792.6AQ 1.4418420 secFIDRES 0.346791 HzSWH 22727.273 HzDS 8NS 8734SOLVENT CDCl3TD 65536PULPROG zgpg30PROBHD 5 mm BBO BB−1HINSTRUM DRX300Time 21.39Date_ 20070821F2 − Acquisition Parameters
PROCNO 1EXPNO 1NAME JTY−1−66−04cCurrent Data Parameters
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Figure S18. 13C NMR spectrum of compound 11b
3.54.04.55.05.56.0 ppm3.
743
3.77
93.
897
3.92
23.
957
4.20
34.
229
4.45
24.
477
4.49
84.
520
4.59
44.
623
4.66
74.
785
4.79
3
6.11
56.
129
1.04
1
1.09
0
1.00
0
1.12
2
1.12
5
0.94
8
PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 300.1299723 MHzSI 32768F2 − Processing parameters
SFO1 300.1319508 MHzPL1 0.00 dBP1 11.00 usecNUC1 1H======== CHANNEL f1 ========
TD0 1D1 1.79999995 secTE 298.5 KDE 158.64 usecDW 111.200 usecRG 1149.4AQ 7.2876530 secFIDRES 0.068610 HzSWH 4496.403 HzDS 2NS 32SOLVENT D2OTD 65536PULPROG zg30PROBHD 5 mm BBO BB−1HINSTRUM DRX300Time 11.21Date_ 20090224F2 − Acquisition Parameters
PROCNO 1EXPNO 1NAME JTY−2−39−19Current Data Parameters
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Figure S19. 1H NMR spectrum of compound 2a
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
67.4
8267
.545
72.7
6072
.842
73.1
3173
.204
74.3
5375
.274
125.
286
142.
967
143.
052
PC 1.40GB 0LB 1.00 HzSSB 0WDW EMSF 75.4675478 MHzSI 32768F2 − Processing parameters
SFO2 300.1312005 MHzPL13 19.00 dBPL12 19.00 dBPL2 0.00 dBPCPD2 85.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========
SFO1 75.4783145 MHzPL1 0.00 dBP1 8.95 usecNUC1 13C======== CHANNEL f1 ========
TD0 1DELTA 0.20000002 secd11 0.03000000 secD1 0.30000001 secTE 298.5 KDE 6.00 usecDW 22.000 usecRG 2298.8AQ 1.4418420 secFIDRES 0.346791 HzSWH 22727.273 HzDS 8NS 31808SOLVENT CDCl3TD 65536PULPROG zgpg30PROBHD 5 mm BBO BB−1HINSTRUM DRX300Time 9.24Date_ 20090225F2 − Acquisition Parameters
PROCNO 1EXPNO 1NAME JTY−2−39−19cCurrent Data Parameters
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Figure S20. 13C NMR spectrum of compound 2a
−30−25−20−15−10−555 50 45 40 35 30 25 20 15 10 5 0 ppm
1.22
01.
228
PC 1.40GB 0LB 3.00 HzSSB 0WDW EMSF 121.4947576 MHzSI 32768F2 − Processing parameters
SFO2 300.1312005 MHzPL13 17.55 dBPL12 17.55 dBPL2 −3.00 dBPCPD2 80.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========
SFO1 121.4960659 MHzPL1 −3.00 dBP1 7.65 usecNUC1 31P======== CHANNEL f1 ========
TD0 1DELTA 1.89999998 secd11 0.03000000 secD1 2.00000000 secTE 298.2 KDE 6.00 usecDW 43.200 usecRG 16384AQ 0.7078388 secFIDRES 0.706425 HzSWH 11574.074 HzDS 2NS 128SOLVENT D2OTD 16384PULPROG zgpg30PROBHD 5 mm QNP 1H/1INSTRUM DPX300Time 12.01Date_ 20090226F2 − Acquisition Parameters
PROCNO 1EXPNO 3NAME JTY−2−39−1Current Data Parameters
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Figure S21. 31P NMR spectrum of compound 2a
3.54.04.55.05.56.06.5 ppm3.
597
3.63
23.
658
3.68
23.
717
4.28
64.
307
4.35
34.
373
4.39
54.
419
4.50
54.
533
4.57
44.
678
4.72
54.
792
4.85
84.
925
5.86
4
PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 300.1299718 MHzSI 32768F2 − Processing parameters
SFO1 300.1319508 MHzPL1 0.00 dBP1 11.00 usecNUC1 1H======== CHANNEL f1 ========
TD0 1D1 1.79999995 secTE 297.9 KDE 158.64 usecDW 111.200 usecRG 2580.3AQ 7.2876530 secFIDRES 0.068610 HzSWH 4496.403 HzDS 2NS 32SOLVENT D2OTD 65536PULPROG zg30PROBHD 5 mm BBO BB−1HINSTRUM DRX300Time 23.46Date_ 20080701F2 − Acquisition Parameters
PROCNO 1EXPNO 1NAME JTY−1−69−45Current Data Parameters# Supplementary Material (ESI) for Organic & Biomolecular Chemistry
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Figure S22. 1H NMR spectrum of compound 2b
190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm
67.2
2567
.286
74.0
0077
.283
77.3
3377
.893
78.3
0678
.372
128.
113
139.
885
139.
969
PC 1.40GB 0LB 1.00 HzSSB 0WDW EMSF 75.4675247 MHzSI 32768F2 − Processing parameters
SFO2 300.1312005 MHzPL13 19.00 dBPL12 19.00 dBPL2 0.00 dBPCPD2 85.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========
SFO1 75.4783145 MHzPL1 0.00 dBP1 8.95 usecNUC1 13C======== CHANNEL f1 ========
TD0 1DELTA 0.20000002 secd11 0.03000000 secD1 0.30000001 secTE 295.6 KDE 6.00 usecDW 22.000 usecRG 2580.3AQ 1.4418420 secFIDRES 0.346791 HzSWH 22727.273 HzDS 8NS 19000SOLVENT CDCl3TD 65536PULPROG zgpg30PROBHD 5 mm BBO BB−1HINSTRUM DRX300Time 9.12Date_ 20070914F2 − Acquisition Parameters
PROCNO 1EXPNO 1NAME JTY−1−69−31cCurrent Data Parameters
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Figure S23. 13C NMR spectrum of compound 2b
−30−25−20−15−10−555 50 45 40 35 30 25 20 15 10 5 0 ppm
0.96
31.
376
PC 1.60GB 0LB 3.00 HzSSB 0WDW EMSF 121.4947599 MHzSI 32768F2 − Processing parameters
SFO2 300.1312005 MHzPL13 17.55 dBPL12 17.55 dBPL2 −3.00 dBPCPD2 80.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========
SFO1 121.4960659 MHzPL1 −3.00 dBP1 7.65 usecNUC1 31P======== CHANNEL f1 ========
TD0 1DELTA 1.89999998 secd11 0.03000000 secD1 2.00000000 secTE 298.2 KDE 6.00 usecDW 43.200 usecRG 26008AQ 0.7078388 secFIDRES 0.706425 HzSWH 11574.074 HzDS 2NS 128SOLVENT D2OTD 16384PULPROG zgpg30PROBHD 5 mm QNP 1H/1INSTRUM DPX300Time 15.45Date_ 20080702F2 − Acquisition Parameters
PROCNO 1EXPNO 1NAME JTY−1−69−01Current Data Parameters
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Figure S24. 31P NMR spectrum of compound 2b
3
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# Supplementary Material (ESI) for Organic & Biomolecular Chemistry# This journal is (c) The Royal Society of Chemistry 2010
