Supplementary Information
Characterization of 10-hydroxygeraniol Dehydrogenase from Catharanthus roseus
Reveals Cascaded Enzymatic Activity in Iridoid Biosynthesis
Ramakrishnan Krithika, Prabhakar Lal Srivastava, Bajaj Rani, Swati P. Kolet, Manojkumar
Chopade, Mantri Soniya, and Hirekodathakallu V. Thulasiram*
1
Supplemental Tables
Table S1: Transcripts from Transcriptome Analysis of Vinca
Transcript ID
Length of ORF
Vinca_1853
2151
Vinca_1617
1425
BLAST Results
MEP Pathway Transcripts
Catharanthus roseus mRNA for 1deoxyxylulose 5-phosphate synthase
Catharanthus roseus 1-deoxy-D-xylulose-5phosphate reductoisomerase (dxr) mRNA,
% Similarity
99%
99%
complete cds
Catharanthus roseus 4-diphosphocytidylVinca_9009
936
methylerythritol 2-phosphate synthase
99%
mRNA, complete cds
Catharanthus roseus 4-diphosphocytidyl-2Vinca_2073
1227
C-methyl-D-erythritol kinase (CMK)
99%
mRNA, complete cds
Catharanthus roseus 2C-methyl-DVinca_2177
711
erythritol 2,4-cyclodiphosphate synthase
98%
(MECS) mRNA, complete cds
Vinca_481
2223
Catharanthus roseus GCPE protein mRNA,
complete cds
99%
Catharanthus roseus 1-hydroxy-2-methylVinca_465
1389
butenyl 4-diphosphate reductase mRNA,
99%
complete cds
Catharanthus roseus plastid isopentenyl
Vinca_775
939
pyrophosphate:dimethylallyl pyrophosphate
isomerase isoform 1 mRNA, complete cds;
99%
nuclear gene for plastid product
Vinca_1158
1218
Vinca_2418
1392
MVA Pathway Transcripts
Catharanthus roseus acetoacetyl-CoA
thiolase mRNA, complete cds
98%
Catharanthus roseus
hydroxymethylglutaryl-CoA synthase
2
99%
mRNA, complete cds
Vinca_1892
1614 (3’ and 5’
ends missing)
Vinca_4366
1163
Vinca_9276
1497
Camellia sinensis cultivar Longjing 43
HMG-CoA reductase (HMGR1) mRNA,
79%
complete cds
Catharanthus roseus mevalonate kinase
99%
mRNA, complete cds
Catharanthus roseus 5-phosphomevalonate
99%
kinase mRNA, complete cds
Catharanthus roseus mevalonate 5-
Vinca_8419
1265
diphosphate decarboxylase mRNA,
99%
complete cds
Catharanthus roseus plastid isopentenyl
Vinca_775
939
pyrophosphate:dimethylallyl pyrophosphate
99%
isomerase isoform 1 mRNA, complete cds;
nuclear gene for plastid product
Vinca_5406
1263
Geranyl diphosphate synthase (CrGDS)
NCBI
GenBank
Accession
Numbers
KF561462
Vinca_2171,
Vinca_3238
1770
Geraniol synthase (CrGS)
KF561459
Vinca_742
1482
Geraniol 10-hydroxylase (CrG10H)
KF561461
Vinca_536,
Vinca_18357
1083
10-hydroxygeraniol dehydrogenase
(Cr10HGO)
KF561458
Vinca_2280
1161
Iridoid synthase (CrIDS)
KF561460
Monoterpene Iridoid Pathway Transcripts
Table S2: Product ratio studies of Cr10HGO with various acyclic compounds
S. No.
1
2
3
4
5
6
Substrate
Lavandulol
Geranygeraniol
β - Citronellol
Linalool
Geranyl acetate
Linalyl acetate
3
% Product
Formed*
0
0
0
0
0
0
7
Farnesol
8
Geraniol
9
10-hydroxygeraniol
10
10-hydroxygeranial
11
10-oxogeraniol
12
10-oxogeranial
13
Nerol
14
Trans- Nerolidol
15
α - Bisabolol
16
Eugenol
17
Dihydromyrcenol
18
Menthol
19
Chrysanthemyl alcohol
20
α - Santalol
21
Geranyllinalool
*Rest of the % is unreacted substrate
8
17
75.2
59
62.3
73.5
7.5
0
0
0
0
0
0
0
0
Table S3: Strains and plasmids used in this study.
Strains / Plasmids
Characteristics
Sources
Strains
E.coli
TOP 10
Host strain for cloning
Invitrogen
BL21 (DE3)
Host strain for expression
Novagen
Rosetta2 (DE3)
Host strain for expression
Novagen
Host strain for yeast expression
Invitrogen
Zero Blunt
Kanr, cloning vector
Invitrogen
pRSETb
Ampr, expression vector
Invitrogen
pET32a
Ampr, expression vector
Novagen
S.cerevisiae
INVSc1
Plasmids
r
pET28a
Kan , expression vector
Novagen
pYES2
Yeast expression vector
Invitrogen
Table S4: Cloning strategies for Cr10HGO, CrGDS, CrGS, CrG10H and CrIDS.
Gene
Oligonucleotide Primer Sequences
Cr10HGO
Fwd Primer: 5'- ATGGCGAAATCACCGGAAGTCGAGC-3’
4
Rev Primer: 5'-TTATGCAGATTTCAGTGTGTTGGCT-3'
Fwd Primer: 5'- ATGTTGTTTTCCAGAGGATTGTATA -3’
CrGDS
Rev Primer: 5'- TCACTTTCTTCTTGTAATAACGCGT -3'
Fwd Primer: 5'- ATGGCAGCCACAATTAGTAACCTTT-3’
CrGS
Rev Primer: 5'-TTAAAAACAAGGTGTAAAAAACAAAGC -3'
Forward Primer: 5'- ATGGATTACCTTACCATAATATTAA -3’
CrG10H
Reverse Primer: 5'- TTAAAGGGTGCTTGGTACAGCACGC -3'
Fwd Primer: 5'-ATGAGTTGGTGGTGGAAGAGGTCCA-3’
CrIDS
Rev Primer: 5'-CTAAGGAATAAACCTATAATCCCTC-3'
Table S5: Bacterial Expression
Gene
Expression Vector
Host strain for
expression
Induction conditions
Cr10HGO
pRSETb
BL21 (DE3)
1 mM IPTG, 30 ᵒC for 6 hours
CrGDS
pET28a
Rosetta2 (DE3)
0.5 mM IPTG, 16 ᵒC for 18 hours
CrGS
pET32a
Rosetta2 (DE3)
1 mM IPTG, 16ᵒC for 12 hours
CrIDS
pET32a
Rosetta2 (DE3)
1 mM IPTG, 16ᵒC for 12 hours
Supplemental Figures
1 2 3 4 5
Legend
Lane 1 : DNA Ladder
Lane 2 : PCR product of Cr10HGO
from White Stem cDNA
Lane 2 : PCR product of Cr10HGO
from White Leaf cDNA
Lane 2 : PCR product of Cr10HGO
from White Root cDNA
Lane 5 : Negative Control
Figure S1: PCR amplification of 10-hydroxygeraniol dehydrogenase from cDNA
5
Figure S2: SDS-PAGE of protein purification of 10-hydroxygeraniol dehydrogenase
6
Figure S3. Total ion chromatograms (TICs) for 10-hydroxygeraniol dehydrogenase (Cr10HGO) catalyzed
reactionswith (a) 10-hydroxygeraniol substrate control, (b) 10-hydroxygeraniol and NADP+, (c) 10oxogeraniol standard, (d) 10-oxogeranial standard, (e) 10-hydroxygeranial standard, (f) 10-oxogeraniol
and NADP+, (g) 10-oxogeraniol and NADPH, (h) 10-oxogeranial and NADPH, (i) 10-hydroxygeranial
and NADP+, (j) 10-hydroxygeranial and NADPH. The peaks represent (2) 10-hydroxygeraniol, (3) 10oxogeraniol, (4) 10-hydroxygeranial, and (5) 10-oxogeranial.
7
Figure S4: TICs of reaction of Cr10HGO with Geraniol (A), Farnesol (B) and Nerol (C) showing
formation of geranial, farnesal and neral, respectively
Figure S5: Michaelis-Menten Plot for 10-hydroxygeraniol kinetics
Figure S6: Michaelis-Menten Plot for NADP+kinetics
8
Figure S7: Michaelis-Menten Plot for 10-oxogeraniolkinetics
Figure S8: Michaelis-Menten Plot for NADPH kinetics
Figure S9: Michaelis-Menten Plot for 10-hydroxygeranial kinetics
9
Figure S10: Michaelis-Menten Plot for 10-oxogeranial kinetics
Figure S11: Michaelis-Menten Plot for 10-hydroxynerol kinetics
1
2
3
4
5
Legend
Lane 1 : Negative Control
Lane 2 : PCR product of CrIDS
from White Stem cDNA
Lane 2 : PCR product of CrIDS
from White Leaf cDNA
Lane 2 : PCR product of CrIDS
from White Root cDNA
Lane 5 : DNA Ladder
Figure S12: PCR amplification of Iridoid synthase from cDNA
10
Figure S13: SDS-PAGE of protein purification of Iridoid synthase
Figure S14: TIC of assay extract of CrIDS. 0.1 mg of protein in 0.5 mL MOPS buffer (20 mM MOPS,
pH 7.0, 10% v/v Glycerol), in the presence of 200 µM NADPH at 30 °C for 30 minutes with 0.2 mM of
10-oxogeranial.
11
Figure S15: Comparison of TICs of (a) Cr10HGO+CrIDS assay, (b) Acetylated Cr10HGO+CrIDS assay
mixture, (c) Acetylated mixture of synthesized cis-trans nepetalactol and (d) Co-injection of acetylated
mixture of synthesized cis-trans nepetalactol and acetylated Cr10HGO+CrIDS assay mixture showing the
peaks, cis-trans-nepetalactol(6), acetylated (1R, 4aS, 7S, 7aR)-nepetalactol (8), acetylated (1S, 4aS, 7S,
7aR)-nepetalactol (9).
In chromatogram (b), there is a small peak, which seems to be in the same position as 9. However, mass
fragmentation analyses indicated that this is not corresponds to acetylated nepetalactol (9) as observed M+
is 152 instead of 210 ( Supplementary Fig. S16 and S17).
12
Figure S16: EI MS of: (A) nepetalactol from Cr10HGO+CrIDS assay and (B) synthesised nepetalactol (6)
Figure S17: EI MS of (A) acetylated (1R, 4aS, 7S, 7aR)-nepetalactol from acetylation of synthesized nepetalactol (8) and (B) (1R, 4aS, 7S, 7aR)nepetalactol from acetylation of Cr10HGO+CrIDS assay.
13
Figure S18: TICs of the CH2Cl2 extracts of the time course of the enzymatic reactions of
Cr10HGO and CrIDS on 10-hydroxygeraniol and NADP+ as substrates.
1
2 3 4 5
Legend
Lane 1 : DNA Ladder
Lane 2 : Negative Control
Lane 3 : PCR product of CrGDS
from White Root cDNA
Lane 4 : PCR product of CrGDS
from White Leaf cDNA
Lane 5 : PCR product of CrGDS
from White Stem cDNA
Figure S19: PCR amplification of Geranyldiphosphate synthase from cDNA
1
2
3 4 5
14
Figure S20: SDS-PAGE of protein purification of Geranyl diphosphate synthase
Geraniol
Figure S21:TIC of assay extract of CrGDS. 0.2 mg of protein in 0.5 mL of MOPSObuffer (50 mM
MOPSO, pH 7.0, 10% v/v Glycerol, 2 mM DTT, 10 mM MgCl2), incubated at 30 °C for 6 hourswith 0.2
mM each of IPP and DMAPP. Further incubation at 30 °C, overnight, with 5 U Alkaline phosphatase to
hydrolyze the GPP formed, so that it may be detected by GC / GC-MS.
15
1
2
3 4
5
Legend
Lane 1 : DNA Ladder
Lane 2 : PCR product of CrGS
from White Stem cDNA
Lane 2 : PCR product of CrGS
from White Leaf cDNA
Lane 2 : PCR product of CrGS
from White Root cDNA
Lane 5 : Negative Control
Figure S22: PCR amplificationof Geraniol synthase from cDNA
Figure S23: SDS-PAGE of protein purification of Geraniol synthase
Geraniol
Figure S24: TIC of assay extract of CrGS. 0.1 mg of protein in 0.5 mL HEPES buffer (100mM HEPESKOH, pH 7.0, 1 mM MgCl2, 100µM MnCl2, 10% v/v Glycerol) incubated at 30 °C for 1 hour with 0.2
mM of GPP.
16
1
2
3
4
5
Legend
Lane 1 : DNA Ladder
Lane 2 : Negative Control
Lane 3 : PCR product of CrG10H
from White Root cDNA
Lane 4 : PCR product of CrG10H
from White Leaf cDNA
Lane 5 : PCR product of CrG10H
from White Stem cDNA
Figure S25: PCR amplificationof Geraniol 10-hydroxylase from cDNA
1
2
3
4
5
Legend
Lane 1
: DNA Ladder
Lanes 2-5 : CrG10H Clones 1-4
Figure S26: Colony PCR amplificationof Geraniol 10-hydroxylase Clones in pYES2
10-Hydroxygeraniol
Figure S27: TIC of assay extract of CrG10H. 1 mg of microsomal fraction in 1 ml potassium
phosphatebuffer (0.1 M K2HPO4, pH 7.6, 1 mM EDTA, 1 mM DTT), in the presence of 1 mM NADPH,
2.5 µM Glucose 6-phosphate, 1U Glucose 6-phosphate dehydrogenase, 10 µM FAD, 10 µM FMN, with
the addition of 0.5 mM Geraniol was incubated at 30 °C for 1 hour.
17
Figure S28: (A) TIC of CrGDS assay in comparison with the control and standard, (B) TIC of CrGS
assay in comparison with the control and standard, (C) TIC of CrG10H assay in comparison with the
control and standard, (D) TIC of CrGDS+CrGS+CrG10H assay in comparison with the control and
standard; (1)-Geraniol, (2)- 10-hydroxygeraniol, (7)-Nerol; Substrate Control - buffer+substrate+co-factor
(wherever applicable), Enzyme Assay - buffer+substrate+co-factor (wherever applicable)+enzyme. The
unlabelled peaks were observed in controls also.
18
Figure S29: Comparison of TICs of CrGDS, CrGS, CrG10H and CrGDS+CrGS+CrG10H assays; 1Geraniol, 2- 10-hydroxygeraniol, 3-Nerol; all unlabelled peaks were observed in controls also; (1)Geraniol, (2)- 10-hydroxygeraniol, (7)-nerol.
19
Figure S30: Comparison of TICs of CrIDS, Cr10HGO+CrIDS and CrG10H+Cr10HGO+CrIDS assays
with substrate control (buffer + Geraniol) and enzyme control (buffer+CrG10H+Cr10HGO+CrIDS); (1)geraniol, (6)-(1R, 4aS, 7S, 7aR)-nepetalactol, on Astec CHIRALDEXTM B-DA column with a temperature
gradient from 60 to 140 °C at 4 °C per min, followed by a temperature gradient from 140 to 190 °C at 2.5
°C per min with a He flow rate of 1 mL/ min.
20
Figure S31: Comparison of TICs of CrIDS, Cr10HGO+CrIDS and CrG10H+Cr10HGO+CrIDS assays
with standard (1R, 4aS, 7S, 7aR)-nepetalactol on Astec CHIRALDEXTM B-DA column with a
temperature gradient from 60 to 160 °C at 1 °C per min, followed by a temperature gradient from 160 to
220 °C at 10 °C per min with a He flow rate of 1 mL/ min.
21
1 2 3 4 5 6 7
Legend
Lane 1 : RNA from White Leaves
Lane 2 : RNA from White Stem
Lane 3 : RNA from White Roots
Lane 4 : RNA from Pink Leaves
Lane 5 : RNA from Pink Stem
Lane 6 : RNA from Pink Roots
Lane 7 : DNA Ladder
Figure S32: RNA from different tissues of Catharanthus roseus
Figure S33: Graph representing the number of transcripts assigned unique KO numbers.
This study identifies the probable number of unique transcripts, eliminating
the multiple copy numbers.
22
Figure S34: Graph representing the number of ORFs obtained (after translation to amino acid sequences)
when the nucleotide sequences were submitted to Virtual Ribosome. These ORFs were used in further
annotational studies
23
Scheme S1: Synthesis of 10-hydroxygeraniol (2), 10-oxogeraniol (3), 10-hydroxygeranial (4), 10oxogeranial (5)and 10- hydroxynerol (12)
24
O
OH
a
+
O
O
O
(S)-Citronellal
13
14, 49%
b
c
d
e
O
NPhMe
15, 72%
O
OH
6, 80%
O
OAc
8, 85%
Reagents and conditions: (a) SeO2, t-BuOOH, Salicylic acid, CH2Cl2, r.t.;
(b) IBX, DMSO, r.t.; (c) NHMePh, Et2O, r.t.; (d) TsOH, THF; (e) Ac2O,
Et3N, DMAP, CH2Cl2
Scheme S2: Synthesis of (1R, 4aS, 7S, 7aR)-nepetalactol (6) and acetylated (1R, 4aS, 7S, 7aR)nepetalactol (8)
Spectral data
10-hydroxygeraniol (2):
60% yield, colourless liquid, 1H NMR (CDCl3, 400 MHz, ppm): δ 5.38-5.35 (m, 2H), 4.15 (d, J
= 6.7 Hz, 2H), 3.97 (s, 2H), 2.15-2.06 (m, 4H), 1.96 (bs, 2H), 1.66 (s, 3H), 1.64 (s, 3H).
13C
NMR (CDCl3, 100 MHz, ppm): δ 138.5, 135.0, 125.1, 123.8, 68.5, 59.0, 38.9, 25.4, 16.0, 13.6.
HRMSm/z: Calculated for C10H18O2Na- 193.1204; found 193.1197 [M+Na]+.
10-oxogeraniol (3):
72% yield, pale yellow liquid, 1H NMR (CDCl3, 400 MHz, ppm):δ 9.38 (s, 1H), 6.50-6.42 (m,
1H), 5.45-5.41 (m, 1H), 4.18 (d, J = 6.7 Hz, 2H), 2.54-2.43 (m, 2H), 2.25-2.17 (m, 2H), 1.74 (s,
3H), 1.70 (s, 3H).13C NMR (CDCl3, 100 MHz, ppm): δ 195.2, 153.7, 139.5, 137.8, 124.5, 59.2,
37.7, 27.0, 16.2, 9.2. HRMSm/z: Calculated for C10H16O2Na- 191.1048; found 191.1039
[M+Na]+.
10-hydroxygeranial (4):
25
37% yield, yellow liquid, 1H NMR (CDCl3, 400 MHz, ppm): δ 9.97 (d, 1H, J = 8.0 Hz), 5.88 (d,
IH, J = 8.0 Hz), 5.34 (m, 1H), 3.97 (s, 2H), 2.25-2.17 (m, 2H), 2.15 (d, 3H, J = l.0 Hz), 2.04-1.97
(m, 2H), 1.64 (s, 3H).
13C
NMR (CDCl3, 100 MHz, ppm): 191.5, 164.0, 135.9, 127.1, 123.3,
67.9, 40.0, 25.0, 17.4, 13.5. HRMS m/z: Calculated for C10H16O2Na-191.1048; found 191.1039
[M+Na]+.
10-oxogeranial (5):
88% yield, pale yellow liquid, 1H NMR (CDCl3, 400 MHz, ppm): δ 10.02 (d, J = 8.1 Hz 1H,),
9.39 (s, 1H), 6.45-6.28 (m, 1H), 5.93 (d, J = 6.7 Hz, 1H), 2.60-2.53 (m, 2H), 2.45-2.38 (m, 2H),
2.20 (s, 3H), 1.76 (s, 3H).
13C
NMR (CDCl3, 100 MHz, ppm): δ194.6, 190.8, 161.3, 151.4,
140.0, 127.5, 38.5, 26.2, 17.4, 9.1. HRMSm/z: Calculated for C10H14O2Na- 189.0891; found
189.0883 [M+Na]+.
(1R, 4aS, 7S, 7aR)-nepetalactol (6):
80% yield, colourless liquid, 1H NMR (CDCl3, 400 MHz, ppm): δ 6.00 (s, 1H), 4.83 (m, 1H),
3.28 (s, 1H), 2.44 (m, 1H), 1.97-1.82 (m, 3H) 1.66 (m, 1H), 1.54 (s, 3H), 1.36 (m, 1H), 1.16-1.10
(m, 1H), 1.08 (d, J = 6.8 Hz, 3H). 13C NMR (CDCl3, 100 MHz, ppm): δ 134.1, 113.7, 94.4, 50.4,
38.7, 35.8, 33.3, 30.8, 20.6, 16.3. HRMSm/z: Calculated for C10H16O2Na- 191.1048; found
191.1041 [M+Na]+. Rotation: [α] -48.85 (c 0.92, CHCl3).
10- hydroxynerol (12):
69% yield, pale yellow liquid, 1H NMR (CDCl3, 400 MHz, ppm): δ 5.46-5.39 (m, 2H), 4.08 (d, J
= 6.7 Hz, 2H), 3.98 (s, 2H), 2.14 (m, 4H), 1.96 (bs, 2H), 1.75 (s, 3H), 1.65 (s, 3H).
13C
NMR
(CDCl3, 100 MHz, ppm): δ 138.4, 135.6, 124.9, 124.8, 68.4, 58.8, 31.3, 25.2, 23.3, 13.7.
HRMSm/z: Calculated for C10H18O2Na- 193.1204; found 193.1197 [M+Na]+.
26
10
9
8
7
6
5
Chemical Shift (ppm)
4
Figure S35:1H NMR spectrum of 2 in CDCl3
27
3
2
1.66
1.64
6.13
2.15
2.12
2.06
4.00
2.13
2.07
2.04
5.38
5.37
5.35
7.26
4.15
4.11
3.97
CHLOROFORM-d
1H-3.ESP
1
0
200
180
160
140
120
100
Chemical Shift (ppm)
80
Figure S36:13C NMR spectrum of 2 in CDCl3
28
60
40
16.0
13.6
25.4
38.9
59.0
77.0
125.1
123.8
138.5
135.0
220
68.5
CHLOROFORM-d
13C-3.esp
20
0
220
200
180
160
140
120
100
Chemical Shift (ppm)
80
Figure S37: DEPT-135 NMR spectrum of 2 in CDCl3
29
60
40
16.0
13.6
25.4
38.9
59.0
68.4
125.0
123.8
DEPT-3.esp
20
0
10
9
8
7
6
5
Chemical Shift (ppm)
30
4
Figure S38:1H NMR spectrum of 3 in CDCl3
3
3.03
3.01
2.03
2.02
1.98
0.96
1.00
0.95
2.54
2.51
2.47
2.43
2.25
2.21
2.17
1.74
1.70
4.18
4.15
5.45
5.44
5.41
6.50
6.46
6.42
7.26
9.38
1H-4.esp
CHLOROFORM-d
2
1
0
200
180
160
140
120
100
Chemical Shift (ppm)
80
Figure S39:13C NMR spectrum of 3in CDCl3
31
60
40
20
9.2
16.2
27.0
37.7
77.0
124.5
139.5
137.8
153.7
195.2
220
59.2
CHLOROFORM-d
13C-4.esp
0
220
200
180
160
140
120
100
Chemical Shift (ppm)
80
Figure S40: DEPT-135 NMR spectrum of 3 in CDCl3
32
60
40
20
9.2
16.2
27.1
37.8
59.2
124.5
153.8
195.3
DEPT-4.esp
0
9
8
7
6
5
Chemical Shift (ppm)
4
Figure S41:1H NMR spectrum of 4 in CDCl3
33
2.25
2.15
2.04
1.98
1.97
1.64
2.2
2.7
1.2
1.0
3.4
3.97
5.34
1.1
2.3
5.88
5.84
7.26
1.0
9.97
9.93
9.86
9.82
10
1.1
CHLOROFORM-d
1H-6.esp
3
2
1
0
200
180
160
140
120
100
Chemical Shift (ppm)
80
Figure S42:13C NMR spectrum of 4 in CDCl3
34
60
40
17.4
13.5
25.0
40.0
77.0
127.1
123.3
135.9
164.0
191.5
220
67.9
CHLOROFORM-d
13C-6.esp
20
0
220
200
180
160
140
120
100
Chemical Shift (ppm)
80
Figure S43: DEPT-135 NMR spectrum of 4 in CDCl3
35
60
40
17.5
13.5
25.0
32.1
40.0
67.8
127.1
123.4
191.5
DEPT-6.esp
20
0
10
9
8
7
6
5
Chemical Shift (ppm)
36
4
Figure S44:1H NMR spectrum of 5 in CDCl3
3
3.18
1.85
1.90
2.95
1.19
0.84
9.39
0.91
2.60
2.57
2.53
2.45
2.41
2.38
2.20
1.76
5.93
5.88
6.45
6.41
6.28
7.26
10.02
9.98
0.99
1H-5.ESP
CHLOROFORM-d
2
1
0
200
180
160
140
120
100
Chemical Shift (ppm)
80
Figure S45:13C NMR spectrum of 5 in CDCl3
37
60
40
20
9.1
17.4
26.2
77.0
127.5
140.0
151.4
161.3
194.6
190.8
220
38.5
CHLOROFORM-d
13C-5.esp
0
220
200
180
160
140
120
100
Chemical Shift (ppm)
80
Figure S46: DEPT-135 NMR spectrum of 5in CDCl3
38
60
40
20
9.1
17.4
26.1
38.5
127.5
151.5
194.7
190.9
DEPT-5.esp
0
10
9
8
7
6
5
Chemical Shift (ppm)
39
4
Figure S47:1H NMR spectrum of 6 in CDCl3
3
2
1.2
1.2
3.0
3.4
0.9
3.2
1.0
3.28
2.45
2.43
1.99
1.97
1.91
1.87
1.82
1.67
1.65
1.54
1.16
1.10
1.08
1.06
4.83
1.0
0.9
6.00
1.1
7.26
1H-11.esp
CHLOROFORM-d
1
0
200
180
160
140
120
100
Chemical Shift (ppm)
80
Figure S48:13C NMR spectrum of6 in CDCl3
40
60
40
20.6
16.3
38.7
35.8
33.3
30.8
50.4
94.4
113.7
134.1
220
77.3
77.0
76.7
CHLOROFORM-d
13C-11.esp
20
0
220
200
180
160
140
120
100
Chemical Shift (ppm)
80
Figure S49: DEPT-135 NMR spectrum of 6 in CDCl3
41
60
40
20.6
16.3
38.8
35.8
33.3
30.8
50.4
94.4
134.1
DEPT-11.esp
20
0
10
9
8
7
6
5
Chemical Shift (ppm)
4
Figure S50:1H NMR spectrum of 12 in CDCl3
42
2.14
1.96
1.75
1.65
3
2.2
2.9
3.1
4.3
4.08
4.05
3.98
2.1
1.8
2.0
7.26
5.46
5.39
5.39
CHLOROFORM-d
1H-7.esp
2
1
0
200
180
160
140
120
100
Chemical Shift (ppm)
80
Figure S51:13C NMR spectrum of 12 in CDCl3
43
60
40
20
13.7
31.3
25.2
23.3
58.8
77.0
138.4
135.6
124.9
124.8
220
68.4
CHLOROFORM-d
13C-7.esp
0
220
200
180
160
140
120
100
Chemical Shift (ppm)
80
Figure S52: DEPT-135 NMR spectrum of 12 in CDCl3
44
60
40
20
13.7
31.2
25.2
23.2
58.8
68.4
124.8
DEPT-7.esp
0