Supporting Information for Combinatorial Generation of Chemical Diversity by Redox Enzymes in Chaetoviridin Biosynthesis Michio Sato, † Jaclyn M. Winter, ‡,§ Hiroshi Noguchi, † Yi Tang, ‡, ∥ and Kenji Watanabe *,† † Department of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan. ‡ Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, United States. ∥ Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, California 90095, United States. § Present Addresses: Department of Medicinal Chemistry, University of Utah, Utah 84112, USA. *Correspondence e-mail: [email protected]
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Supporting Information for
Combinatorial Generation of Chemical Diversity by Redox Enzymes in
Chaetoviridin Biosynthesis
Michio Sato,† Jaclyn M. Winter,‡,§ Hiroshi Noguchi,† Yi Tang,‡, ∥ and Kenji Watanabe*,†
†Department of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan. ‡Department of Chemistry and Biochemistry, University of California, Los Angeles, California
90095, United States.∥Department of Chemical and Biomolecular Engineering, University of California, Los Angeles,
California 90095, United States.
§Present Addresses: Department of Medicinal Chemistry, University of Utah, Utah 84112, USA.
1.1. Strains and general techniques for DNA manipulation.
For the construction of a disruption cassette and confirmation of the modified genotype, the
genomic DNA isolated from Chaetomium globosum, CGKW14 or their transformants1 was
analyzed by PCR. Genomic DNA from those strains was prepared using the CTAB isolation
buffer at pH 8.0 (20 g/L CTAB, 1.4 M sodium chloride and 20 mM EDTA). The gene-specific
primers are listed in Table S1. PCR was performed using KOD Plus Neo (TOYOBO Co., Ltd.).
Sequences of PCR products were confirmed through DNA sequencing (Macrogen Japan
Corporation). Escherichia coli XL1-Blue (Stratagene) was used for plasmid propagation. DNA
restriction enzymes were used as recommended by the manufacturer (Fermentas).
1.2. In silico analysis of genome sequence.
Protein sequences obtained by translating the caz genes in silico were used for BLASTP2 query
analyses performed against the NCBI GenBank database (http://www.ncbi.nlm.nih.gov) and the
Broad Institute database (http://www.broadinstitute.org).
1.3. Spectroscopic analyses.
NMR spectra were obtained with a JEOL JNM-ECA 500 MHz spectrometer (1H 500 MHz, 13C
125 MHz) and a Bruker BioSpin AVANCE 400 MHz spectrometer (1H 400 MHz, 13C 100 MHz). 1H NMR chemical shifts are reported in parts/million (ppm) using the proton resonance of
residual solvent as reference: CDCl3 7.26 and CD3OD 3.31.3 13C NMR chemical shifts are
reported relative to CDCl3 77.16 and CD3OD 49.0.3 Mass spectra were recorded with a
Thermo SCIENTIFIC ACCELA Exactive liquid chromatography mass spectrometer by using
both positive and negative ESI. LC–MS was conducted with a Thermo SCIENTIFIC ACCELA
Exactive liquid chromatography mass spectrometer by using positive electrospray ionization.
Samples were separated for analysis on an ACQUITY UPLC 1.8 µm, 2.1 x 50 mm C18 reversed-
phase column (Waters) using a linear gradient of 5–100% (v/v) MeCN in H2O supplemented
with 0.05% (v/v) formic acid at a flow rate of 0.5 mL/min. Optical rotations were measured on a
JASCO P-2200 digital polarimeter. Infrared spectra were collected by an attenuated total
2.1. Chaetoviridin biosynthetic gene cluster in C. globosum.8
Figure S9. The caz gene cluster found in the C. globosum genome. Predicted function of the translation product of each of the gene found in the caz gene cluster is given in Table S2.
Table S2. Deduced functions of cazI, L, O and P in the chaetoviridin biosynthetic gene cluster from C. globosum.
Deduced function of the ORFs was determined based on the sequence similarity/identity to
known proteins as determined by Protein BLAST (BLASTP) search2 against the NCBI non-
redundant database.
Locus ID Gene name Deduced function Species, NCBI accession number
The steady-state kinetic parameters were determined for CazL using 1 or 2 as a substrate. A
series of reactions (100 μL) containing 80 nM CazL, 0.2 mM NADPH and 1 at seven different
concentrations ranging 10–100 μM or 2 at three different concentrations ranging 50–100 μM in
100 mM sodium phosphate (pH 7.4) were carried out at 30 °C. The reactions were carried out in
triplicate. After incubating for 30 min, all of the reactions were terminated by extraction with
ethyl acetate (100 µL). Each of the extracts was dried in vacuo, and the dried residue was
dissolved in N,N-dimethylformamide (50 µL). The resulting solution was subjected to LC–MS
analysis. The obtained data were fitted to the Michaelis–Menten equation by nonlinear
regression. Km and kcat values for 1 as 73.04 ± 9.93 µM and 30.88 ± 2.30 min–1, respectively. Km
and kcat values for 2 as 68.12 ± 22.10 µM and 51.58 ± 7.80 min–1, respectively.
Figure S11. Michaelis–Menten kinetics for a pyranoquinone formation from substrates 1 (▲)
and 2 (■) catalyzed by CazL. Each data point is a mean of triplicate measurements. The standard
deviation is given in the plot as an error bar at each data point.
- S21 -
2.4. Spontaneous transformation of 8 in a neutral buffer.
Figure S12. Spontaneous transformation of 8 in a neutral buffer. 100 uL of 20 mM HEPES
buffer (pH7.4) at room temperature containing 60 µM of 8 was incubated at room temperature
for (i) 10 min and (ii) 240 min and analyzed by LC–MS following essentially the same procedure
used for characterizing in vitro assays as described earlier.
- S22 -
2.5. UV spectra and ESIHRMS data of the substrate 10 and the reaction products 11 and 13.
Figure S13. In vitro characterization of the dehydrogenase CazP from the chaetoviridin
biosynthetic pathway for the formation of an intermediate 13 and chaetoviridin U 11. The HPLC
traces of the reaction are given in Figure 3c in the main text. UV spectra of the substrate 10 (a-i)
and the reaction products 11 (b-i) and 13 (c-i). ESIHRMS data of 10 (a-ii, m/z+ 399 [M+H]+), 11
(b-ii, m/z+ 401 [M+H]+) and 13 (c-ii, m/z+ 401 [M+H]+).
- S23 -
2.6. Chemical characterization of 1.
Table S3. NMR data of compound 114 in CDCl3. The molecular formula of 1 was established by mass data [ESI-MS: m/z 275 (M-H)–; HRESIMS: m/z 275.1294 (M-H)–, calcd. for C16H19 O4
–, 275.1289, = 0.50 mmu]; []D
20: –220.0 (c 0.06, DMSO).
Position H [ppm] mult. (J in Hz) HMBC C [ppm]
1 9.82 1H s 1,2 192.72 113.03 161.54 111.25 164.56 6.15 1H s 3,4,9 110.87 137.28 4.05 2H s 5,6,10 43.69 197.410 6.2 1H d (16.0) 10,13 127.311 6.94 1H dd (16.4, 8.4) 10,13,16 155.612 2.28 1H m 38.713 1.45 2H m 13,15,16 28.914 0.9 3H t (7.2) 13,14 11.84-Me 2.04 3H s 2,3,4 7.112-Me 1.09 3H d (7.2) 12,13,14 19.03-OH 12.7 1H s 2,35-OH 6.5 1H br1H and 13C NMR spectra were recorded at 400 MHz and 100 MHz, respectively.
- S24 -
Fig. S14. 1H NMR spectrum of 1 in CDCl3 (400 MHz).
H [ppm]
Fig. S15. 13C NMR spectrum of 1 in CDCl3 (100 MHz).
C [ppm]
HO
OH O
O12
3
6 7
4
912 14
HO
OH O
O12
3
6 7
4
912 14
- S25 -
Fig. S16. 1H-1H COSY spectrum of 1 in CDCl3 (400 MHz). COSY, correlated spectroscopy.
Fig. S17. HMBC spectrum of 1 in CDCl3 (400 MHz). HMQC, heteronuclear multiple quantum coherence.
- S26 -
2.7. Chemical characterization of 2.
Table S4. NMR data of compound 2 in CDCl3. The molecular formula of 2 was established by mass data [ESI-MS: m/z 309 (M–H)–; HRESIMS: m/z 309.0901(M–H)–, calcd. for C16H18 ClO4
-, 309.0899, = 0.18 mmu]; []D
20: –18.7 (c 0.01, DMSO).
Position H [ppm] mult. (J in Hz) HMBC C [ppm]
1 9.86 1H s 3 194.82 113.83 162.74 113.55 156.26 112.77 1348 4.33 2H s 6,7,9 39.99 193.210 6.20 1H d.(16.0) 9 127.411 6.94 1H dd.(16.4, 8.4) 9 155.112 2.28 1H m 38.613 1.45 2H m 11 28.914 0.9 3H t.(7.2) 12,13 11.84-Me 2.17 3H s 3,4,5 8.212-Me 1.09 3H d.(7.2) 12,13,14 193-OH 12.6 1H s1H and 13C NMR spectra were recorded at 400 MHz and 100 MHz, respectively.
- S27 -
Fig. S18. 1H NMR spectrum of 2 in CDCl3 (400 MHz).
H [ppm]
Fig. S19. 13C NMR spectrum of 2 in CDCl3 (100 MHz).
C [ppm]
HOCl
OH O
O12
3
6 7
4
912 14
HOCl
OH O
O12
3
6 7
4
912 14
- S28 -
Fig. S20. 1H-1H COSY spectrum of 2 in CDCl3 (400 MHz).
Fig. S21. HMBC spectrum of 2 in CDCl3 (400 MHz).
- S29 -
2.8. Chemical characterization of 4.
Table S5. NMR data of compound 411 in CDCl3. The molecular formula of 4 was established by mass data [ESI-MS: m/z 433 (M+H)+; HRESIMS: m/z 433.1412 (M+H)+, calcd. for C23H26 ClO6
+, 433.1414, = 0.21 mmu]; []25
D: +64.6 (c 1.00, CHCl3).
Position H [ppm] mult. (J in Hz)11 H [ppm] mult. (J in Hz) C [ppm]11 C [ppm]1 8.80 s 8.70 s 151.5 151.53 157.1 157.24 6.56 s 6.50 s 105.3 105.44a 139.7 140.05 108.9 108.76 183.4 183.57 87.5 87.58 162.6 162.38a 110.4 110.49 6.10 d (15.7) 6.05 d (15.7) 119.7 119.810 6.62 dd (15.7, 8.3) 6.57 dd (15.7, 7.8) 148.0 148.1
- S30 -
11 2.30 m 2.24 m 38.9 38.912 1.45 m 1.40 dq (6.9, 7.4) 30.1 29.113 0.92 t (7.4) 0.86 t (7.4) 11.6 11.77-CH3 1.70 s 1.62 s 26.2 26.311-CH3 1.10 d (6.6) 1.04 d (6.9) 19.2 19.31' 167.9 167.92' 125.1 125.33' 201.1 201.34' 3.64 m 3.57 m 51.0 51.05' 3.86 m 3.78 m 70.8 70.86' 1.17 d (6.6) 1.09 d (6.9) 13.4 13.44'-CH3 1.17 d (6.6) 1.09 d (6.4) 21.4 21.35'-OH 2.28 brs 2.40 brs
1H and 13C NMR spectra were recorded at 400 MHz and 100 MHz, respectively.
- S31 -
Fig. S22. 1H NMR spectrum of 4 in CDCl3 (400 MHz).
H [ppm]
Fig. S23. 13C NMR spectrum of 4 in CDCl3 (100 MHz).
C [ppm]
O
O
1
4a 43
56
78
9
10 1213
8a
11
Cl
O
OOHO
1' 2'3'
4' 5'
6'
O
O
1
4a 43
56
78
9
10 1213
8a
11
Cl
O
OOHO
1' 2'3'
4' 5'
6'
- S32 -
2.9. Chemical characterization of 5.
Table S6. NMR data of compound 5 in CDCl3. The molecular formula of 3 was established by mass data [ESI-MS: m/z 275 (M+H)+; HRESIMS: m/z 275.1278 (M+H)+, calcd. for C16H19O4
+, 275.1278, = 0.0 mmu]; []D
20: –272.0 (c 0.08, CH3OH).
Position H [ppm] mult. (J in Hz) HMBC C [ppm]1 7.90 1H s 3, 4a, 8, 8a 152.63 156.24 6.13 1H s 3, 4a, 5, 8a 108.64a 144.35 5.58 1H 4, 7, 8a 106.06 195.87 83.68 196.38a 115.79 5.96 1H d (15.5) 3, 4, 11 119.710 6.48 1H dd (15.5, 8.0) 3 146.511 2.25 1H m 9, 10, 11-CH3, 12 39.012 1.43 1H dq (7.5, 6.9) 10, 11, 11-CH3, 13 29.313 0.90 1H t (7.5) 11, 12 11.87-CH3 1.55 3H s 6, 7, 8 28.811-CH3 1.08 3H d (6.9) 10, 11, 12 19.51H and 13C NMR spectra were recorded at 500 MHz and 125 MHz, respectively.
O
O
OHO
selected HMBC
DQF-COSY
- S33 -
Fig. S24. 1H NMR spectrum of 5 in CDCl3 (500 MHz).
H [ppm]
Fig. S25. 13C NMR spectrum of 5 in CDCl3 (125 MHz).
C [ppm]
O
O
HOO
1
4a 43
56
78
9
10 1213
8a
11
O
O
HOO
1
4a 43
56
78
9
10 1213
8a
11
- S34 -
Fig. S26. 1H-1H COSY spectrum of 5 in CDCl3 (500 MHz).
Fig. S27. HMQC spectrum of 5 in CDCl3 (500 MHz). HMQC, heteronuclear multiple quantum coherence.
X : parts per Million : 1H8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0
Y :
parts
per
Mill
ion
: 1H
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0
abundance0 100.0
abun
danc
e0
100.
0
X : parts per Million : 1H8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0
Y :
parts
per
Mill
ion
150.
014
0.0
130.
012
0.0
110.
010
0.0
90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0
(Thousands)0 1.0 2.0 3.0
abun
danc
e0
100.
0
- S35 -
Fig. S28. HMBC spectrum of 5 in CDCl3 (500 MHz).
X : parts per Million : 1H8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0
Y :
parts
per
Mill
ion
: 13C
210.
0200.
0190
.018
0.01
70.0
160.
0150
.014
0.01
30.0
120.
0110
.010
0.0
90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0
(Thousands)0 1.0
abun
danc
e0
20.0
40.0
- S36 -
2.10. Chemical characterization of 6.
Table S7. NMR data of compound 611 in CDCl3. The molecular formula of 6 was established by mass data [ESI-MS: m/z 435 (M+H)+; HRESIMS: m/z 435.1570 (M+H)+, calcd. for C23H28ClO6
+, 435.1569, = 0.1 mmu]; []25
D: –123.62 (c 0.70, CHCl3).
Position H [ppm] mult. (J in Hz)11 H [ppm] mult. (J in Hz) C [ppm]11 C [ppm]1 7.30 s 7.27 s 145.7 145.73 157.8 157.84 6.55 s 6.55 s 105.0 105.14a 140.6 140.55 110.1 110.26 189.3 189.47 83.9 84.28 3.01 d (10.1) 3.00 d (10.0) 50.5 50.78a 114.4 114.49 6.06 d (15.7) 6.06 d (15.6) 120.2 120.310 6.53 dd (15.7, 8.0) 6.52 dd (15.8, 8.3) 146.9 147.011 2.26 m 2.26 m 38.9 39.112 1.43 m 1.43 m 29.2 29.313 0.91 t (7.4) 0.91 t (7.7) 11.7 11.97-CH3 1.40 s 1.40s 23.4 23.411-CH3 1.08 d (6.6) 1.08 d (6.8) 19.4 19.61' 170.8 170.72' 3.07 d (10.1) 3.07 d (10.0) 58.3 58.43' 104.1 104.34' 1.91 m 1.89 m 45.0 45.15' 4.32 m 4.30 m 77.3 77.46' 1.41 d (5.2) 1.41d (6.3) 18.7 18.94'-CH3 1.14 d (7.2) 1.13 d (7.0) 8.8 8.93-OH 3.19 brs 3.08 brs5'-OH 1.86 brs
1H and 13C NMR spectra were recorded at 400 MHz and 100 MHz, respectively.
- S37 -
Fig. S29. 1H NMR spectrum of 6 in CDCl3 (400 MHz).
H [ppm]
Fig. S30. 13C NMR spectrum of 6 in CDCl3 (100 MHz).
C [ppm]
O
O
1
4a 43
56
78
9
10 1213
8a
11
Cl
2'3'
4' 5'
6'
O
OO
HO H1'
O
O
1
4a 43
56
78
9
10 1213
8a
11
Cl
2'3'
4' 5'
6'
O
OO
HO H1'
- S38 -
2.11. Chemical characterization of 7.
Table S8. NMR data of compound 74 in CD3OD. The molecular formula of 7 was established by mass data [ESI-MS: m/z 476 (M+H)+; HRESIMS: m/z 476.1835 (M+H)+, calcd. for C25H31 ClNO6
+, 476.1834, = 0.1 mmu]; []25
D: +1189.0 (c 0.036, CH3OH).
Position H (ppm) mult. (J in Hz) HMBC C (ppm)1 8.80 1H s 3, 4a, 5, 8, 8a, 1" 143.73 151.34 7.03 1H s 3, 5, 8a, 9 112.04a 148.55 113.56 182.47 89.98 168.48a 99.19 6.63 1H d (15.6) 4, 11 121.610 6.48 1H dd (15.6, 8.0) 3, 11, 11-CH3, 12 150.911 2.38 1H m 9, 10, 11-CH3, 12,
1340.6
12 1.51 2H m 10, 11, 11-CH3, 13 30.213 0.96 3H t (7.4) 11, 12 12.27-CH3 1.66 3H s 6, 7, 8 27.011-CH3 1.14 3H d (6.8) 10, 11, 12 19.81' 170.32' 126.03' 202.1
4
3.56 1H m 3', 4'-CH3, 5', 6' 52.45' 3.77 1H m 4', 6' 71.56' 1.10 3H d (6.8) 3', 4', 5' 13.54'-CH3 1.09 3H d (6.3) 21.15'-OH1" 4.29 1H td (14.8, 4.5) 1 57.9
4.19 1H td (14.6, 5.5) 1, 3, 2"2" 3.91 2H d (5.5) 61.22"-OH1H and 13C NMR spectra were recorded at 400 MHz and 100 MHz, respectively.
- S39 -
Fig. S31. 1H NMR spectrum of 7 in CD3OD (400 MHz).
H [ppm]
Fig. S32.13C NMR spectrum of 7 in CD3OD (100 MHz).
C [ppm]
N
OCl
O
OOHO
OH1
34
4a5
678 8a
911
13
1''
2''
5'4'2'
1'
N
OCl
O
OOHO
OH1
34
4a5
678 8a
911
13
1''
2''
5'4'2'
1'
- S40 -
2.12. Chemical characterization of 8.
Table S9. NMR data of compound 811 in CDCl3. The molecular formula of 8 was established by mass data [ESI-MS: m/z 435 (M+H)+; HRESIMS: m/z 435.1569 (M+H)+, calcd. for C23H28ClO6
+, 435.1569, = 0.1 mmu]; []26
D: –90.6 (c 0.50, CHCl3).
Position H [ppm] mult. (J in Hz)11 H [ppm] mult. (J in Hz) C [ppm]11 C [ppm]1 7.40 s 7.42 s 147.2 147.13 157.8 158.14 6.48 s 6.47 s 105.9 104.94a 140.9 141.55 110.3 109.16 184.4 184.67 84.5 83.68 3.91 d (12.1) 3.89 d (12.0) 53.8 53.08a 114.1 113.79 6.03 d (15.7) 6.02 d (15.8) 120.9 120.210 6.51 dd (15.7, 8.0) 6.51 dd (15.7, 7.8) 146.4 146.711 2.27 m 2.24 m 39.8 38.912 1.43 m 1.40 m 30.2 29.213 0.90 t (7.4) 0.88 t (7.5) 12.8 11.87-CH3 1.60 s 1.57 s 24.6 23.611-CH3 1.25 d (6.3) 1.23 d (6.2) 20.3 19.31' 168.9 169.12' 4.22 d (12.1) 4.26 d (12.2) 59.8 58.83' 207.1 207.34' 3.21 m 3.23 m 44.1 42.55' 3.81 m 3.77 m 74.3 72.76' 1.06 d (6.1) 1.03 d (6.9) 14.1 13.13-OH4'-CH3 1.08 d (7.0) 1.06 d (6.6) 23.4 22.25'-OH 1.31 brs 2.35 brs1H and 13C NMR spectra were recorded at 400 MHz and 100 MHz, respectively.
- S41 -
Fig. S33. 1H NMR spectrum of 8 in CDCl3 (400 MHz).
H [ppm]
Fig. S34. 13C NMR spectrum of 8 in CDCl3 (100 MHz).
C [ppm]
O
O
1
4a 43
56
7
8
9
10 1213
8a
11
Cl
O
OOHO
1' 2'3'
4' 5'
6'
H
H
O
O
1
4a 43
56
7
8
9
10 1213
8a
11
Cl
O
OOHO
1' 2'3'
4' 5'
6'
H
H
- S42 -
2.13. Chemical characterization of 10.
Table S10. NMR data of compound 1013 in CDCl3. The molecular formula of 10 was established by mass data [ESI-MS: m/z 399 (M+H)+; HRESIMS: m/z 399.1806 (M+H)+, calcd. for C23H27O6
+, 399.1802, = 0.4 mmu]; []20
D: –12.1 (c 0.06, CH3OH).
Position H [ppm] mult. (J in Hz)13 H [ppm] mult. (J in Hz) C [ppm]13 C [ppm]1 8.77 s 8.79 s 152.7 152.93 155.4 155.54 6.01 s 6.09 s 107.9 108.14a 146.6 146.85 5.32 s 5.34 d (1.00) 105.7 105.96 190.1 190.27 87.7 87.88 165.2 165.68a 111.0 111.19 5.95 d (15.9) 5.95 d (15.7) 119.5 119.710 6.50 dd (15.9, 8.2) 6.51 dd (15.7, 7.8) 144.3 144.311 2.24 m 2.25 m 38.8 39.012 1.42 m 1.43 dq (7.3, 7.3) 29.1 29.313 0.89 t (7.2) 0.90 t (7.3) 11.6 11.87-CH3 1.67 s 1.69 s 26.4 26.511-CH3 1.06 d (6.2) 1.08 d (6.6) 19.3 19.51' 168.4 168.52' 124.1 124.13' 201.1 201.34' 3.65 m 3.67 dq (6.9, 6.9) 50.8 51.05' 3.87 dq (6.7, 6.1) 3.89 m 70.7 70.96' 1.16 d (6.7) 1.18 d (6.9) 21.3 21.53-OH4'-CH3 1.15 d (6.7) 1.16 d (6.4) 13.5 13.85'-OH 1.81 d (5.9)1H and 13C NMR spectra were recorded at 400 MHz and 100 MHz, respectively.
- S43 -
Fig. S35. 1H NMR spectrum of 10 in CDCl3 (400 MHz).
H [ppm]
Fig. S36. 13C NMR spectrum of 10 in CDCl3 (100 MHz).
C [ppm]
- S44 -
3. Supporting References
1. Binninger, D. M.; Skrzynia, C.; Pukkila, P. J.; Casselton, L. A. EMBO J. 1987, 6, 835–840.
2. Johnson, M.; Zaretskaya, I.; Raytselis, Y.; Merezhuk, Y.; McGinnis, S.; Madden, T. L.
Nucleic Acids Res. 2008, 36, W5.
3. Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem. 1997, 62, 7512–7515