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A unifying paradigm for naphthoquinone-based meroterpenoid (bio)synthesis
Zachary D. Miles,1,4 Stefan Diethelm,1,4 Henry P. Pepper,2,4 David M. Huang,2 Jonathan H.
George,2* Bradley S. Moore1,3*
1Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography,
University of California San Diego, La Jolla, CA 92093, USA.
2Department of Chemistry, University of Adelaide, Adelaide, SA 5005, Australia.
3Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego,
La Jolla, CA 92093, USA.
4These authors contributed equally to this work
*To whom correspondence should be addressed: bsmoore@ucsd.edu or
jonathan.george@adelaide.edu.au
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2829
NATURE CHEMISTRY | www.nature.com/naturechemistry 1
2
Table of Contents
1. General Methods 4 2. Biochemical Methods 6 Heterologous expression and purification of Mcl24 and NapH1 6 Cloning of Streptomyces sp. CNQ-525 napH3 and napT8 6 Expression of Streptomyces sp. CNQ-525 NapH3 and NapT8 6 Purification of NapH3 7 Purification of NapT8 7 pH screen for optimization of the production of 10 by Mcl24 8 Preparative scale production of 10 8 Structure elucidation of 10 9 Supplementary Table 1: 1H- and 13C-NMR chemical shift assignment for 10 10 Mcl24 incorporation of 18OH2 into pre-merochlorin (9) 10 NapT8 in vitro activity assay for conversion of 34 to 33 and requirements 11 NapT8, NapH3, and NapH1 coupled in vitro activity assays 11 NapH3 in vitro activity assays with synthetic substrates 12 Preparative scale production of 33 12 Preparative scale production of naphthomevalin (1) 12 Initial velocity measurements of NapH3 catalyzed and non-enzymatic conversion of
33 to naphthomevalin (1) 13
NapT8 and NapH1/Mcl24 coupled in vitro activity assays 13 3. Chemical Methods 15 Chemical chlorination of pre-merochlorin analogue 17 15 Supplementary Table 2: Chemical chlorination of 17 with varying stoichiometries of
i-Pr2NH/NCS reagent Supplementary Table 3: 1H- and 13C-NMR chemical shift assignment for 18 Supplementary Table 4: 1H-NMR chemical shift comparison of 18 and 5 Supplementary Table 5: 13C-NMR chemical shift comparison of 18 and 5 Supplementary Table 6: 1H- and 13C-NMR chemical shift assignment for 19 Supplementary Table 7: 1H- and 13C-NMR chemical shift assignment for 20
15
16 17 18 19 20
Total synthesis of naphthomevalin 21 Supplementary Table 8: Conditions tested for the thermal a-hydroxyketone
rearrangement of 33 34
Synthesis of proposed biosynthetic intermediates and a-hydroxyketone rearrangement test substrates
36
4. Computational Methods and Data 43 5. Supplementary Figures 56 Supplementary Figure 1: pH screen for optimization of the production of 10 by
Mcl24 56
Supplementary Figure 2: Experimental and calculated circular dichroism spectra of 10.
57
3
Supplementary Figure 3: Incorporation of 18OH2 into 10 58 Supplementary Figure 4: UV/Visible and mass spectra of the substrate, product, and
synthetic standard in the NapT8 in vitro assay 59
Supplementary Figure 5: UV/Visible and mass spectra of the product and synthetic naphthomevalin (1) in the coupled NapH3 in vitro assay
61
Supplementary Figure 6: UV/Visible and mass spectra of the product and napyradiomycin A1 (2) standard in the coupled NapH1 in vitro assay
62
Supplementary Figure 7: Reaction requirements for NapT8 activity 63 Supplementary Figure 8: In vitro assays of NapH3 with synthetic 33 64 Supplementary Figure 9: In vitro assays of NapH3 with other synthetic substrates 65 Supplementary Figure 10: Coupled in vitro assays of NapT8 and NapH1/Mcl24 66 Supplementary Figure 11: Comparison of the initial velocities for the NapH3
catalyzed and non-enzymatic conversion of 33 to naphthomevalin (1) 67
Supplementary Figure 12: Experimental and calculated circular dichroism spectra of 33
68
Supplementary Figure 13: Experimental and calculated circular dichroism spectra of 1
69
Supplementary Figure 14: Multiple sequence alignment of VHPO homologs 70 Supplementary Figure 15: Gene sequences used in this study as predicted by
Genemark 71
Supplementary Figure 16: SDS-PAGE gels (12%) of purified NapH3 and NapT8 72 6. NMR and Compound Characterization 73 Supplementary Table 9: 1H NMR comparison of natural naphthomevalin (1), natural
SF2415B1 (SI-9) and synthetic naphthomevalin (1) in CDCl3 106
Supplementary Table 10: 13C NMR comparison of natural naphthomevalin (1), natural SF2415B1 (SI-9) and synthetic naphthomevalin (1) in CDCl3
107
7. References 116
4
1. General Methods
Chemicals and Solvents:
All chemicals used were purchased from commercial suppliers (Acros, Aldrich, Fluka, Oakwood or Alfa
Aesar) and used as received unless otherwise noted. Pb(OAc)4 was recrystallized from glacial acetic acid
prior to use. For reactions, analytical grade solvents were purchased and used without further purification.
For flash chromatography, technical grade solvents were used without further purification. Labeled 18OH2
(~97% enriched) was purchased from Sigma Aldrich.
Reactions:
All non-aqueous reactions were performed under an inert atmosphere of N2, unless otherwise stated.
Reactions were magnetically stirred and monitored by TLC unless otherwise stated. Thin-layer
chromatography was performed using Merck aluminum sheets silica gel 60 F255 or Merck silica gel 60 F254
TLC glass plates. Visualization was aided by viewing under a UV lamp and staining with ceric
ammonium molybdate or KMnO4 solutions followed by heating. All Rf values were rounded to the
nearest 0.01. All organic extracts were dried over anhydrous magnesium sulfate. Flash chromatography
was performed using Davasil silica gel (40-63 micron grade) or Alfa Aesar silica gel (60 Å pore size)
using the solvents indicated as eluent with 0.3-0.5 bar pressure. The yields given refer to
chromatographically purified and spectroscopically pure compounds unless otherwise stated.
Analysis:
Melting points were recorded on a Reichart Thermovar Kofler microscope apparatus and are uncorrected.
Infrared spectra were recorded using a Perkin Elmer Spectrum BX FT-IR system spectrometer as the neat
compounds. Absorptions are given in wavenumbers (cm-1). 1H- and 13C- NMR spectra as well as 2D-
NMR spectra were recorded on a VARIAN Inova (500 MHz), BRUKER Avance (600 MHz), or Agilent-
500/54/ASC (500 MHz) spectrometer in the solvents indicated. All signals are reported in ppm with the
internal chloroform signal at 7.26 ppm or 77.0 ppm, or the internal DMSO signal at 2.50 ppm or 39.5
ppm as standard. The data is being reported as (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet or
5
unresolved, br=broad signal, coupling constant(s) in Hz, integration). All J values were rounded to the
nearest 0.1 Hz. High-resolution ESI mass spectra were recorded on an Agilent-6230 TOF LC-MS.
Analytical and semi-preparative HPLC were carried out on an Agilent Technologies 1200 Series
system with a diode-array detector. Analytical separation for Mcl24 reactions was accomplished by
reversed-phase chromatography using water + 0.1% trifluoroacetic acid (A) and acetonitrile + 0.1%
trifluoroacetic acid (B) in a gradient of 5% to 95% (B) over 20 minutes at a flow rate of 0.7 mL/min on a
Phenomenex Luna C18(2), 5μm-100 x 4.6 mm column. Preparative HPLC for the isolation of 10 was
carried out using a Waters system with a 600 Controller, 2487 dual wavelength detector, and 600 Pump
connected to a Phenomenex Synergi Hydro- RP, 10μm-250 x 21 mm column using water + 0.1%
trifluoroacetic acid (A) and acetonitrile + 0.1% trifluoroacetic acid (B) in a gradient of 75% to 85% (B)
over 30 minutes with a flow rate of 4 mL/min.
For NapT8, NapH3, and NapH1 assays, analytes were separated by reversed-phase
chromatography on an Agilent Technologies Eclipse XDB-C18, 5μm-150 x 4.6 mm column at a flow
rate of 0.75 mL/min with a mobile phase combination of water + 0.1% formic acid (A) and acetonitrile +
0.1% formic acid (B) using a gradient as follows: 10% to 100% (B), 0 to 20 min; 100% (B), 20 to 24 min;
100% to 10% (B), 24 to 27 min; 10% (B), 27 to 30 min.
LC-MS was carried out on an Agilent Technologies 1200 Series system with a diode-array
detector coupled to an Agilent Technologies 6530 Accurate-Mass Q-TOF mass spectrometer in negative
ion mode using the analytical separation described above for the respective enzyme reaction. Figures for
mass spectral data were created in Mass Hunter (Agilent Technologies). Circular dichroism (CD)
measurements of 10 were obtained on an Aviv CD spectrometer model 62DS using a 1 nm bandwidth in
a 0.5 cm cell at a concentration of 1.0 mM at 25 °C.
6
2. Biochemical Methods
Heterologous expression and purification of Mcl24 and NapH1:
Mcl24 and NapH1 were expressed and purified as previously reported1-3.
Cloning of Streptomyces sp. CNQ-525 napH3 and napT8:
Putative open reading frames in the regions of the originally annotated napH3 and napT8 genes were
predicted using GeneMark4. The open reading frames from 21833 bp to 23629 bp (encoding NapH3) and
19734 to 20642 bp (encoding NapT8)(see Supplementary Fig. 15 for the complete sequences) in the
Streptomyces sp. CNQ-525 nap cluster (GenBank accession number EF397639) were amplified from
genomic DNA by PCR using the following primers: 5’-
ATACATATGACGACATCCGCCCCTGCCCAG-3’ (forward), and 5’-
ATTAAGCTTTCAGTCCTTGACGTCGCCGTTGATG-3’ (reverse) for napH3, and 5’-
ATACATATGACTGACACAGGCATGGAAGG-3’ (forward) and 5’-
ATAGGATCCTCAGCTGCCGGCGCCCGCCGCG-3’ (reverse) for napT8 at annealing temperatures of
60 °C and 53 °C, respectively. The resulting PCR products were digested with restriction enzymes NdeI
and either HindIII (napH3) or BamHI (napT8), and subsequently ligated into a similarly digested pET28a
vector (Novagen) for expression of His6-tagged NapH3 and NapT8. The pET28a:NapH3 F378S mutant
was created using Phusion DNA polymerase (New England Biolabs) according to the manufacturer’s
recommended protocol and the following primers: 5’-
GAGTACCCCTCCGGCTCCACCACCTTGATCGCG-3’ (forward), and 5’-
CGCGATCAAGGTGGTGGAGCCGGAGGGGTACTC-3’ (reverse) at an annealing temperature of 72
°C.
Expression of Streptomyces sp. CNQ-525 NapH3 and NapT8:
Escherichia coli BL21-Gold(DE3) cells (Agilent Technologies) containing the pET28a:napH3 or
pET28a:napT8 vector were grown in 4 L of TB broth containing 50 µg/mL kanamycin at 37 °C until an
OD600 of approximately 0.5, at which time the temperature was lowered to 18 °C. Cells were then grown
7
to an OD600 of approximately 0.7-0.8 and expression was induced by addition of 0.1 mM IPTG (final
concentration). Cells were grown overnight at 18 °C, harvested the next day by centrifugation (5000 × g),
and the pellets reserved and frozen at -80 °C.
Purification of NapH3:
Cells from 4 L of growth were resuspended in 120 mL of buffer containing 50 mM Tris-HCl (pH 8.0), 0.5
M NaCl, and 40 mM imidazole (buffer A1) with an additional 1 mM phenylmethanesulfonyl fluoride
added and sonified using a Branson digital sonifier (40% amplitude). The lysate was centrifuged for 30
min at 18000 × g to pellet insoluble material. The cleared lysate was then loaded onto a 5 mL HisTrap FF
column (GE Healthcare) equilibrated prior in buffer A1. The column was washed with 50 mL of buffer
A1, and NapH3 was eluted in a gradient of 0-100% buffer B1 (buffer A containing 0.5 M imidazole) over
75 mL. Fractions containing NapH3 were identified by SDS-PAGE, pooled, and concentrated to <2 mL
total volume using an Amicon Ultra-15 10 kDa cutoff concentrator (EMD Millipore) by centrifugation at
3500 × g, 4 °C. The concentrated protein was then loaded onto a Superdex 200 gel filtration column (16
cm × 60 cm, GE Healthcare) equilibrated prior in 25 mM HEPES-NaOH (pH 8.0), 300 mM NaCl, and
10% glycerol, and eluted at a constant flow rate of 1.0 mL/min. Fractions containing NapH3 were
identified using SDS-PAGE, pooled, and concentrated as described above. Protein aliquots were frozen
on dry ice and stored at -80 °C (see Supplementary Fig. 16 for gel of purified protein). The protein
concentration was determined using the Bradford method using bovine serum albumin as a standard.
Purification of NapT8:
Cells from 4 L of growth were resuspended in 120 mL of buffer containing 50 mM Tris-HCl (pH 8.0), 0.5
M NaCl, and 25 mM imidazole (buffer A2) with an additional 1 mM phenylmethanesulfonyl fluoride
added and sonified using a Branson digital sonifier (40% amplitude). The lysate was centrifuged for 30
min at 18000 × g to pellet insoluble material. The cleared lysate was then loaded onto a 5 mL HisTrap FF
column (GE Healthcare) equilibrated prior in buffer A2. The column was washed with 50 mL of buffer
A2, and NapT8 was eluted in a gradient of 0-100% buffer B2 (buffer A2 containing 0.5 M imidazole)
8
over 75 mL. Fractions containing NapT8 were identified by SDS-PAGE, pooled, and concentrated to <2
mL total volume using an Amicon Ultra-15 10 kDa cutoff concentrator (Millipore) by centrifugation at
3500 × g, 4 °C. The concentrated protein was then loaded onto a Superdex 75 gel filtration column (16
cm × 60 cm, GE Healthcare) equilibrated prior in 25 mM HEPES-NaOH (pH 8.0), 300 mM NaCl, and
10% glycerol, and eluted at a constant flow rate of 1.0 mL/min. Fractions containing NapT8 were
identified using SDS-PAGE, pooled, and concentrated as described above. Protein aliquots were frozen
on dry ice, and stored at -80 °C (see Supplementary Fig. 16 for gel of purified protein). The protein
concentration was determined using the Bradford method using bovine serum albumin as a standard.
pH screen for optimization of the production of 10 by Mcl24:
The pH screening assay for maximal Mcl24 10 production contained 50 mM HEPES-NaOH buffer (pH
6.5-8.5), 50 mM KCl, 0.1 mM Na3VO4, 50 μg Mcl24, 100 μM pre-merochlorin (9) (added as 10 μL of a
10 mM stock solution in DMSO), and initiated by addition of 1.0 mM H2O2 in a total reaction volume of
1.0 mL. The reaction was incubated at 37°C for 12 h, and then extracted twice with EtOAc. The solvent
was evaporated and the residue was taken up in MeOH. The reaction was analyzed by analytical HPLC as
described in the analysis section.
Preparative scale production of 10:
The reaction (12 mL) contained 50 mM HEPES-NaOH pH 8.0, 150 mM KCl, 0.1 mM Na3VO4, and 10%
glycerol, 200 µL of a stock solution of pre-merochlorin (9) (4 mg in DMSO), 4.17 mM H2O2 (100 µL of a
500 mM stock solution), and initiated by addition of 640 μg of Mcl24 (40 µL, 16 mg/mL stock solution).
The reaction was incubated at 37 °C for 12 h, then extracted three times with EtOAc. The combined
organic layers were washed with brine and dried over MgSO4. The solvent was removed and the residue
was filtered through a plug of SiO2 (eluent: hexanes/EtOAc 4:1). After removal of the solvent, the crude
mixture was subjected to preparative HPLC (Phenomenex Synergi Hydro-RP, 10μm-250 x 21 mm
column, MeCN/H2O; 75% MeCN to 85% MeCN over 30 min, flow rate of 4 mL/min) to give 10 (0.8-1.0
mg).
9
Structure elucidation of 10:
The structure of 10 was elucidated based on 2D NMR analysis (see section 6 for the full spectral data)
and MS data. Shown below (box) are the relevant HMBC correlations leading to the assignment of the
C10 prenylation pattern (main text numbering: C3). The carbon numbering used follows the numbering of
merochlorin D (7)5. The relevant 13C-NMR chemical shifts as well as the HMBC correlations observed for
compound 10 closely fit the data reported for merochlorin D (7), confirming the structural assignment of
compound 10.
(E)-3,3-dichloro-2-(5,9-dimethyl-2-(propan-2-ylidene)deca-4,8-dien-1-yl)-2,5,7-trihydroxy-2,3-
dihydronaphthalene-1,4-dione (10): TLC (hexanes:EtOAc, 4:1 v/v): Rf = 0.45; [α]D24.3°C = -0.6 (c 0.25
in MeOH); 1H-NMR (600 MHz, d6-DMSO): d 7.15 (bs, 2H, OH), 6.87 (d, J = 2.2 Hz, 1H), 6.76 (s, 1H,
OH), 6.66 (d, J = 2.3 Hz, 1H), 4.96 (t, J = 7.2 Hz, 1H), 4.78 (t, J = 7.1 Hz, 1H), 2.81 (dd, J = 14.9, 7.0 Hz,
1H), 2.62 (d, J = 14.1 Hz, 1H), 2.35 (dd, J = 14.8, 7.1 Hz, 1H), 2.27 (d, J = 14.1 Hz, 1H), 1.97-1.93 (m,
2H), 1.88-1.84 (m, 2H), 1.59 (s, 3H), 1.51 (s, 3H), 1.50 (s, 3H), 1.42 (s, 3H), 1.14 (s, 3H); 13C-NMR (150
MHz, d6-DMSO): d 193.2, 184.3, 166.0, 164.4, 134.7, 134.4, 130.6, 130.3, 125.2, 124.0, 122.5, 108.5,
108.0, 106.6, 93.8, 86.5, 39.1, 38.6, 30.7, 26.0, 25.5, 20.6, 20.3, 17.5, 15.6; IR νmax (film)/cm-1: 2967,
2924, 1704, 1654, 1619, 1581, 1460, 1440, 1362, 1259, 1174, 1095, 1023; HRMS (ESI): m/z calculated
for C25H29Cl2O5 ([M-H]-) 479.1398, found 479.1425. UV (MeCN) λmax/nm: 230, 267, 318.
10
Supplementary Table 1. 1H- and 13C-NMR chemical shift assignment for 10.
No. δC δH HMBC signals (C → H) 1 193.2
- 3, C10-OH, 11 2 134.7
- - 3 108.0
6.87 (d, J = 2.2 Hz, 1H)
5 4 166.0
- 3, 5 5 108.5
6.66 (d, J = 2.3 Hz, 1H)
3 6 164.4
- 5 7 106.6
- 3, 5 8 184.3
- - 9 93.8
- C10-OH, 11 10 86.5
- C10-OH, 11, 22, 24 11 38.6
2.62 (d, J = 14.1 Hz, 1H), 2.27 (d, J = 14.1 Hz, 1H)
C10-OH, 13 12 125.2
- 11, 13, 22, 24 13 30.7
2.81 (dd, J = 14.9, 7.0 Hz, 1H), 2.35 (dd, J = 14.8, 7.1 Hz, 1H)
11 14 122.5
4.78 (t, J = 7.1 Hz, 1H)
13, 16, 25 15 134.2
- 13, 16, 17, 25 16 39.1
1.88-1.84 (m, 2H)
17, 20, 21, 25 17 26.0
1.97-1.93 (m, 2H)
16 18 124.0
.
4.96 (t, J = 7.2 Hz, 1H)
16, 17, 20, 21 19 130.6
- 17, 20, 21 20 25.5
1.59 (s, 3H)
21 21 17.5 1.51 (s, 3H) 20 22 20.3
1.50 (s, 3H)
24 23 130.3
- 11, 13, 24 24 20.6
1.14 (s, 3H)
22 25 15.6
1.42 (s, 3H)
16 C4-OH - 7.15 (bs, 2H) C6-OH - 7.15 (bs, 2H)
C10-OH - 6.76 (s, 1H)
Mcl24 incorporation of 18OH2 into pre-merochlorin (9):
Reactions containing 5 µL of 1 M HEPES-NaOH pH 8.0, 5 µL of 1 M KCl, and 1 µL of 10 mM Na3VO4
were placed in a speedvac and as much of the water was removed as possible. Then, 98.3 µL of either
unlabeled water or 18OH2 was used to resuspend the remaining residue, and 1 µL of 25 mM pre-
merochlorin (9) and 0.5 µL of 400 mM H2O2 were added. The reactions were initiated by addition of 0.2
µL of 715 µM Mcl24. The final reaction mixture (0.1 mL) contained 50 mM HEPES-NaOH pH 8.0, 150
mM KCl, 0.1 mM Na3VO4, 2.0 mM H2O2, 250 µM pre-merochlorin (9), and 1.5 µM enzyme. The
reaction was incubated at room temperature for 8 h, and quenched by EtOAc extraction (1.0 mL, twice).
The solvent was evaporated and the residue was brought up in 0.1 mL MeOH. 20 µL was injected onto
the LC-MS and analyzed as described in the analysis section.
11
NapT8 in vitro activity assay for conversion of 34 to 33 and determination of requirements:
Assays (0.1 mL) contained 50 mM HEPES-NaOH pH 8.0, 150 mM KCl, 5.0 mM MgCl2, 1.0 mM of
either dimethylallyl pyrophosphate (DMAPP), isopentenyl pyrophosphate (IPP), or geranyl
pyrophosphate (GPP), 1.0 mM racemic synthetic substrate 34 (from a 50 mM stock dissolved in DMSO),
and initiated by addition of 10 µM enzyme. DMAPP was used as the isoprene substrate when either
enzyme or MgCl2 was omitted. The reaction was left to incubate at room temperature for 30 min, and
quenched by EtOAc extraction (1.0 mL, twice). The solvent was evaporated and the residue was brought
up in 0.1 mL MeOH. 25 µL was injected onto the LC-MS and analyzed as described in the analysis
section. Assays contained indicated components unless otherwise noted. Control reactions were
conducted with water in place of enzyme.
NapT8, NapH3, and NapH1 coupled in vitro activity assays:
Assays (0.1 mL) contained 50 mM HEPES-NaOH pH 8.0, 150 mM KCl, 5.0 mM MgCl2, 1.0 mM of
dimethylallyl pyrophosphate (DMAPP), 1.0 mM racemic synthetic substrate 34 (from a 50 mM stock
dissolved in DMSO), and initiated by addition of 10 µM enzyme. The reaction was left to incubate at
room temperature for 30 min, and quenched by EtOAc extraction (1.0 mL, twice). The residue from the
reaction was either resuspended in 0.1 mL MeOH if analyzed by LC-MS, or resuspended in 2 µL of
DMSO for the subsequent reaction. Next, NapH3 assays (0.1 mL) were conducted and contained 50 mM
HEPES-NaOH (pH 8.0), 150 mM KCl, NapT8 reaction extract resuspended in 2 µL of DMSO, and
initiated by addition of 20 µM enzyme. Reactions were allowed to incubate for 2 h at room temperature,
and quenched by EtOAc extraction (1.0 mL, twice). The solvent was evaporated and the residue was
brought up in 0.1 mL MeOH if analyzed by LC-MS, or resuspended in 2 µL of DMSO for the subsequent
reaction. Next, NapH1 assays (0.1 mL) were conducted and contained 50 mM HEPES-NaOH (pH 8.0),
150 mM KCl, 0.1 mM Na3VO4, 2.0 mM H2O2, NapH3 reaction extract resuspended in 2 µL of DMSO,
and initiated by addition of 20 µM enzyme. Reactions were allowed to incubate for 2 h at room
temperature, and quenched by EtOAc extraction (1.0 mL, twice). The solvent was evaporated and the
12
residue was brought up in 0.1 mL MeOH. 25 µL was injected onto the LC-MS, and analyzed as described
in the analysis section. All control reactions were conducted with water in place of enzyme and standards
were dissolved in MeOH at the same concentration as described for 34 in a typical assay
NapH3 in vitro activity assays with synthetic substrates:
Assays (0.1 mL) contained 50 mM HEPES-NaOH (pH 8.0), 150 mM KCl, 0.5 mM racemic synthetic
substrate (from a 50 mM stock dissolved in DMSO), and initiated by addition of 20 µM enzyme.
Reactions were allowed to incubate for 2 h at room temperature, and quenched by EtOAc extraction (1.0
mL, twice). The solvent was evaporated and the residue was brought up in 0.1 mL MeOH. 25 µL was
injected onto the LC-MS, and analyzed as described in the analysis section. All control reactions were
conducted with water in place of enzyme.
Preparative scale production of 33:
A 10 mL reaction containing 50 mM HEPES-NaOH pH 8.0, 150 mM KCl, 5 mM MgCl2, 26 mg of solid
dimethylallyl pyrophosphate ammonium salt, 8 mg of synthetic compound 34 dissolved in 200 µL
DMSO, and initiated by addition of ~5 mg of NapT8. The reaction was then gently stirred at room
temperature for 2.5 h. The reaction was quenched by extraction with EtOAc (3x) and the solvent was
removed. The crude mixture was resuspended in methanol and purified by reversed-phase
chromatography on a semi-preparative HPLC using a Phenomenex Luna C18(2), 5μm-250 x 10 mm
column at a flow rate of 2.5 mL/min with a mobile phase combination of water + 0.1% trifluoroacetic
acid (A) and acetonitrile + 0.1% trifluoroacetic acid (B) using a gradient as follows: 10% to 100% (B), 0
to 20 min; 100% (B), 20 to 27 min; 100% to 10% (B), 27 to 30 min; 10% (B), 30 to 33 min.
Preparative scale production of naphthomevalin (1):
A 10 mL reaction containing 50 mM HEPES-NaOH pH 8.0, 150 mM KCl, 10% glycerol, 7 mg of
synthetic compound 33 dissolved in 200 µL DMSO, and initiated by addition of ~20 mg of NapH3. The
reaction was then gently stirred at room temperature for 2 h. The reaction was quenched by extraction
with EtOAc (3x) and the solvent was removed. The crude mixture was resuspended in methanol and
13
purified by reversed-phase chromatography as described in the preparative scale reaction of compound
33. The reisolated naphthomevalin (1) was obtained from a preparative NapH1 reaction conducted in the
same manner as previously described3.
Initial velocity measurements of NapH3 catalyzed and non-enzymatic conversion of 33 to
naphthomevalin (1):
A 50 µL reaction containing 50 mM HEPES-NaOH pH 8.0, 150 mM KCl, and 0.5 mM synthetic 33 or
0.25 mM NapT8-produced 33 was initiated by addition of either 1, 5, or 10 µM NapH3. For non-
enzymatic measurements NapH3 was omitted. The reactions were quenched at various time intervals with
an equal part ice-cold methanol and placed on dry ice to aid in enzyme precipitation. The reactions were
then centrifuged at 21000 × g, 4 °C for 15 min to pellet precipitated enzyme and 40 µL was injected on
the HPLC and separated as described in the analysis section. Initial velocities were obtained from the
linear portion of the reaction by integration of the naphthomevalin (1) product peak and comparison to a
standard set of synthetic naphthomevalin (1) concentrations. The initial naphthomevalin (1) present at the
zero time-point is a result of the purification of compound 33 in aqueous conditions. The concentrations
of substrate were confirmed to be saturating and the rate of NapH3 catalysis increased linearly with
increased enzyme concentration (1, 5, and 10 µM). All measurements were conducted in at least
triplicate.
NapT8 and NapH1/Mcl24 coupled in vitro activity assays:
Assays (0.1 mL) contained 50 mM HEPES-NaOH pH 8.0, 150 mM KCl, 5.0 mM MgCl2, 1.0 mM of
dimethylallyl pyrophosphate (DMAPP), 0.5 mM racemic synthetic substrate 34 (from a 50 mM stock
dissolved in DMSO), and initiated by addition of 10 µM enzyme. The reaction was left to incubate at
room temperature for 30 min, and quenched by EtOAc extraction (1.0 mL, twice). The residue from the
reaction was either resuspended in 0.1 mL MeOH if analyzed by LC-MS, or resuspended in 2 µL of
DMSO for the subsequent reaction. Next, NapH1/Mcl24 assays (0.1 mL) were conducted and contained
50 mM HEPES-NaOH (pH 8.0), 150 mM KCl, 0.1 mM Na3VO4, 2.0 mM H2O2, NapT8 reaction extract
14
resuspended in 2 µL of DMSO, and initiated by addition of 20 µM enzyme. Reactions were allowed to
incubate for 2 h at room temperature, and quenched by EtOAc extraction (1.0 mL, twice). The solvent
was evaporated and the residue was brought up in 0.1 mL MeOH. 25 µL was injected onto the LC-MS,
and analyzed as described in the analysis section.
15
3. Chemical Methods
Chemical chlorination of pre-merochlorin analogue 17:
A solution of naphthol 17 (80 mg, 0.22 mmol) (prepared as previously reported2) in CH2Cl2 (5 mL) was
cooled to −78 °C. Diisopropylamine (0.9-10 equiv., see table below) was added to the reaction followed
by N-chlorosuccinimide (0.9-10 equiv., see table below). The mixture was allowed to warm to 0 °C and
stirred for 1 h. After removal of the solvent under reduced pressure, the residue was subjected to flash
column chromatography (hexanes/EtOAc 40:1 → 9:1) to give products 18, 19 and 20. The following
yields were obtained using different stoichiometries of chlorinating reagent:
Supplementary Table 2. Chemical chlorination of 17 with varying stoichiometries of i-Pr2NH/NCS
reagent.
entry reagent product yield
i-Pr2NH/NCS 18 19 20
1b 0.9 equiv. 16% - -
2a,b 2.0 equiv. - 17% 21%
3 5.0 equiv. - 3% 58%
4 10.0 equiv. - - 64%
a) 165 mg (0.45 mmol) of substrate 17 was used. b) The reaction was accompanied by formation of a complex mixture of oxidized byproducts that could not be further characterized.
(±)-(3aR,4S,5S,10bS)-5-Chloro-4-methyl-4-(4-methylpent-3-en-1-yl)-2-(propan-2-ylidene)-
1,2,3,3a,4,5-hexahydro-6H-5,10b-methanobenzo[e]azulene-6,11-dione (18): TLC (hexanes:EtOAc,
9:1 v/v): Rf = 0.32; 1H-NMR (600 MHz, d6-DMSO): δ 8.06 (dd, J = 7.8, 1.4 Hz, 1H), 7.78 (td, J = 7.6,
O
O
Cl
18
O
O
Cl
diagnostic NOE signals:H15 ∩ H25H9 ∩ H16
H25
15
169
16
1.5 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.55 (t, J = 7.6 Hz, 1H), 4.98-4.94 (m, 1H), 3.07-2.98 (m, 2H),
2.50-2.41 (m, 2H), 2.29 (dd, J = 9.6, 3.8 Hz, 1H), 2.11 (td, J = 13.9, 13.2, 6.5 Hz, 1H), 1.82 (td, J = 13.6,
13.2, 6.4 Hz, 1H), 1.75 (s, 3H), 1.64 (s, 3H), 1.59 (s, 3H), 1.52 (s, 3H), 1.44 (td, J = 13.0, 4.8 Hz, 1H),
1.12 (td, J = 13.0, 4.6 Hz, 1H), 0.91 (s, 3H); 13C-NMR (150 MHz, d6-DMSO): d 200.0, 190.5, 147.0,
136.0, 132.2, 131.3, 131.0, 128.9, 128.3, 123.8, 123.0, 122.7, 91.9, 60.9, 58.7, 43.8, 39.8, 31.5, 28.4, 25.4,
22.1, 21.0, 20.6, 17.4, 16.3; IR νmax (neat)/cm-1: 2972, 2920, 2856, 1774, 1696, 1595, 1453, 1379, 1287;
HRMS (ESI): m/z calculated for C25H30ClO2 ([M+H]+) 397.1923, found 397.1927.
Supplementary Table 3. 1H- and 13C-NMR chemical shift assignment for 18.
No. δC δH HMBC signals (C →
H) 1 190.5 - 3, 6 2 131.0 - 4, 6 3 128.9 8.06 (dd, J = 7.8, 1.4 Hz, 1H) 5, 4 4 128.3 7.55 (t, J = 7.6 Hz, 1H) 6 5 136.0 7.78 (td, J = 7.6, 1.5 Hz, 1H) 3, 4 6 123.0 7.69 (d, J = 7.8 Hz, 1H) 4 7 147.0 9, 3, 5 8 60.9 - 6, 9, 13, 15 9 58.7 2.29 (dd, J = 9.6, 3.8 Hz, 1H) 13, 15, 16, 25
10 43.8 - 9, 15, 16, 25 11 91.9 - 16, 25 12 200.0 - 9, 13 13 28.4 3.07-2.98 (m, 2H) 15, 22, 24 14 132.2 - 9, 13, 15, 22, 24 15 31.5 2.50-2.41 (m, 2H) 9, 13, 22 16 39.8 1.44 (td, J = 13.0, 4.8 Hz, 1H), 1.12 (td, J = 13.0, 4.6 Hz, 1H) 9, 15, 17, 18, 20 17 22.1 2.11 (td, J = 13.9, 13.2, 6.5 Hz, 1H), 1.82 (td, J = 13.6, 13.2, 6.4 Hz, 1H) 16, 18 18 123.8 4.98-4.94 (m, 1H) 17, 20, 21 19 131.3 - 20, 21 20 17.4 1.52 (s, 3H) 18, 21 21 25.4 1.59 (s, 3H) 18, 20 22 21.0 1.75 (s, 3H) 24 23 122.7 - 13, 15, 22, 24 24 20.6 1.64 (s, 3H) 22 25 16.3 0.91 (s, 3H) 9, 16
17
Supplementary Table 4. 1H-NMR chemical shift comparison of 18 and 5.
No. δH for 18 δH for 5 1 - - 2 - - 3 8.06, dd (7.8, 1.4) 6.16, d (2.0)
4 7.55, t (7.6) - 5 7.78, td (7.6, 1.5) 6.38, d (2.0)
6 7.69, d (7.8) - 7 - - 8 - - 9 2.29, dd (9.6, 3.8) 2.24, dd (9.4, 4.0)
10 - - 11 - - 12 - - 13 3.07-2.98, m 2.87, d (13.0); 2.65, d (13.0) 14 - - 15 2.50-2.41, m 2.36, dd (14.0, 4.0); 2.33, dd (14.0, 9.4) 16 1.44, td (13.0, 4.8); 1.12, td (13.0, 4.6) 1.40, dt (14.8, 4.8); 1.14, q (6.0) 17 2.11, td (13.9, 13.2, 6.5); 1.82, td (13.6, 13.2, 6.4) 2.03, m; 1.75, m 18 4.98-4.94, m 4.92, t (6.5) 19 - - 20 1.52, s 1.45, s 21 1.59, s 1.53, s 22 1.75, s 1.56, s 23 - - 24 1.64, s 1.65, s 25 0.91, s 0.81, s
18
Supplementary Table 5. 13C- NMR chemical shift comparison of 18 and 5.
No. δC for 18 δC for 5 1 190.5 193.2 2 131.0 109.8 3 128.9 165.4 4 128.3 102.1 5 136.0 166.5 6 123.0 103.7 7 147.0 150.5 8 60.9 61.5 9 58.7 58.8
10 43.8 45.3 11 91.9 91.3 12 200.0 200.1 13 28.4 29.3 14 132.2 132.1 15 31.5 31.9 16 39.8 39.2 17 22.1 22.8 18 123.8 124.2 19 131.3 131.6 20 17.4 18.1 21 25.4 26.1 22 21.0 20.9 23 122.7 123.1 24 20.6 21.1 25 16.3 16.5
(E)-2,2,4-Trichloro-4-(5,9-dimethyl-2-(propan-2-ylidene)deca-4,8-dien-1-yl)naphthalene-
1,3(2H,4H)-dione (19): TLC (hexanes:EtOAc, 9:1 v/v): Rf = 0.65; 1H-NMR (600 MHz, CDCl3): d 8.01
(d, J = 8.0 Hz, 2H), 7.76 (td, J = 7.4, 1.5 Hz, 1H), 7.59 (td, J = 7.5, 1.0 Hz, 1H), 4.99 (tp, J = 7.0, 1.4 Hz,
1H), 4.56 (tdd, J = 6.0, 2.7, 1.3 Hz, 1H), 3.50 (d, J = 13.5 Hz, 1H), 2.74 (d, J = 13.4 Hz, 1H), 2.11 (dd, J
= 15.3, 6.4 Hz, 1H), 1.99-1.95 (m, 2H), 1.88-1.84 (m, 2H), 1.65 (s, 3H), 1.56 (s, 3H), 1.50 (dd, J = 21.4,
6.1 Hz, 1H), 1.44 (s, 3H), 1.32 (s, 3H), 1.27 (s, 3H); 13C-NMR (150 MHz, CDCl3): d 189.7, 182.0, 139.2,
19
136.6, 136.1, 135.2 (2C), 131.4, 129.7, 129.2, 128.5, 124.1, 124.0, 121.6, 81.0, 70.3, 49.7, 39.5, 31.1,
26.4, 25.7, 21.2, 20.7, 17.7, 16.0; IR νmax (film)/cm-1: 2964, 2922, 2856, 1787, 1753, 1723, 1597, 1450,
1378, 1291, 1267, 1238; HRMS (ESI): m/z calculated for C25H28Cl3O2 ([M-H]-) 465.1160, found
465.1185.
Supplementary Table 6. 1H- and 13C-NMR chemical shift assignment for 19.
No. δC δH HMBC signals (C → H) 1 70.3 - 3, 11, 22, 24 2 139.2 - 4, 6, 11 3 129.2 8.01 (d, J = 8.0 Hz, 2H) 5 4 135.2 7.76 (td, J = 7.4, 1.5 Hz, 1H) 6.5 5 129.7 7.59 (td, J = 7.5, 1.0 Hz, 1H) 3 6 128.5 8.01 (d, J = 8.0 Hz, 2H) 4 7 135.2 - 3, 5 8 182.0 - 6 9 81.0 - 11
10 189.7 - 11 11 49.7 3.50 (d, J = 13.5 Hz, 1H), 2.74 (d, J = 13.4 Hz, 1H) 13 12 136.6 - 11, 22, 24 13 31.1 2.11 (dd, J = 15.3, 6.4 Hz, 1H), 1.50 (dd, J = 21.4, 6.1 Hz, 1H) 11, 14 14 121.6 4.56 (tdd, J = 6.0, 2.7, 1.3 Hz, 1H) 13, 16, 25 15 136.1 - 16, 25 16 39.5 1.88-1.84 (m, 2H) 14, 17, 25 17 26.4 1.99-1.95 (m, 2H) 16 18 124.1 4.99 (tp, J = 7.0, 1.4 Hz, 1H) 16, 17, 20, 21 19 131.4 - 17, 20, 21 20 17.7 1.56 (s, 3H) 21 21 25.7 1.65 (s, 3H) 20 22 21.2 1.27 (s, 3H) 24 23 124.0 - 11, 22, 24 24 20.7 1.44 (s, 3H) 22 25 16.0 1.32 (s, 3H) 14, 16
20
(E)-3-(2,2-Dichloroacetyl)-3-(5,9-dimethyl-2-(propan-2-ylidene)deca-4,8-dien-1-yl)isobenzofuran-
1(3H)-one (20): TLC (hexanes:EtOAc, 9:1 v/v): Rf = 0.48 1H-NMR (600 MHz, CDCl3): d 7.88 (d, J =
7.6 Hz, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.72 (td, J = 7.5, 1.1 Hz, 1H), 7.59 (td, J = 7.5, 1.0 Hz, 1H), 6.48 (s,
1H), 5.01 (tdd, J = 5.6, 3.0, 1.5 Hz, 1H), 4.78 (dddt, J = 7.6, 6.3, 2.7, 1.4 Hz, 1H), 3.29 (d, J = 14.5 Hz,
1H), 2.67 (d, J = 14.3 Hz, 1H), 2.65 (dd, J = 15.6, 5.9 Hz, 1H), 2.49 (dd, J = 15.4, 7.4 Hz, 1H), 2.03-1.99
(m, 2H), 1.95-1.91 (m, 2H), 1.65 (s, 3H), 1.62 (s, 3H), 1.62 (s, 3H), 1.57 (s, 3H), 1.55 (s, 3H); 13C-NMR
(150 MHz, CDCl3): d 193.4, 168.3, 147.6, 136.1, 134.7, 133.9, 131.3, 130.4, 125.8, 124.7, 124.2, 123.8,
123.0, 122.1, 91.7, 66.7, 41.0, 39.6, 31.7, 26.5, 25.6, 21.6, 20.7, 17.6, 16.1; IR νmax (film)/cm-1: 3567 (br),
2922, 1785, 1749, 1655, 1462, 1284, 1241, 1059; HRMS (ESI): m/z calculated for C25H29Cl2O3 ([M-H]-)
447.1499, found 447.1499.
Supplementary Table 7. 1H- and 13C-NMR chemical shift assignment for 20.
No. δC δH HMBC signals (C → H) 1 91.7 - 3, 11, 22, 24 2 147.6 - 6, 4, 11 3 123.0 7.80 (d, J = 7.6 Hz, 1H) 5 4 134.7 7.72 (td, J = 7.5, 1.1 Hz, 1H) 6 5 130.4 7.59 (td, J = 7.5, 1.0 Hz, 1H) 3 6 125.8 7.88 (d, J = 7.6 Hz, 1H) 4 7 124.7 - 5, 3 8 168.3 6, 5 9 66.7 6.48 (s, 1H) 11
10 193.4 9, 11 11 41.0 3.29 (d, J = 14.5 Hz, 1H), 2.67 (d, J = 14.3 Hz, 1H) 13 12 133.9 - 11, 13, 22, 24 13 31.7 2.65 (dd, J = 15.6, 5.9 Hz, 1H), 2.49 (dd, J = 15.4, 7.4 Hz, 1H) 11, 24 14 122.1 4.78 (dddt, J = 7.6, 6.3, 2.7, 1.4 Hz, 1H) 13, 16, 25 15 136.1 - 16, 25 16 39.6 1.95-1.91 (m, 2H) 17, 25 17 26.5 2.03-1.99 (m, 2H) 16 18 124.2 5.01 (tdd, J = 5.6, 3.0, 1.5 Hz, 1H) 16, 20 19 131.3 - 20, 21 20 17.6 1.55 (s, 3H) 21 21 25.6 1.62 (s, 3H) 20 22 21.6 1.65 (s, 3H) 24 23 123.8 - 11, 22, 24 24 20.7 1.62 (s, 3H) 22 25 16.1 1.57 (s, 3H) 16
21
Total synthesis of naphthomevalin:
Methyl 2-(2-acetyl-3,5-dimethoxyphenyl)acetate SI-1: To a solution of methyl 3,5-
dimethoxyphenylacetate (25) (12.6 g, 60.0 mmol) in Ac2O (50.0 mL) was added 70% HClO4 (0.2 mL).
The mixture was stirred at RT temperature for 36 h before being diluted with Et2O (200 mL) and addition
of Na2CO3 (20.0 g). The resultant mixture was filtered through celite and concentrated in vacuo. The
residue was purified by flash chromatography (petroleum ether/EtOAc 4:1 → 2:1) to give SI-1 (12.6 g,
49.9 mmol, 83%) as a pale yellow solid. Data for SI-1 matched that of published data6.
mp: 58−60 °C; TLC (petroleum ether/EtOAc, 2:1 v/v): Rf = 0.30; 1H-NMR (600 MHz, CDCl3): δ 6.42
(d, J = 2.2 Hz, 1H), 6.36 (d, J = 2.2 Hz, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 3.70 (s, 2H), 3.68 (s, 3H), 2.51 (s,
3H); 13C-NMR (150 MHz, CDCl3): d 203.7, 171.7, 161.6, 159.4, 135.0, 123.7, 108.3, 97.5, 55.6, 55.4,
52.0, 39.1, 32.2; IR νmax (neat)/cm-1: 2950, 2842, 1718, 1666, 1593, 1578, 1196, 1160, 843.
25
MeO
MeOCO2Me
Ac2O, HClO4, RT
MeO
MeOCO2Me
O
83%
SI-1
22
Methyl 2-(2-acetyl-3,5-dihydroxyphenyl)acetate 26: To a solution of SI-1 (28.2 g, 111 mmol) in
CH2Cl2 (500 mL), was added AlCl3 (76.2 g, 555 mmol). The resultant mixture was stirred at RT for 16 h.
The mixture was cooled to 0 °C and quenched carefully with 1 M HCl (300 mL). The precipitate was
isolated under reduced pressure rinsing with cold H2O and cold CH2Cl2 to yield 26 (17.9 g, 79.8 mmol,
72%) as a white solid. Data for 26 matched that of published data7.
mp: 135−137 °C; TLC (petroleum ether/EtOAc, 1:1 v/v): Rf = 0.23; 1H-NMR (500 MHz, d6-acetone): δ
6.36 (d, J = 2.3 Hz, 1H), 6.32 (d, J = 2.3 Hz, 1H), 3.79 (s, 2H), 3.63 (s, 3H), 2.51 (s, 3H); 13C-NMR (125
MHz, d6-acetone): d 203.5, 172.1, 161.8, 161.5, 137.9, 119.6, 112.4, 102.8, 52.0, 40.5, 32.2; IR νmax
(neat)/cm-1: 3179, 1705, 1607, 1574, 1351, 1231, 1167, 1022, 845, 699.
MeO
MeOCO2Me
O HO
HOCO2Me
O
26
AlCl3, CH2Cl2, RT
72%
SI-1
23
Methyl 2-(2-acetyl-3,5-bis(methoxymethoxy)phenyl)acetate SI-2: To a suspension of 26 (15.0 g, 66.9
mmol) in CH2Cl2 (300 mL) at 0 °C were added MOMCl (15.2 mL, 200.7 mmol) and
diisopropylethylamine (35.0 mL, 200.7 mmol). The mixture was stirred at RT temperature for 2 h. The
mixture was quenched with 0.5 M HCl (400 mL) and extracted with CH2Cl2 (200 mL). The combined
organic layers were washed with brine (400 mL), dried over anhydrous MgSO4, filtered and concentrated
in vacuo. The residue was purified by flash chromatography (petroleum ether/EtOAc 4:1) to yield SI-2
(19.0 g, 60.8 mmol, 91%) as a colorless oil.
TLC (petroleum ether/EtOAc, 3:1 v/v): Rf = 0.15; 1H-NMR (500 MHz, CDCl3): δ 6.78 (d, J = 2.2 Hz,
1H), 6.57 (d, J = 2.2 Hz, 1H), 5.19 (s, 2H), 5.16 (s, 2H), 3.69 (s, 2H), 3.68 (s, 3H), 3.48 (s, 3H), 3.47 (s,
3H), 2.54 (s, 3H); 13C-NMR (125 MHz, CDCl3): d 203.8, 171.6, 159.0, 156.6, 134.5, 125.3, 112.1, 102.1,
94.7, 94.3, 56.4, 56.2, 51.9, 38.8, 32.2; IR νmax (neat)/cm-1: 2954, 1736, 1683, 1602, 1435, 1144, 1020,
997, 922; HRMS (ESI): m/z calculated for C15H21O7 313.1287 [M+H]+, found 313.1276.
MOMCl, i-Pr2NEt, CH2Cl2, RT
CO2Me
OHO
HO26
91%CO2Me
OMOMO
MOMO
SI-2
24
6,8-Bis(methoxymethoxy)naphthalene-1,3-diol 27: To a solution of SI-2 (12.9 g, 41.3 mmol) in DMF
(200 mL) was added NaH (60% dispersion in mineral oil, 5.87 g, 145 mmol). The mixture was stirred at
room temperature for 1 h. The mixture was quenched with 0.5 M HCl (300 mL) and extracted with
EtOAc (4 x 150 mL). The combined organic layers were washed with brine (4 x 200 mL), dried over
anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography
(petroleum ether/EtOAc 3:1) to yield 27 (9.36 g, 33.4 mmol, 81%) as a yellow solid.
mp: 93−95 °C; TLC (petroleum ether/EtOAc, 3:1 v/v): Rf = 0.11; 1H-NMR (500 MHz, CDCl3): δ 9.21
(s, 1H), 6.85 (d, J = 2.1 Hz, 1H), 6.65 (d, J = 2.2 Hz, 1H), 6.56 (d, J = 2.4 Hz, 1H), 6.39 (d, J = 2.4 Hz,
1H), 5.41 (s, 2H), 5.24 (s, 2H), 5.03 (s, 1H), 3.58 (s, 3H), 3.52 (s, 3H); 13C-NMR (125 MHz, CDCl3): d
155.9, 155.6, 155.5, 154.8, 138.3, 106.9, 103.2, 101.3, 100.2, 99.2, 95.7, 94.4, 56.9, 56.2; IR νmax
(neat)/cm-1: 3331, 1636, 1620, 1605, 1380, 1140, 1031, 905, 836; HRMS (ESI): m/z calculated for
C14H17O6 281.1025 [M+H]+, found 281.1016.
OHMOMO
MOMO OH
27
CO2Me
OMOMO
MOMO
SI-2
NaH, DMF, RT
81%
25
(E)-4-(3,7-Dimethylocta-2,6-dien-1-yl)-6,8-bis(methoxymethoxy)naphthalene-1,3-diol 29: A solution
of 27 (7.50 g, 26.7 mmol), ethyl geranyl carbonate (28) (9.06 g, 40.0 mmol) and Pd(PPh3)4 (1.56 g, 1.35
mmol) in THF (100 mL) was degassed. Et3B (1.0 M in THF, 40.0 mL, 40.0 mmol) was then added and
the resultant mixture was stirred at 50 °C for 2 h. The mixture was cooled, quenched with sat. NH4Cl
solution (100 mL) and extracted with Et2O (2 x 100 mL). The combined organic layers were washed with
brine (100 mL) dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was
purified by flash chromatography (petroleum ether/EtOAc 5:1 → 3:1) to yield 29 (3.28 g, 7.9 mmol,
29%) as a brown gum along with recovered starting material (3.46 g, 12.3 mmol, 46%).
TLC (petroleum ether/EtOAc, 3:1 v/v): Rf = 0.19; 1H-NMR (500 MHz, CDCl3): δ 9.24 (s, 1H), 7.08 (d, J
= 2.1 Hz, 1H), 6.71 (d, J = 2.1 Hz, 1H), 6.40 (s, 1H), 5.41 (s, 2H), 5.25 (s, 2H), 5.21 (t, J = 6.7 Hz, 1H),
5.06 (t, J = 6.8 Hz, 1H), 3.59 (d, J = 6.6 Hz, 1H), 3.58 (s, 3H), 3.52 (s, 3H), 2.13 – 1.99 (m, 4H), 1.88 (s,
3H), 1.66 (s, 3H), 1.58 (s, 3H); 13C-NMR (125 MHz, CDCl3): d 155.6, 155.3, 154.0, 153.3, 137.1, 136.7,
131.7, 124.0, 122.6, 109.7, 107.4, 101.1, 100.5, 98.8, 95.8, 94.6, 56.9, 56.1, 39.7, 26.6, 25.6, 24.5, 17.7,
16.3; IR νmax (neat)/cm-1: 3378, 2915, 1622, 1602, 1378, 1287, 1138, 1019, 990, 909, 816; HRMS (ESI):
m/z calculated for C24H33O6 417.2277 [M+H]+, found 417.2268.
OHMOMO
MOMO OH
OHMOMO
MOMO OH
Pd(PPh3)4, Et3BTHF, 50 °C
29%(+46% 27 recovered)
27
29
EtO2OC28
26
(E)-3,3-Dichloro-1-(3,7-dimethylocta-2,6-dien-1-yl)-5,7-bis(methoxymethoxy)-2,4-dioxo-1,2,3,4-
tetrahydronaphthalen-1-yl acetate 30: To a solution of 29 (234 mg, 0.576 mmol) in CHCl3 (8 mL) at
−40 °C was added Pb(OAc)4 (268 mg, 0.604 mmol) in small portions. The mixture was stirred at −40 °C
for 5 min before NCS (147 mg, 1.04 mmol) was added portion wise. The mixture was stirred at −40 °C
for a further 20 min before Na2S2O3 (20 mg) was added. The mixture was warmed to RT, filtered through
a short pad of SiO2 and concentrated in vacuo. The residue was purified by flash chromatography
(petroleum ether/EtOAc 5:1) to yield 30 (166 mg, 0.305 mmol, 53%) as a yellow oil.
TLC (petroleum ether/EtOAc, 3:1 v/v): Rf = 0.22; 1H-NMR (500 MHz, CDCl3): δ 6.90 (d, J = 2.2 Hz,
1H), 6.72 (d, J = 2.2 Hz, 1H), 5.30 (d, J = 6.8 Hz, 1H), 5.28 (d, J = 6.8 Hz, 1H), 5.23 (d, J = 7.0 Hz, 1H),
5.20 (d, J = 7.0 Hz, 1H), 5.02 (t, J = 6.8 Hz, 1H), 4.92 (t, J = 7.3 Hz, 1H), 3.53 (s, 3H), 3.48 (s, 3H), 2.96
(dd, J = 14.2, 8.0 Hz, 1H), 2.73 (dd, J = 14.3, 7.4 Hz, 1H), 2.14 (s, 3H), 2.04 – 1.90 (m, 4H), 1.66 (s, 3H),
1.58 (s, 3H), 1.41 (s, 3H); 13C-NMR (125 MHz, CDCl3): d 190.7, 178.5, 169.2, 162.8, 159.8, 143.4,
141.9, 131.7, 123.8, 113.7, 113.6, 105.4, 104.0, 94.9, 94.4, 82.1, 81.4, 56.7, 56.6, 40.4, 39.8, 26.0, 25.7,
20.4, 17.6, 16.3; IR νmax (neat)/cm-1: 2917, 1753, 1719, 1599, 1324, 1225, 1147, 1019, 972, 924; HRMS
(ESI): m/z calculated for C26H33Cl2O8 543.1552 [M+H]+, found 543.1554.
OHMOMO
MOMO OH
30
Pb(OAc)4, CHCl3 -40 °C; then NCS
OMOMO
MOMO O
Cl
OAc
Cl
29
53%
27
(E)-3-Chloro-1-(3,7-dimethylocta-2,6-dien-1-yl)-1,4-dihydroxy-5,7-
bis(methoxymethoxy)naphthalen-2(1H)-one 31: To a solution of 30 (681 mg, 1.25 mmol) in THF (25
mL) at −78 °C was added LDA (2.0 M solution in THF, 1.25 mL, 2.50 mmol). The mixture was stirred at
−78 °C for 1 h. The mixture was quenched with 0.5 M HCl (30 mL) and extracted with Et2O (3 x 20 mL).
The combined organic layers were washed with brine (50 mL), dried over anhydrous MgSO4, filtered and
concentrated in vacuo. The residue was purified by flash chromatography (petroleum ether/EtOAc 2:1) to
give a 3:1 inseparable mixture of SI-3 and 31 (470 mg) as a colorless oil which was used in the next step
without further purification. To a solution of the 3:1 mixture of SI-3 and 31 (470 mg) in MeOH (20 mL)
was added KOH (207 mg, 3.69 mmol). The mixture was heated at reflux for 1 h. The mixture was cooled,
quenched with 0.5 M HCl (30 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers
were washed with brine (50 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. The
residue was purified by flash chromatography (petroleum ether/EtOAc 2:1) to yield 31 (313 mg, 0.67
mmol, 53% over 2 steps) as a pale yellow solid.
mp: 66−68 °C; TLC (petroleum ether/EtOAc, 1:1 v/v): Rf = 0.19; 1H-NMR (500 MHz, CDCl3): δ 10.60
(s, 1H), 7.15 (d, J = 2.3 Hz, 1H), 6.87 (d, J = 2.3 Hz, 1H), 5.41 (s, 2H), 5.27 (d, J = 6.8 Hz, 1H), 5.21 (d, J
= 6.9 Hz, 1H), 5.07 (t, J = 6.8 Hz, 1H), 4.93 (t, J = 7.9 Hz, 1H), 3.96 (s, 1H), 3.58 (s, 3H), 3.49 (s, 3H),
2.52 (dd, J = 13.5, 8.3 Hz, 1H), 2.45 (dd, J = 13.6, 7.7 Hz, 1H), 2.06 – 1.90 (m, 4H), 1.68 (s, 3H), 1.60 (s,
3H), 1.36 (s, 3H); 13C-NMR (125 MHz, CDCl3): d 194.3, 163.6, 160.3, 155.2, 146.6, 141.3, 131.6, 124.1,
115.9, 109.1, 107.4, 105.2, 102.5, 96.5, 94.3, 79.9, 57.4, 56.5, 46.3, 39.9, 26.8, 25.6, 17.6, 15.9; IR νmax
KOH, MeOH, 60 °CLDA, THF, −78 °C
OMOMO
MOMO O
Cl
30
OAc
ClOHMOMO
MOMO O
Cl
SI-3
OAc53% over 2 steps
OHMOMO
MOMO O
Cl
31
OH
28
(neat)/cm-1: 3351, 2924, 1599, 1275, 1148, 1077, 1012, 826; HRMS (ESI): m/z calculated for C24H30ClO7
465.1686 [M−H]−, found 465.1687.
29
2-Chloro-4-((E)-3,7-dimethylocta-2,6-dien-1-yl)-4-hydroxy-6,8-bis(methoxymethoxy)-2-(3-
methylbut-2-en-1-yl)naphthalene-1,3(2H,4H)-dione 32: To a solution of 31 (324 mg, 0.694 mmol) in
DMF (15 mL) was added NaH (60% dispersion in mineral oil, 31 mg, 0.36 mmol). The mixture was
stirred at room temperature for 20 minutes before cooling to 0 °C. Prenyl bromide (0.13 mL, 0.46 mmol)
was added at 0 °C and the mixture was stirred at 0 °C for a further 2 h. The mixture was quenched with
0.5 M HCl (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers were washed
with brine (3 x 50 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was
purified by flash chromatography (petroleum ether/EtOAc 7:1) to yield 32 (205 mg, 0.38 mmol, 55%) as
a yellow oil.
TLC (petroleum ether/EtOAc, 3:1 v/v): Rf = 0.31; 1H-NMR (500 MHz, CDCl3): δ 7.03 (d, J = 2.3 Hz,
1H), 6.81 (d, J = 2.2 Hz, 1H), 5.26 (d, J = 6.8 Hz, 1H), 5.24 (s, 2H), 5.23 (d, J = 6.7 Hz, 1H), 5.08 – 5.00
(m, 2H), 4.67 (t, J = 7.6 Hz, 1H), 3.85 (s, 1H), 3.54 (s, 3H), 3.50 (s, 3H), 3.02 – 2.89 (m, 2H), 2.65 (dd, J
= 14.7, 8.4 Hz, 1H), 2.53 (dd, J = 14.6, 6.1 Hz, 1H), 2.10 – 1.95 (m, 4H), 1.70 (s, 3H), 1.60 (s, 3H), 1.53
(s, 3H), 1.47 (s, 3H), 1.46 (s, 3H); 13C-NMR (125 MHz, CDCl3): d 201.2, 188.3, 162.6, 157.8, 144.6,
141.7, 137.6, 131.7, 124.0, 115.7, 115.4, 115.3, 105.4, 103.6, 95.2, 94.2, 79.9, 70.0, 56.6, 56.5, 43.3, 39.8,
38.2, 26.4, 25.7, 25.6, 17.9, 17.7, 16.5; IR νmax (neat)/cm-1: 3481, 2915, 1735, 1698, 1599, 1576, 1296,
1224, 1143, 1019, 923, 866; HRMS (ESI): m/z calculated for C29H40ClO7 535.2463 [M+H]+, found
535.2461.
OHMOMO
MOMO O
Cl
OH
31
OMOMO
MOMO O
Cl
OH
32
NaH, DMF, 0 °C
55%
Br
30
2-Chloro-4-((E)-3,7-dimethylocta-2,6-dien-1-yl)-4,6,8-trihydroxy-2-(3-methylbut-2-en-1-
yl)naphthalene-1,3(2H,4H)-dione 33: To a solution of 32 (416 mg, 0.779 mmol) in MeOH (10 mL), was
added 32% HCl (1.0 mL) at RT. The mixture was stirred at RT for 20 h. The mixture was diluted with 1
M HCl (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with
brine, dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified by flash
chromatography (6:1 petroleum ether/EtOAc) to yield 33 (205 mg, 0.460 mmol, 59%) as a yellow oil.
TLC (petroleum ether/EtOAc, 3:1 v/v): Rf = 0.27; 1H-NMR (500 MHz, CDCl3): δ 12.01 (s, 1H), 6.75 (d,
J = 2.3 Hz, 1H), 6.59 (s, 1H), 6.38 (d, J = 2.3 Hz, 1H), 5.08 (t, J = 7.6 Hz, 1H), 5.02 (t, J = 7.6 Hz, 1H),
4.50 (t, J = 7.6 Hz, 1H), 3.92 (s, 1H), 3.13 (qd, J = 13.3, 7.8 Hz, 2H), 2.67 (qd, J = 14.5, 7.4 Hz, 2H), 2.11
– 2.01 (m, 4H), 1.72 (s, 3H), 1.61 (s, 3H), 1.56 (s, 3H), 1.52 (s, 3H), 1.42 (s, 3H); 13C-NMR (125 MHz,
CDCl3): d 200.6, 193.9, 165.1, 164.6, 146.0, 142.8, 138.9, 132.0, 123.9, 115.6, 115.0, 109.2, 106.0, 102.7,
79.3, 64.4, 45.5, 39.9, 37.5, 26.3, 25.7, 25.7, 17.9, 17.7, 16.4; IR νmax (neat)/cm-1: 3372, 2916, 1731, 1623,
1451, 1317, 1161, 1009; HRMS (ESI): m/z calculated for C25H30ClO5 445.1787 [M−H]−, found 445.1785.
33
OMOMO
MOMO O
Cl
OH
HCl, MeOH, RT
32
OHO
HO O
Cl
OH59%
31
(±)-Naphthomevalin 1: A solution of 33 (23 mg, 0.052 mmol) in PhMe (1 mL) was heated at reflux for
16 h. The solution was then cooled and concentrated in vacuo to yield (±)-naphthomevalin (1) (23 mg,
0.052 mmol, quant.) as a colorless oil.
TLC (petroleum ether/EtOAc, 3:1 v/v): Rf = 0.27; 1H-NMR (500 MHz, CDCl3): δ 11.97 (s, 1H), 7.06 (d,
J = 2.0 Hz, 1H), 6.81 (s, 1H), 6.73 (d, J = 2.0 Hz, 1H), 5.02 (t, J = 6.3 Hz, 1H), 4.94 (t, J = 7.2 Hz, 1H),
4.84 (t, J = 7.6 Hz, 1H), 4.18 (s, 1H), 3.03 – 2.93 (m, 2H), 2.52 (dd, J = 14.9, 8.0 Hz, 1H), 2.31 (dd, J =
14.6, 8.2 Hz, 1H), 2.03 – 1.85 (m, 4H), 1.71 (s, 3H), 1.59 (s, 3H), 1.57 (s, 3H), 1.31 (s, 3H), 1.30 (s, 3H);
13C-NMR (125 MHz, CDCl3): d 196.5, 195.4, 164.7, 163.4, 141.4, 138.2, 134.3, 131.8, 123.8, 116.4,
115.4, 110.5, 109.2, 107.3, 84.4, 82.9, 39.7, 38.3, 37.3, 26.3, 25.8, 25.7, 17.7, 16.1; IR νmax (neat)/cm-1:
3349, 2917, 1702, 1614, 1583, 1451, 1237, 1171, 867, 732; HRMS (ESI): m/z calculated for C25H30ClO5
445.1787 [M−H]−, found 445.1795.
(±)-naphthomevalin (1)33
PhMe, 110 °C, 16 h
OHO
HO O
Cl
OH
OHO
HOO
OH
Cl
quant.
32
(±)-Naphthomevalin 1: A supension of 33 (10 mg, 0.022 mmol) in H2O (2 mL) was heated at 60 °C and
stirred vigorously for 16 h. The solution was then cooled and diluted with H2O (5 mL) and extracted with
EtOAc (3 x 5 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous
MgSO4, filtered and concentrated in vacuo to yield (±)-naphthomevalin (1) (10 mg, 0.022 mmol, quant.)
as a colorless oil.
TLC (petroleum ether/EtOAc, 3:1 v/v): Rf = 0.27; 1H-NMR (500 MHz, CDCl3): δ 11.97 (s, 1H), 7.06 (d,
J = 2.0 Hz, 1H), 6.81 (s, 1H), 6.73 (d, J = 2.0 Hz, 1H), 5.02 (t, J = 6.3 Hz, 1H), 4.94 (t, J = 7.2 Hz, 1H),
4.84 (t, J = 7.6 Hz, 1H), 4.18 (s, 1H), 3.03 – 2.93 (m, 2H), 2.52 (dd, J = 14.9, 8.0 Hz, 1H), 2.31 (dd, J =
14.6, 8.2 Hz, 1H), 2.03 – 1.85 (m, 4H), 1.71 (s, 3H), 1.59 (s, 3H), 1.57 (s, 3H), 1.31 (s, 3H), 1.30 (s, 3H);
13C-NMR (125 MHz, CDCl3): d 196.5, 195.4, 164.7, 163.4, 141.4, 138.2, 134.3, 131.8, 123.8, 116.4,
115.4, 110.5, 109.2, 107.3, 84.4, 82.9, 39.7, 38.3, 37.3, 26.3, 25.8, 25.7, 17.7, 16.1; IR νmax (neat)/cm-1:
3349, 2917, 1702, 1614, 1583, 1451, 1237, 1171, 867, 732; HRMS (ESI): m/z calculated for C25H30ClO5
445.1787 [M−H]−, found 445.1795.
(±)-naphthomevalin (1)33
OHO
HO O
Cl
OH
OHO
HOO
OH
Cl
quant.
H2O, 60 °C, 16 h
33
(±)-Naphthomevalin 1: A solution of 33 (43 mg, 0.096 mmol) in 1:1 MeOH:H2O (2 mL) was heated at
60 °C for 40 h. The solution was then cooled and diluted with H2O (5 mL) and extracted with EtOAc (3 x
5 mL). The combined organic layers were washed with brine (10 mL), dried over anhydrous MgSO4,
filtered and concentrated in vacuo to yield (±)-naphthomevalin (1) (43 mg, 0.096 mmol, quant.) as a
colorless oil.
TLC (petroleum ether/EtOAc, 3:1 v/v): Rf = 0.27; 1H-NMR (500 MHz, CDCl3): δ 11.97 (s, 1H), 7.06 (d,
J = 2.0 Hz, 1H), 6.81 (s, 1H), 6.73 (d, J = 2.0 Hz, 1H), 5.02 (t, J = 6.3 Hz, 1H), 4.94 (t, J = 7.2 Hz, 1H),
4.84 (t, J = 7.6 Hz, 1H), 4.18 (s, 1H), 3.03 – 2.93 (m, 2H), 2.52 (dd, J = 14.9, 8.0 Hz, 1H), 2.31 (dd, J =
14.6, 8.2 Hz, 1H), 2.03 – 1.85 (m, 4H), 1.71 (s, 3H), 1.59 (s, 3H), 1.57 (s, 3H), 1.31 (s, 3H), 1.30 (s, 3H);
13C-NMR (125 MHz, CDCl3): d 196.5, 195.4, 164.7, 163.4, 141.4, 138.2, 134.3, 131.8, 123.8, 116.4,
115.4, 110.5, 109.2, 107.3, 84.4, 82.9, 39.7, 38.3, 37.3, 26.3, 25.8, 25.7, 17.7, 16.1; IR νmax (neat)/cm-1:
3349, 2917, 1702, 1614, 1583, 1451, 1237, 1171, 867, 732; HRMS (ESI): m/z calculated for C25H30ClO5
445.1787 [M−H]−, found 445.1795.
(±)-naphthomevalin (1)33
OHO
HO O
Cl
OH
OHO
HOO
OH
Cl
quant.
MeOH-H2O, 60 °C, 40 h
34
Supplementary Table 8. Conditions tested for the thermal a-hydroxyketone rearrangement of 33
Solvent Temperature Time Starting Material : Product Ratio
PhMe 110 °C 16 h 0 1
H2O RT 16 h 1 0
1:1 H2O:MeOH RT 16 h 5 1
1:1 H2O:acetone RT 16 h 10 1
1:1 H2O:DMF RT 16 h 13 1
1:1 H2O:THF RT 16 h 30 1
H2O 50 °C 16 h 2 1
MeOH 50 °C 16 h 12 1
PhMe 50 °C 16 h 12 1
H2O 60 °C 16 h 0 1
H2O 60 °C 4 h 5 1
MeOH 60 °C 16 h 1 3
No Solvent 60 °C 16 h 1 1
1:1 H2O:MeOH 60 °C 40 h 0 1
(±)-naphthomevalin (1)33
OHO
HO O
Cl
OH
OHO
HOO
OH
Clconditions
α-hydroxyketonerearrangement
35
(±)-A80915G (SI-4): To a solution of (±)-naphthomevalin (1) (31 mg, 0.069 mmol) in MeOH (2 mL) was
added NaOH (8 mg, 0.12 mmol) at RT. The mixture was stirred at RT for 1 h. The mixture was then
quenched with 1 M HCl (7 mL) and extracted with EtOAc (3 x 5 mL). The combined organics were
washed with brine (10 mL), dried over MgSO4, filtered and concentrated in vacuo. The residue was
purified by flash chromatography on SiO2 (petrol/EtOAc, 8:1 as eluent) to yield pure A80915G (SI-4) (23
mg, 0.056 mmol, 82%) as a colorless oil.
TLC (petroleum ether/EtOAc, 3:1 v/v): Rf = 0.44; 1H NMR (500 MHz, CDCl3) δ 11.82 (s, 1H), 7.05 (d,
J = 2.4 Hz, 1H), 6.63 (d, J = 2.4 Hz, 1H), 6.35 (s, 1H), 5.15 (t, J = 7.0 Hz, 1H), 5.15 (t, J = 7.0 Hz, 1H),
5.05 (t, J = 6.9 Hz, 1H), 3.24 (dd, J = 15.3, 7.1 Hz, 1H), 3.10 (dd, J = 15.3, 7.0 Hz, 1H), 2.55 (dd, J =
15.3, 6.7 Hz, 1H), 2.42 (dd, J = 15.3, 6.5 Hz, 1H), 2.10 – 1.95 (m, 4H), 1.73 (s, 3H), 1.72 (s, 3H), 1.72 (s,
3H), 1.64 (s, 3H), 1.57 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 195.4, 191.5, 164.5, 162.9, 139.0, 135.5,
134.3, 131.6, 124.0, 116.9, 116.8, 109.3, 108.8, 108.0, 67.7, 67.4, 39.7, 26.5, 25.8, 25.7, 25.5, 25.3, 18.2,
17.7, 16.6; IR νmax (neat)/cm-1: 3412, 2918, 1696, 1638, 1616, 1450, 1378, 1321, 1160 cm-1; HRMS
(ESI): m/z calculated for C25H31O5 411.2171 [M+H]+, found 411.2163.
The facile synthesis of A80915G (SI-4) from naphthomevalin (1) under mild basic conditions is
supporting evidence in favor of the relative configuration of naphthomevalin.
(±)-A80915G (SI-4)
OHO
HOO
82%
NaOH, MeOH, RT, 1 h
(±)-naphthomevalin (1)
OHO
HOO
OH
ClO
36
Synthesis of proposed biosynthetic intermediates and a-hydroxyketone rearrangement test
substrates:
(E)-1-(3,7-Dimethylocta-2,6-dien-1-yl)-4-hydroxy-5,7-bis(methoxymethoxy)-2-oxo-1,2-
dihydronaphthalen-1-yl acetate SI-5: To a solution of 29 (2.05 g, 5.04 mmol) in CHCl3 (40 mL) was
added Pb(OAc)4 (2.35 g, 5.30 mmol) portion-wise at -20 °C. The mixture was stirred at -20 °C for 5 min
before slowly warming to RT. The mixture was filtered through a short pad of SiO2 and concentrated in
vacuo. The residue was purified by flash chromatography on SiO2 (petroleum ether/EtOAc, 2:1 as eluent)
to yield SI-5 (1.40 g, 2.95 mmol, 58%) as a yellow oil.
TLC (petroleum ether/EtOAc, 1:1 v/v): Rf = 0.22; 1H-NMR (500 MHz, CDCl3) δ 9.81 (s, 1H), 6.85 (d, J
= 2.3 Hz, 1H), 6.82 (d, J = 2.3 Hz, 1H), 5.66 (s, 1H), 5.36 (s, 2H), 5.19 (d, J = 7.0 Hz, 1H), 5.17 (d, J =
7.0 Hz, 1H), 5.04 (t, J = 6.8 Hz, 1H), 4.92 (t, J = 7.4 Hz, 1H), 3.56 (s, 3H), 3.47 (s, 3H), 2.69 (dd, J =
13.5, 7.9 Hz, 1H), 2.60 (dd, J = 13.5, 7.7 Hz, 1H), 2.13 (s, 3H), 1.98 – 1.85 (m, 4H), 1.67 (s, 3H), 1.57 (s,
3H), 1.31 (s, 3H). 13C-NMR (125 MHz, CDCl3) δ 195.1, 169.2, 167.6, 159.9, 155.6, 147.0, 140.8, 131.5,
124.0, 115.0, 109.5, 107.9, 103.0, 102.2, 96.4, 94.3, 81.8, 57.2, 56.4, 40.9, 39.9, 26.8, 25.6, 21.0, 17.6,
15.9. IR νmax (neat)/cm-1: 3316, 2919, 1744, 1634, 1600, 1232, 1149, 1016, 966 HRMS (ESI): m/z
calculated for C26H35O8 475.2332 [M+H]+, found 475.2331.
OHMOMO
MOMO OH
29
Pb(OAc)4, CHCl3, −20 °C
58%
OHMOMO
MOMO OOAc
SI-5
37
(E)-1-(3,7-Dimethylocta-2,6-dien-1-yl)-1,4-dihydroxy-5,7-bis(methoxymethoxy)naphthalen-2(1H)-
one SI-6: To a solution of SI-5 (1.40 g, 2.95 mmol) in MeOH (40 mL) was added KOH (662 mg, 11.8
mmol). The mixture was heated at reflux for 2 h. The mixture was cooled, quenched with 0.5 M HCl (50
mL) and extracted with EtOAc (3 x 40 mL). The combined organic layers were washed with brine (100
mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified by flash
chromatography (petroleum ether/EtOAc, 2:1) to yield SI-6 (888 mg, 2.05 mmol, 69%) as a yellow oil.
TLC (petroleum ether/EtOAc, 1:1 v/v): Rf = 0.33; major tautomer (enol) 1H-NMR (500 MHz, CDCl3) δ
9.89 (s, 1H), 7.14 (d, J = 2.0 Hz, 1H), 6.83 (d, J = 2.0 Hz, 1H), 5.57 (s, 1H), 5.38 (s, 2H), 5.26 (d, J = 5.8
Hz, 1H), 5.21 (d, J = 6.8 Hz, 1H), 5.07 (t, J = 7.6 Hz, 1H), 5.01 (t, J = 7.8 Hz, 1H), 4.02 (s, 1H), 3.56 (s,
3H), 3.49 (s, 3H), 2.51 – 2.41 (m, 2H), 2.07 – 1.90 (m, 4H), 1.68 (s, 3H), 1.59 (s, 3H), 1.35 (s, 3H); all
peaks 13C-NMR (125 MHz, CDCl3) δ 204.1, 200.7, 188.8, 168.7, 162.7, 160.1, 159.0, 155.6, 148.8,
148.6, 140.7, 140.3, 131.8, 131.4, 124.1, 123.7, 116.8, 116.4, 109.1, 108.2, 105.9, 103.7, 102.1, 100.0,
96.3, 95.2, 94.2, 94.1, 80.4, 78.9, 57.2, 56.6, 56.5, 56.4, 50.6, 45.8, 43.0, 39.9, 39.7, 26.8, 26.3, 25.6, 25.6,
17.7, 17.6, 16.3, 15.9; IR νmax (neat)/cm-1: 3440, 3313, 1629, 1597, 1425, 1147, 1015, 964; HRMS (ESI):
m/z calculated for C34H31O7 431.2075 [M-H]-, found 431.2081.
KOH, MeOH, 60 °C
OHMOMO
MOMO O
SI-5
OAc69%
OHMOMO
MOMO
SI-6
OHO
38
(E)-1-(3,7-Dimethylocta-2,6-dien-1-yl)-1,4,5,7-tetrahydroxynaphthalen-2(1H)one SI-7: To a solution
of SI-6 (98 mg, 0.23 mmol) in MeOH (4 mL) was added 32% HCl (0.4 mL). The mixture was stirred at
room temperature for 16 h. The mixture was diluted with H2O (15 mL) and extracted with EtOAc (3 x 10
mL). The combined organic layers were washed with brine (20 mL) dried over anhydrous MgSO4, filtered
and concentrated in vacuo. The residue was purified by flash chromatography (petroleum ether/EtOAc,
2:1) to yield pure SI-7 (52 mg, 0.15 mmol, 66%) as a brown solid.
Mp: 100 – 104 °C; TLC (petroleum ether/EtOAc, 1:1 v/v): Rf = 0.07; major tautomer 1H-NMR (500
MHz, acetone-d6) δ 13.24 (s, 1H), 12.53 (s, 1H), 6.76 (d, J = 2.2 Hz, 1H), 6.23 (d, J = 2.2 Hz, 1H), 5.55
(s, 1H), 4.98 (t, J = 6.7 Hz, 1H), 4.69 (t, J = 7.4 Hz, 1H), 2.91 (dd, J = 13.1, 8.2 Hz, 1H), 2.55 (dd, J =
13.1, 7.4 Hz, 1H), 1.91 – 1.78 (m, 4H), 1.62 (s, 3H), 1.53 (s, 3H), 1.40 (s, 3H), all peaks 13C-NMR (125
MHz, acetone-d6) δ 192.7, 178.4, 166.1, 165.4, 151.2, 142.3, 133.6, 126.7, 120.0, 119.8, 119.5, 111.0,
108.7, 107.7, 107.4, 105.3, 104.7, 104.4, 104.1, 76.1, 52.6, 46.2, 45.4, 45.2, 42.4, 29.2, 29.0, 27.5, 19.5,
18.2, 17.9; IR νmax (neat)/cm-1: 3254, 2915, 1590, 1231, 1157, 1007; HRMS (ESI): m/z calculated for
C20H23O5 343.1551 [M−H]−, found 343.1161.
HCl, MeOH, RT
OHMOMO
MOMO O
SI-6
OH66%
OHHO
HO
SI-7
OHO
39
(E)-2,2-Dichloro-4-(3,7-dimethylocta-2,6-dien-1-yl)-4-hydroxy-6,8-
bis(methoxymethoxy)naphthalene-1,3(2H,4H)-dione SI-8: To a solution of SI-6 (888 mg, 2.05 mmol)
in THF (20 mL), a solution of NCS (548 mg, 4.10 mmol) in THF (20 mL) was added drop wise at −78
°C. The mixture was stirred at −78 °C for 20 min before Na2S2O3 (50 mg) was added. The mixture was
warmed to RT, filtered through celite and concentrated in vacuo. The residue was purified by flash
chromatography (petroleum ether/EtOAc, 6:1) to yield SI-8 (771 mg, 1.54 mmol, 75%) as a colorless oil.
TLC (petroleum ether/EtOAc, 3:1 v/v): Rf = 0.25; 1H-NMR (500 MHz, CDCl3) δ 7.07 (d, J = 2.2 Hz,
1H), 6.88 (d, J = 2.2 Hz, 1H), 5.33 (d, J = 6.8 Hz, 1H), 5.28 (d, J = 6.8 Hz, 1H), 5.28 – 5.24 (m, 2H), 5.04
(t, J = 6.9 Hz, 1H), 4.99 (t, J = 6.9 Hz, 1H), 3.75 (s, 1H), 3.52 (s, 3H), 3.50 (s, 3H), 2.66 (dd, J = 14.8, 8.5
Hz, 1H), 2.48 (dd, J = 14.8, 6.2 Hz, 1H), 2.09 – 1.97 (m, 4H), 1.71 (s, 3H), 1.60 (s, 3H), 1.47 (s, 3H); 13C-
NMR (125 MHz, CDCl3) δ 195.0, 178.0, 163.4, 159.3, 144.2, 142.9, 131.9, 123.8, 114.8, 111.9, 105.7,
103.8, 94.8, 94.2, 79.8, 78.8, 56.7, 56.6, 43.1, 39.8, 26.3, 25.7, 17.7, 16.5; IR νmax (neat)/cm-1: 3464, 2918,
1751, 1715, 1598, 1574, 1296, 1225, 1144, 1019, 923, 796; HRMS (ESI): m/z calculated for C24H29Cl2O7
499.1296 [M-H]-, found 499.1305.
NCS, THF, −78 °C
OHMOMO
MOMO O
SI-6
OH75%
OMOMO
MOMO
SI-8
OHO
ClCl
40
(E)-2,2-Dichloro-4-(3,7-dimethylocta-2,6-dien-1-yl)-4,6,8-trihydroxynaphthalene-1,3(2H,4H)-dione
35: To a solution of SI-8 (167 mg, 0.335 mmol) in MeOH (8 mL) was added 32% HCl (0.80 mL). The
mixture was stirred at RT for 16 h. The mixture was diluted with H2O (15 mL) and extracted with EtOAc
(3 x 10 mL). The combined organic layers were washed with brine (15 mL), dried over anhydrous
MgSO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (petroleum
ether/EtOAc 8:1 → 4:1) to yield 35 (64 mg, 0.155 mmol, 47%) as a colorless oil.
TLC (petroleum ether/EtOAc, 3:1 v/v): Rf = 0.19; 1H-NMR (500 MHz, CDCl3): δ 11.70 (s, 1H), 6.95 (s,
1H), 6.82 (d, J = 2.4 Hz, 1H), 6.46 (d, J = 2.4 Hz, 1H), 5.07 (t, J = 6.5 Hz, 1H), 5.00 (t, J = 7.0 Hz, 1H),
3.86 (s, 1H), 2.73 (dd, J = 14.6, 7.0 Hz, 1H), 2.65 (dd, J = 14.6, 6.1 Hz, 1H), 2.15 – 2.01 (m, 4H), 1.72 (s,
3H), 1.62 (s, 3H), 1.53 (s, 3H); 13C-NMR (125 MHz, CDCl3): d 193.9, 186.2, 166.6, 166.0, 145.7, 143.7,
132.2, 123.7, 115.0, 106.9, 105.4, 103.4, 79.5, 77.9, 45.5, 39.8, 26.2, 25.7, 17.7, 16.5; IR νmax (neat)/cm-1:
3376, 2918, 1748, 1621, 1585, 1453, 1309, 1258, 1164, 856; HRMS (ESI): m/z calculated for
C20H23Cl2O5 413.0923 [M+H]+, found 413.0898
HCl, MeOH, RT
47%
35
OHO
HO OOH
ClCl
SI-8
OMOMO
MOMO OOH
ClCl
41
(E)-3,3-Dichloro-2-(3,7-dimethylocta-2,6-dien-1-yl)-2,5,7-trihydroxy-2,3-dihydronaphthalene-1,4-
dione 36: A solution of 35 (35 mg, 0.084 mmol) in PhMe (1 mL) was heated at reflux for 5 h. The
solution was then cooled and concentrated in vacuo. The residue was purified by flash chromatography
(petroleum ether/EtOAc 5:1) to yield 36 (24 mg, 0.058 mmol, 68%) as a colorless oil.
TLC (petroleum ether/EtOAc, 3:1 v/v): Rf = 0.29; 1H-NMR (500 MHz, CDCl3): δ 11.46 (s, 1H), 7.03 (d,
J = 2.0 Hz, 1H), 6.75 (d, J = 1.9 Hz, 1H), 6.64 (s, 1H), 6.56 (s, 1H), 5.00 (t, J = 6.7 Hz, 1H), 4.87 (t, J =
6.8 Hz, 1H), 4.44 (s, 1H), 2.81 (dd, J = 14.5, 6.2 Hz, 1H), 2.37 (dd, J = 14.2, 8.5 Hz, 1H), 2.03 – 1.83 (m,
4H), 1.70 (s, 3H), 1.59 (s, 3H), 1.24 (s, 3H); 13C-NMR (125 MHz, CDCl3): d 193.7, 186.9, 165.7, 163.9,
141.6, 133.7, 131.9, 123.6, 114.9, 109.4, 108.5, 107.7, 90.4, 85.7, 39.7, 36.3, 26.2, 25.7, 17.7, 16.1; IR
νmax (neat)/cm-1: 3377, 2919, 1712, 1656, 1613, 1580, 1350, 1248, 1172, 1095, 810; HRMS (ESI): m/z
calculated for C20H23Cl2O5 413.0923 [M+H]+, found 413.0914
35
OHO
HOO
OH
OHO
HO OOH
36
ClCl
ClClPhMe, 110 °C
68%
42
(E)-3-Chloro-1-(3,7-dimethylocta-2,6-dien-1-yl)-1,4,5,7-tetrahydroxynaphthalen-2(1H)-one 34: To a
solution of 31 (326 mg, 0.698 mmol) in MeOH (8 mL), was added 32% HCl (0.8 mL). The mixture was
stirred at RT for 16 h. The mixture was diluted with H2O (20 mL) and extracted with EtOAc (3 x 15 mL).
The combined organic layers were washed with brine (30 mL), dried over anhydrous MgSO4, filtered and
concentrated in vacuo. The residue was triturated with cold CH2Cl2 to yield 34 (163 mg, 0.430 mmol,
59%) as an off white solid.
mp: 172−175 °C; TLC (CH2Cl2:MeOH, 6:1 v/v): Rf = 0.05; 1H-NMR (500 MHz, d6-acetone): δ 13.02 (s,
1H), 6.77 (d, J = 2.3 Hz, 1H), 6.27 (d, J = 2.3 Hz, 1H), 4.99 (t, J = 6.9 Hz, 1H), 4.62 (t, J = 7.5 Hz, 1H),
2.94 (dd, J = 13.1, 8.6 Hz, 1H), 2.52 (dd, J = 13.1, 7.4 Hz, 1H), 1.93 – 1.75 (m, 4H), 1.62 (s, 3H), 1.54 (s,
3H), 1.38 (s, 3H); 13C-NMR (125 MHz, d6-acetone): d 183.7, 164.0, 163.7, 148.1, 141.4, 131.8, 124.9,
118.0, 117.2, 108.6, 105.9, 102.6, 76.3, 44.4, 40.6, 27.5, 25.7, 17.6, 16.0; IR νmax (neat)/cm-1: 3340, 3159,
2925, 1645, 1604, 1587, 1455, 1323, 1206, 1147, 1009, 829; HRMS (ESI): m/z calculated for C20H22ClO5
377.1161 [M−H]−, found 377.1170.
HCl, MeOH, RT
59%
34
OHHO
HO OOH
Cl
31
OHMOMO
MOMO OOH
Cl
43
4. Computational Methods and Data
Molecular geometries, energies, and free energies of reactants, products, and transition states were
computed by density functional theory using the Gaussian 09 (Revision D.01) software package8.
Geometry optimizations of reactants and products were initiated from approximate global minimum-
energy structures identified by molecular dynamics simulated annealing with the classical Amber
classical force field9 using Gabedit (version 2.4.7)10. Geometries were optimized in the gas phase at the
M06-2X/6-31G(d) level11. The optimized geometries were identified as stable or transition states by the
number of imaginary frequencies (0 or 1, respectively) in gas-phase vibrational-frequency calculations at
the M06- 2X/6-31G(d) level. The steepest-descent path from each of the transition states identified was
computed12,13 at the same level of theory to verify that the transition state connected the correct reactant
and product states. Single-point gas-phase energy calculations of the optimized geometries were carried
out at the M06-2X/6-311+G(d,p) level11. Unscaled zero-point energies and thermodynamic corrections
obtained from the frequency calculations at the M06-2X/6-31G(d) level were added to these energies to
compute the gas-phase ZPE-corrected free energy, ∆Egas, and reaction Gibbs free energy at 298.15 K and
1 atm, ∆Ggas. The Gibbs free energy of solvation in toluene, computed using the SMD continuum
solvation model14 at the M06-2X/6-31(d) level, was added to ∆Ggas to compute the reaction Gibbs free
energy change in solution at 298.15 K and 1 mol/L concentration, ∆Gsoln. A similar procedure was used to
compute the gas-phase activation energy, ∆E‡gas, gas-phase activation free energy, ∆G‡
gas, and activation
free energy in toluene, ∆G‡soln.
The geometries of all species optimized at the M06-2X/6-31G(d) level are given in Cartesian coordinates
(in units of Angstrom) below. Listed below the coordinates of each species are the following energies and
free energies (in units of Hartree):
• M06-2X/6-31G(d) electronic potential energy (EM06-2X/6-31G(d))
• M06-2X/6-31G(d) zero-point energy (ZPEM06-2X/6-31G(d))
44
• Thermal correction to M06-2X/6-31G(d) energy at 298.15 K, which comprises translational,
rotational, and vibrational energies, assuming an ideal gas and the harmonic oscillator–rigid rotor
model (∆Ethermal,M06-2X/6-31G(d))
• Thermal correction to M06-2X/6-31G(d) enthalpy at 298.15 K (∆Hthermal,M06-2X/6-31G(d))
• Thermal correction to M06-2X/6-31G(d) Gibbs free energy at 298.15 K and 1 atm (∆Gthermal,M06-
2X/6-31G(d))
• M06-2X/6-311+G(d,p) electronic potential energy (EM06-2X/6-311+G(d,p))
• Sum of M06-2X/6-31G(d) electronic potential energy and solvation free energy in toluene at
298.15 K and 1 mol/L (GM06-2X/6-31G(d),toluene)
The electronic circular dichroism (ECD) spectra of compounds 1, 10, and 33 were calculated as the
weighted average of the spectra of 5 different conformers of each compound (weighted by exp(-∆Gsoln
/kBT), where ∆Gsoln is the free energy of solvation, calculated using the methods described above). The
geometries of the conformers were obtained by DFT energy minimization (using the method described
above) of structures extracted from a high-temperature (1000 K) classical molecular dynamics simulation
using the AMBER force field9. The ECD spectra were calculated using time-dependent density functional
theory (TD-DFT) in Gaussian 09 (Revision D.01). The CAM-B3LYP functional15, which has previously
been shown to model ECD spectra well for organic compounds16, was used, along with the 6-311+G(d,p)
basis set. The methanol solvent used to measure the experimental spectrum was modelled implicitly using
the integral equation formalism polarizable continuum model (IEFPCM)17. 25 excited states (singlets
only) were used in each calculation. The ECD spectrum was constructed from the calculated excitation
energies and rotatory strengths using a Gaussian line broadening of 0.333 eV. To compare with the
experimental spectra, the calculated ECD spectra were scaled vertically to match the peak heights in the
experimental spectra approximately. As TD-DFT does not generally predict excitation energies with
quantitative accuracy better than tenths of an eV, the spectra were shifted along the wavelength axis by an
45
amount determined by matching the peak positions of the calculated and experimental UV/vis absorption
spectra (by 35 nm, 32 nm, and 25 nm for compounds 1, 10, and 33, respectively).
The reactions calculated are as follows:
OHO
HO O
Cl
O
Cl
OHHO
HO O
Cl
OH
OHO
HOO
OH
ClCl
PhMe, 110 °Cα-ketol
rearrangement
34
OHO
HO O
Cl
O
H
H
ΔE‡gas = +36.3 kcal mol-1ΔG‡gas = +37.8 kcal mol-1ΔG‡soln = +36.9 kcal mol-1
ΔEgas = −10.0 kcal mol-1ΔG°gas = −9.1 kcal mol-1ΔG°soln = −8.6 kcal mol-1
68%
OHO
HOO
OH
ClPhMe, 110 °C
α-ketol rearrangement
ΔE‡gas = +36.3 kcal mol-1ΔG‡gas = +38.0 kcal mol-1ΔG‡soln = +37.1 kcal mol-1
ΔEgas = −7.1 kcal mol-1ΔG°gas = −5.8 kcal mol-1ΔG°soln = −5.4 kcal mol-1
quant.
OHHO
HOO
OH
Cl
PhMe, 110 °C no reaction
ΔE‡gas = +34.0 kcal mol-1ΔG‡gas = +35.6 kcal mol-1ΔG‡soln = +35.7 kcal mol-1
ΔEgas = +11.8 kcal mol-1ΔG°gas = +12.0 kcal mol-1ΔG°soln = +11.4 kcal mol-1
36
37
(±)-naphthomevalin (1)
35
33
46
Compound 33 (prenyl) C 0.00000000 0.00000000 0.00000000 C 0.00000000 0.00000000 1.39290901 C 1.20965578 0.00000000 2.07532285 C 2.43789775 -0.02062611 1.39192049 C 2.43554892 0.00510561 0.01355678 C 1.22690444 0.05399948 -0.70983354 H 3.38090024 -0.06027348 1.92823240 H -0.93786867 -0.01748508 1.93461864 C 1.21651219 0.18789799 -2.16544843 C 2.44459279 0.83263872 -2.82036449 C 3.72971916 0.72859835 -1.99061801 C 3.72986330 -0.15296741 -0.74995624 O -1.18153998 -0.02816153 -0.62609839 O 1.15459139 -0.00280658 3.42455768 O 4.73663705 1.30948301 -2.32022375 O 4.81047403 0.18969371 0.06763495 H 5.53213252 0.44150302 -0.53624045 C 3.88198952 -1.63417023 -1.24534599 H 2.99532855 -1.92203955 -1.82236630 H 3.89411742 -2.24405015 -0.33653879 C 5.12462641 -1.78646046 -2.07185393 H 4.99730789 -1.60971287 -3.14062381 C 6.36944709 -1.99601042 -1.62518211 C 6.73738333 -2.24907650 -0.18731299 H 7.33619574 -3.16439626 -0.11175778 H 5.86898989 -2.34101102 0.46603679 H 7.35491486 -1.43280816 0.20692079 C 7.52109925 -1.88926647 -2.59386840 H 7.15181874 -1.99136013 -3.62005559 H 8.25306304 -2.69096879 -2.43112023 C 8.22457662 -0.51752284 -2.46935108 H 8.76208601 -0.46355378 -1.51572228 H 7.44833061 0.25705763 -2.43812810 C 9.17840670 -0.25999797 -3.60528308 H 10.22299976 -0.52061878 -3.43661091 C 8.83514381 0.21525120 -4.80707517 C 9.85781841 0.40794657 -5.89530858 H 10.86040856 0.12464828 -5.56430167 H 9.88576529 1.45488310 -6.22175479 H 9.60479150 -0.19067352 -6.77917826 C 7.42626105 0.58361729 -5.19467327 H 7.38799564 1.61536732 -5.56453795 H 6.70820912 0.49320436 -4.37598107 H 7.08206119 -0.05581567 -6.01756704 O 0.23090697 -0.05726153 -2.84874624 C 2.15866498 2.34918440 -3.01731390 H 2.05199964 2.78126908 -2.01549440 H 3.07798332 2.76231271 -3.44444795 C 0.97916268 2.66436072 -3.88680645 H 1.16226721 2.61190997 -4.95806439 C -0.25024822 2.98002590 -3.46704741 C -0.67800717 3.03597590 -2.02519532 H 0.13058589 2.83640407 -1.31769583 H -1.46507562 2.29592656 -1.83731511 H -1.10332241 4.01816111 -1.78710658 C -1.34859902 3.27494867 -4.45274086 H -0.99203524 3.22548936 -5.48425062 H -1.77266880 4.27122629 -4.27813090 H -2.16815561 2.55558180 -4.33676068 Cl 2.75775412 0.05223797 -4.40765750 H -1.00853418 -0.12349107 -1.58951861 H 2.05358082 -0.00807587 3.78404505
EM06-2X/6-31G(d) -1807.00614010 ZPEM06-2X/6-31G(d) 0.519865 ΔEthermal,M06-2X/6-31G(d) 0.552720 ΔHthermal,M06-2X/6-31G(d) 0.553664 ΔGthermal,M06-2X/6-31G(d) 0.453740 EM06-2X/6-311+G(d,p) -1807.43490088 GM06-2X/6-31G(d),toluene -1807.03255879
47
Transition state 33→1 (naphthomevalin): C 0.00000000 0.00000000 0.00000000 C 0.00000000 0.00000000 1.39732386 C 1.19947905 0.00000000 2.09684703 C 2.42844375 -0.00899058 1.41951956 C 2.42316462 0.00708693 0.03519608 C 1.22351172 0.03833317 -0.71044878 H 3.37294461 -0.03118882 1.95588878 H -0.94260602 -0.00927848 1.93142773 C 1.22196538 0.10339020 -2.18423136 C 2.52574786 0.49753117 -2.88529884 C 3.77412990 0.00757514 -2.15194559 C 3.66847077 0.01513879 -0.71225334 O -1.18402189 -0.00758619 -0.61444301 O 1.12676802 -0.00248880 3.44469910 O 4.95526413 0.28540714 -2.57010933 O 4.83108680 0.08705800 -0.15145977 H 5.40035713 0.22787680 -1.02480368 C 3.60282698 -1.86831301 -1.93762505 H 3.24104506 -1.98083579 -2.96195462 H 2.84560344 -2.21124383 -1.23390500 C 4.95953941 -2.37682495 -1.74272845 H 5.61200763 -2.31002710 -2.61066818 C 5.49524908 -2.79033374 -0.58029617 C 4.74615738 -2.90217616 0.71843129 H 4.68834184 -3.95252104 1.02978728 H 3.72972076 -2.50495491 0.67162148 H 5.28247569 -2.36399304 1.50894689 C 6.96680676 -3.11095478 -0.51008207 H 7.32663255 -3.48673629 -1.47420341 H 7.15760655 -3.89363047 0.23542783 C 7.78368726 -1.85082418 -0.14575767 H 7.38265403 -1.41797144 0.77961473 H 7.62027699 -1.10410039 -0.93016027 C 9.24469660 -2.15833287 0.02942927 H 9.52988983 -2.54210637 1.00978785 C 10.20098009 -2.05660761 -0.89938081 C 11.62744295 -2.43082065 -0.59357616 H 11.74578802 -2.77228821 0.43799291 H 12.29653005 -1.57598472 -0.75195110 H 11.97288416 -3.22841349 -1.26286924 C 9.96971962 -1.57899223 -2.30870370 H 10.57951991 -0.69159621 -2.51804694 H 8.92707694 -1.33210422 -2.51657499 H 10.28289474 -2.34953780 -3.02366305 O 0.18937844 -0.04123510 -2.82540252 C 2.60359261 2.05558998 -2.90070256 H 2.62655667 2.38462845 -1.85466888 H 3.58125124 2.28525572 -3.33506581 C 1.49369502 2.71516409 -3.66340130 H 1.63054465 2.75640394 -4.74186786 C 0.35834970 3.20411993 -3.15298872 C -0.00977645 3.18019440 -1.69264270 H 0.82576814 2.93106650 -1.03410205 H -0.80543246 2.44444280 -1.51549855 H -0.40527797 4.15374035 -1.38127502 C -0.69286560 3.80362679 -4.04813859 H -0.39609727 3.77074813 -5.09906946 H -0.89038363 4.84707211 -3.77422764 H -1.64063581 3.26206187 -3.94114033 Cl 2.51872683 -0.09875635 -4.57199417 H -1.01621559 -0.06932121 -1.58449874 H 2.01839676 -0.00015614 3.82183229
EM06-2X/6-31G(d) -1806.94442280 ZPEM06-2X/6-31G(d) 0.516946 ΔEthermal,M06-2X/6-31G(d) 0.549021 ΔHthermal,M06-2X/6-31G(d) 0.549966 ΔGthermal,M06-2X/6-31G(d) 0.452822 EM06-2X/6-311+G(d,p) -1807.37335289 GM06-2X/6-31G(d),toluene -1806.97227434
48
Compound 1 (naphthomevalin): C 0.00000000 0.00000000 0.00000000 C 0.00000000 0.00000000 1.39631952 C 1.20015222 0.00000000 2.09668981 C 2.42695580 0.00332829 1.41691933 C 2.42141268 -0.02320980 0.03613754 C 1.22657489 -0.01754843 -0.70948482 H 3.37507081 0.00993877 1.94710991 H -0.94373098 0.00509950 1.92917764 C 1.23031491 0.01848319 -2.18290961 C 2.58493121 0.21334633 -2.87418335 C 3.68837876 -0.57837287 -2.12615825 C 3.71834759 -0.06871026 -0.68244619 O -1.18356452 0.03464572 -0.61585755 O 1.13171096 -0.00546387 3.44489463 O 4.93861217 -0.35752921 -2.70723371 O 4.77222824 0.24782231 -0.16803003 H 5.51539265 -0.01910865 -1.99769530 C 3.38765728 -2.10360422 -2.09809753 H 3.69240513 -2.47646288 -3.08068286 H 2.31255127 -2.27814534 -1.99964164 C 4.16153869 -2.76964496 -0.99786939 H 5.24535395 -2.72511717 -1.11351595 C 3.67592081 -3.29624695 0.13272380 C 2.21484322 -3.43478516 0.47313703 H 1.95294084 -4.49515071 0.57194331 H 1.54717670 -2.98939054 -0.26709362 H 1.99545724 -2.96342549 1.43884359 C 4.63293398 -3.73762100 1.21506419 H 5.62583951 -3.92163377 0.79025590 H 4.29766645 -4.67727771 1.67422542 C 4.77103572 -2.65660979 2.30901377 H 3.79230705 -2.49962848 2.78396349 H 5.03569218 -1.71359066 1.81824738 C 5.78126599 -3.03026383 3.35584281 H 5.44355171 -3.75539138 4.09752994 C 7.04676822 -2.60489481 3.43445297 C 7.96277277 -3.08880600 4.52802035 H 7.45804907 -3.78657219 5.20110449 H 8.33824389 -2.24680747 5.12273212 H 8.84160402 -3.59168452 4.10617975 C 7.68570172 -1.63789649 2.47181436 H 8.04450186 -0.74798890 3.00354464 H 7.01813826 -1.30954064 1.67255756 H 8.56599262 -2.09925146 2.00773679 O 0.19169800 -0.03287903 -2.82653534 C 2.91413449 1.72860029 -2.89541291 H 2.99889348 2.06815611 -1.85530041 H 3.90827299 1.81120428 -3.34511398 C 1.91088503 2.55409141 -3.65074927 H 2.05842961 2.59511162 -4.72765068 C 0.84734443 3.18007962 -3.13610264 C 0.46913236 3.17974093 -1.67737374 H 1.27347762 2.84583333 -1.01729792 H -0.39443083 2.52117932 -1.51094740 H 0.16777403 4.18430406 -1.36027386 C -0.11076211 3.92647581 -4.02545539 H 0.18492013 3.87103474 -5.07567661 H -0.17091805 4.98238917 -3.73532536 H -1.12188415 3.51234983 -3.93035645 Cl 2.47944224 -0.38166756 -4.56214684 H -1.01764896 -0.01486007 -1.58614914 H 2.02627831 -0.01004094 3.81605891
EM06-2X/6-31G(d) -1807.01800958 ZPEM06-2X/6-31G(d) 0.521238 ΔEthermal,M06-2X/6-31G(d) 0.553404 ΔHthermal,M06-2X/6-31G(d) 0.554348 ΔGthermal,M06-2X/6-31G(d) 0.456528 EM06-2X/6-311+G(d,p) -1807.44687272 GM06-2X/6-31G(d),toluene -1807.04389948
49
Compound 35 (dichloride): C 0.00000000 0.00000000 0.00000000 C 0.00000000 0.00000000 1.39249041 C 1.20715571 0.00000000 2.07576038 C 2.43604062 -0.02381892 1.39186438 C 2.44179905 -0.01737360 0.01487484 C 1.23240183 0.01775026 -0.71688655 H 3.37665700 -0.04296756 1.93322871 H -0.93988553 0.00337649 1.93068755 C 1.19562340 0.14718927 -2.16233469 C 2.50629461 0.50462619 -2.90908665 C 3.76541933 0.59429417 -2.02451153 C 3.76132630 -0.17187781 -0.70508151 O -1.18259876 0.00445049 -0.61175228 O 1.15365703 0.00849194 3.42236524 O 4.74649822 1.18640689 -2.38856084 O 4.78728864 0.32598769 0.10553848 H 5.55095635 0.46881275 -0.48124494 C 4.02331422 -1.68675857 -1.01199316 H 3.18460649 -2.09831835 -1.58343799 H 4.02841586 -2.17832264 -0.03448551 C 5.31622744 -1.85581501 -1.75378102 H 5.24426070 -1.79305965 -2.83998244 C 6.54067586 -1.96769556 -1.22175000 C 6.83490387 -2.06741103 0.25166107 H 7.38941410 -2.99052568 0.45790783 H 5.93725379 -2.04962680 0.87028676 H 7.47302659 -1.23741816 0.57803523 C 7.74474537 -1.92000678 -2.13057421 H 7.44824235 -2.16464436 -3.15654791 H 8.49746134 -2.65866705 -1.82592757 C 8.38238213 -0.51194745 -2.13663536 H 8.79338863 -0.29032877 -1.14482937 H 7.58492339 0.21916048 -2.31685655 C 9.46672083 -0.38245659 -3.17246351 H 10.48325554 -0.59089291 -2.83933894 C 9.27783653 -0.07861429 -4.46081612 C 10.43205344 -0.00159179 -5.42441557 H 11.38376383 -0.22596930 -4.93577638 H 10.50061949 0.99870400 -5.86956234 H 10.29607301 -0.70628971 -6.25400170 C 7.92898509 0.19936680 -5.07158182 H 7.92467154 1.17824720 -5.56580740 H 7.11060031 0.18370186 -4.34793701 H 7.70722722 -0.54357348 -5.84805770 O 0.17783609 0.05300947 -2.83295695 Cl 2.81454199 -0.77437605 -4.12496106 H -1.03290076 -0.01870104 -1.58335894 H 2.05209479 0.01647664 3.78353941 Cl 2.25459975 2.06847978 -3.71017736
EM06-2X/6-31G(d) -2071.31237811 ZPEM06-2X/6-31G(d) 0.390979 ΔEthermal,M06-2X/6-31G(d) 0.418500 ΔHthermal,M06-2X/6-31G(d) 0.419444 ΔGthermal,M06-2X/6-31G(d) 0.330635 EM06-2X/6-311+G(d,p) -2071.72100736 GM06-2X/6-31G(d),toluene -2071.33682074
50
Transition state 35→36 (dichloride): C 0.00000000 0.00000000 0.00000000 C 0.00000000 0.00000000 1.39922018 C 1.19717977 0.00000000 2.09895191 C 2.42852114 -0.01472756 1.42084098 C 2.42491544 0.00066114 0.03886188 C 1.22421357 0.03300444 -0.70689081 H 3.37182411 -0.04307857 1.95892991 H -0.94441469 -0.00690289 1.93026111 C 1.22274419 0.08878543 -2.17465759 C 2.55521321 0.45050646 -2.87867836 C 3.79920383 -0.05409553 -2.14347244 C 3.67379106 -0.01975574 -0.70366073 O -1.18511550 -0.01516308 -0.60693553 O 1.12694910 -0.00332139 3.44569691 O 4.97862836 0.16711621 -2.57301950 O 4.83074450 0.00696118 -0.13066694 H 5.42604644 0.11930340 -0.97585168 C 3.53539422 -1.92511733 -1.92697394 H 3.15651038 -2.04056077 -2.94489882 H 2.77827352 -2.23392795 -1.20778189 C 4.87703900 -2.47518740 -1.74262782 H 5.52114316 -2.43574273 -2.61819772 C 5.41547551 -2.89251177 -0.58252136 C 4.68145514 -2.97244178 0.72677604 H 4.62349939 -4.01510587 1.06200971 H 3.66592111 -2.57248465 0.68194020 H 5.22876099 -2.41838352 1.49858886 C 6.88043203 -3.24562463 -0.53245398 H 7.20786057 -3.66912016 -1.48833196 H 7.07327497 -4.00065317 0.23993760 C 7.73278118 -1.98929152 -0.24267405 H 7.38600390 -1.52704490 0.69009817 H 7.54123120 -1.26103265 -1.03895868 C 9.19832329 -2.31145459 -0.13749330 H 9.55677461 -2.58781124 0.85431679 C 10.07524205 -2.33721333 -1.14658288 C 11.51732934 -2.70642298 -0.91937199 H 11.71924651 -2.92492838 0.13238320 H 12.18107121 -1.89227156 -1.23556811 H 11.79283588 -3.58624552 -1.51388627 C 9.72808839 -2.01697748 -2.57660144 H 10.32992031 -1.17509678 -2.93960599 H 8.67492273 -1.76718439 -2.72120971 H 9.96371432 -2.87178735 -3.22241033 O 0.21027283 -0.06268083 -2.83676990 Cl 2.54256219 -0.07607447 -4.56407313 H -1.03796509 -0.05975948 -1.57717948 H 2.01860872 0.01319065 3.82260888 Cl 2.61043985 2.25313620 -2.81318831
EM06-2X/6-31G(d) -2071.25080387 ZPEM06-2X/6-31G(d) 0.388132 ΔEthermal,M06-2X/6-31G(d) 0.415009 ΔHthermal,M06-2X/6-31G(d) 0.415953 ΔGthermal,M06-2X/6-31G(d) 0.329646 EM06-2X/6-311+G(d,p) -2071.65973548 GM06-2X/6-31G(d),toluene -2071.27672947
51
Compound 36 (rearranged dichloride): C 0.00000000 0.00000000 0.00000000 C 0.00000000 0.00000000 1.39733317 C 1.19913525 0.00000000 2.09726793 C 2.42784038 -0.01145226 1.41733742 C 2.42470670 -0.04476823 0.03922074 C 1.22859869 -0.02278622 -0.70656938 H 3.37505828 -0.01802448 1.94847034 H -0.94410919 0.00823495 1.92913128 C 1.23273905 0.01016009 -2.17217681 C 2.61521768 0.12185423 -2.86569458 C 3.68903281 -0.69307552 -2.10410456 C 3.72604444 -0.15007988 -0.66842730 O -1.18403324 0.02169002 -0.61124201 O 1.13189379 -0.00302453 3.44409213 O 4.94359746 -0.56416956 -2.68257651 O 4.78650317 0.13480243 -0.15619102 H 5.49040288 -0.04287619 -2.06773169 C 3.29774307 -2.19588956 -2.07872645 H 3.64058455 -2.59719572 -3.03759806 H 2.21135798 -2.31283619 -2.04368463 C 3.96425406 -2.89008175 -0.92674390 H 5.05381554 -2.90319953 -0.97327089 C 3.37188096 -3.38844061 0.16529492 C 1.88508342 -3.43615946 0.40305177 H 1.57145139 -4.46866012 0.59635857 H 1.29434641 -3.05684386 -0.43256749 H 1.61301460 -2.85012814 1.29004946 C 4.22172745 -3.89244437 1.30802257 H 5.22591064 -4.14635670 0.95092083 H 3.79113674 -4.80660760 1.73722664 C 4.36257202 -2.82880844 2.41794174 H 3.36476895 -2.56664577 2.79625659 H 4.77334469 -1.92133445 1.96102974 C 5.22236774 -3.30416839 3.55580434 H 4.71893349 -3.91430880 4.30650762 C 6.53514668 -3.09494373 3.70091480 C 7.28912770 -3.65730806 4.87715829 H 6.63386191 -4.21633891 5.55006235 H 7.76926978 -2.85529484 5.45133253 H 8.09063852 -4.32674029 4.54168459 C 7.38297407 -2.31038402 2.73399194 H 7.86232478 -1.46436316 3.24148509 H 6.82443490 -1.92189479 1.87983745 H 8.19250689 -2.94158368 2.34772771 O 0.21360377 -0.02161262 -2.84043557 Cl 2.50486897 -0.40930436 -4.54741495 H -1.03371721 0.00665017 -1.58185641 H 2.02617241 -0.00670226 3.81655303 Cl 3.05481715 1.86646450 -2.80800222
EM06-2X/6-31G(d) -2071.32843109 ZPEM06-2X/6-31G(d) 0.391513 ΔEthermal,M06-2X/6-31G(d) 0.418765 ΔHthermal,M06-2X/6-31G(d) 0.419709 ΔGthermal,M06-2X/6-31G(d) 0.332382 EM06-2X/6-311+G(d,p) -2071.73718503 GM06-2X/6-31G(d),toluene -2071.35216888
52
Compound 34 (chloroenol): C 0.00000000 0.00000000 0.00000000 C 0.00000000 0.00000000 1.39286800 C 1.19856203 0.00000000 2.09110813 C 2.42314900 -0.02771649 1.41106513 C 2.42778409 -0.03617245 0.03157501 C 1.22648971 -0.01661881 -0.71143762 H 3.36791762 -0.02700362 1.94698312 H -0.94544630 0.01758416 1.92125292 C 1.31810037 0.09973674 -2.16176807 C 2.49780877 0.29328878 -2.81865600 C 3.75009677 0.39621470 -2.09534795 C 3.74239901 -0.19823413 -0.68776142 O -1.21593677 0.02284567 -0.57667256 O 1.13119497 0.02290045 3.44115850 O 4.79434804 0.81268848 -2.56453013 O 4.78386697 0.36209319 0.05904915 H 5.48657025 0.54971594 -0.59111089 C 3.99627450 -1.74359323 -0.86561728 H 3.13572115 -2.19120709 -1.37426332 H 4.02484648 -2.14367183 0.15232519 C 5.26521509 -2.00090942 -1.61927692 H 5.16981128 -2.04350686 -2.70438898 C 6.50074681 -2.06165238 -1.10699420 C 6.82532607 -1.99358507 0.36205836 H 7.48566504 -2.82398945 0.63894483 H 5.93814691 -2.02148054 0.99573482 H 7.36094665 -1.06680803 0.59968990 C 7.68580382 -2.10416564 -2.03971018 H 7.35807363 -2.38530591 -3.04662083 H 8.41891109 -2.85403242 -1.71357836 C 8.36972707 -0.72081882 -2.12533711 H 8.81811891 -0.47494666 -1.15526057 H 7.59167363 0.02801635 -2.31282350 C 9.42831479 -0.67656637 -3.19187833 H 10.41770322 -1.02899253 -2.89851300 C 9.25263095 -0.29529891 -4.46142632 C 10.39075546 -0.32369071 -5.44743546 H 10.15656079 -0.97894272 -6.29565899 H 11.31778128 -0.67555187 -4.98711077 H 10.57091596 0.67555010 -5.86292685 C 7.94256245 0.18274800 -5.03164282 H 7.64405768 -0.45632632 -5.87214227 H 8.04969011 1.19716465 -5.43532826 H 7.12408661 0.19159441 -4.30865650 O 0.14849615 0.05119166 -2.83299343 H 0.31024832 0.18611386 -3.78453445 Cl 2.50523577 0.56851656 -4.53611482 H -1.12844280 0.02908958 -1.54272408 H 2.02768701 0.03065252 3.80619005
EM06-2X/6-31G(d) -1611.74860185 ZPEM06-2X/6-31G(d) 0.401235 ΔEthermal,M06-2X/6-31G(d) 0.427768 ΔHthermal,M06-2X/6-31G(d) 0.428713 ΔGthermal,M06-2X/6-31G(d) 0.341378 EM06-2X/6-311+G(d,p) -1612.12979305 GM06-2X/6-31G(d),toluene -1611.77197847
53
Transition state 34→37 (chloroenol): C 0.00000000 0.00000000 0.00000000 C 0.00000000 0.00000000 1.38434609 C 1.20374212 0.00000000 2.09362390 C 2.42577271 -0.01690343 1.42783178 C 2.43820572 -0.02884597 0.04152177 C 1.23621465 -0.01267854 -0.71880225 H 3.37877977 -0.00440233 1.94992457 H -0.94505397 0.01349975 1.91350048 C 1.31936534 0.03979807 -2.15268157 C 2.52691818 0.07743757 -2.82038934 C 3.71863806 0.07202417 -2.08013774 C 3.74776459 -0.10482361 -0.63574543 O -1.21049418 0.00843202 -0.58862355 O 1.11326846 0.02031239 3.44149426 O 4.90297199 0.24456266 -2.60359402 O 4.87059876 0.11960232 -0.07940043 H 5.47146399 0.27607752 -1.77004763 C 3.67212194 -2.07348067 -1.03162096 H 2.72076135 -2.30346952 -1.50902420 H 3.66602694 -2.27092850 0.03787590 C 4.87125093 -2.45009351 -1.74413787 H 4.78216922 -2.54747334 -2.82617497 C 6.11005026 -2.55518530 -1.21200589 C 6.43555746 -2.43290561 0.24992377 H 7.04891070 -3.28649821 0.56538939 H 5.54737836 -2.37266544 0.87948470 H 7.01008148 -1.52079064 0.43906991 C 7.28399440 -2.78444809 -2.12721258 H 6.93081909 -3.04577693 -3.13104422 H 7.89323832 -3.62476085 -1.76694750 C 8.18290081 -1.53218443 -2.24025833 H 8.64266011 -1.32125854 -1.26874386 H 7.54594285 -0.67251168 -2.47629034 C 9.25513482 -1.70042325 -3.28248496 H 10.20833217 -2.09895715 -2.93503832 C 9.12070669 -1.44760486 -4.58841305 C 10.25638988 -1.67583476 -5.55055352 H 9.97402814 -2.40006461 -6.32461904 H 11.14961004 -2.04750350 -5.04185151 H 10.51874421 -0.74606292 -6.07035082 C 7.85512103 -0.92922463 -5.22079648 H 7.50488598 -1.62994595 -5.98902979 H 8.04373208 0.02360542 -5.73054469 H 7.04089516 -0.77820932 -4.50824568 O 0.15490091 0.04454646 -2.84154408 H 0.33467889 0.09265802 -3.79700759 Cl 2.57016323 0.16760759 -4.55898360 H -1.11527458 0.02244216 -1.55399267 H 2.00437207 0.02981121 3.82022647
EM06-2X/6-31G(d) -1611.68977585 ZPEM06-2X/6-31G(d) 0.398616 ΔEthermal,M06-2X/6-31G(d) 0.424640 ΔHthermal,M06-2X/6-31G(d) 0.425585 ΔGthermal,M06-2X/6-31G(d) 0.340679 EM06-2X/6-311+G(d,p) -1612.07241626 GM06-2X/6-31G(d),toluene -1611.71292480
54
Compound 37 (rearranged chloroenol): C 0.00000000 0.00000000 0.00000000 C 0.00000000 0.00000000 1.39436089 C 1.18890450 0.00000000 2.11843479 C 2.40452440 -0.01200025 1.44354221 C 2.39747810 -0.03305890 0.05444214 C 1.21909590 -0.00423151 -0.71146955 H 3.35833569 -0.01533894 1.96504852 H -0.93860476 -0.00058698 1.94218890 C 1.32180955 0.04790435 -2.18659335 C 2.49767597 -0.19473045 -2.79539549 C 3.73093282 -0.63561862 -2.05426335 C 3.70628890 -0.06211226 -0.64295646 O -1.16884200 -0.00992086 -0.67816041 O 1.09032981 0.00243571 3.47001139 O 4.89884132 -0.22381539 -2.70535905 O 4.74614184 0.27734847 -0.11324583 H 5.49978740 0.08012475 -2.00123464 C 3.72775819 -2.19637819 -1.91552426 H 3.80958546 -2.56829702 -2.94361101 H 2.75965242 -2.51305966 -1.51819736 C 4.87660069 -2.66136052 -1.07536207 H 5.86112550 -2.48627418 -1.50885820 C 4.81860497 -3.15693912 0.16656285 C 3.54777983 -3.44905931 0.92163212 H 3.57804984 -4.46719692 1.32835140 H 2.64868215 -3.35486519 0.31014401 H 3.43622904 -2.76986151 1.77539562 C 6.09261949 -3.37291312 0.94372180 H 6.95398378 -3.24435681 0.27921107 H 6.14436209 -4.39426623 1.34507219 C 6.21714035 -2.36134875 2.10787936 H 5.52425106 -2.63814397 2.91080259 H 5.89454679 -1.37958817 1.73807441 C 7.61893183 -2.28639281 2.65309568 H 7.83702627 -2.88591654 3.53667340 C 8.61603299 -1.58037419 2.11018007 C 9.99976955 -1.58806022 2.70321234 H 10.73609630 -1.94630448 1.97308799 H 10.05646204 -2.22597386 3.58916671 H 10.30735523 -0.57429617 2.98832103 C 8.46268445 -0.73799442 0.87017523 H 9.10179023 -1.12783696 0.06756183 H 8.79289665 0.29037535 1.06128513 H 7.43602826 -0.70081152 0.49689964 O 0.18830755 0.36714131 -2.84391821 H 0.38320948 0.37600888 -3.79588523 Cl 2.59214540 -0.18406843 -4.53313403 H -1.89842345 -0.00742983 -0.04202238 H 1.97808366 0.02574206 3.85529796
EM06-2X/6-31G(d) -1611.72817700 ZPEM06-2X/6-31G(d) 0.400395 ΔEthermal,M06-2X/6-31G(d) 0.427201 ΔHthermal,M06-2X/6-31G(d) 0.428146 ΔGthermal,M06-2X/6-31G(d) 0.341136 EM06-2X/6-311+G(d,p) -1612.11040003 GM06-2X/6-31G(d),toluene -1611.75254900
55
Compound 10 (rearranged dichloride): C 0.00000000 0.00000000 0.00000000 C 0.00000000 0.00000000 1.39645381 C 1.19962228 0.00000000 2.09633034 C 2.42902487 -0.01650832 1.41652550 C 2.42576140 -0.01869569 0.03707902 C 1.22899702 0.00493091 -0.70659457 H 3.37709072 -0.03289665 1.94693023 H -0.94393719 0.00459192 1.92845587 C 1.22826216 0.04973675 -2.17089860 C 2.59912976 0.24413459 -2.86270883 C 3.71405031 -0.54631708 -2.13451477 C 3.72900898 -0.07103313 -0.67480727 O -1.18488855 0.00192097 -0.60988890 O 1.12825442 0.00741056 3.44280857 O 4.95261362 -0.28672861 -2.71447649 O 4.78117821 0.22600125 -0.15236881 H 5.46982141 0.22448142 -2.06529026 C 3.39934968 -2.07176100 -2.18668227 H 3.87611871 -2.43277560 -3.10639835 H 2.32494727 -2.22459785 -2.30952049 C 3.94481464 -2.83517576 -0.99885225 C 3.16744573 -3.37618361 -0.04369574 C 1.66066095 -3.29913144 0.00106533 H 1.33611434 -2.74980517 0.89551542 H 1.23872333 -4.30785939 0.08345876 H 1.20206565 -2.81711086 -0.86191085 C 3.71518282 -4.15580692 1.12715817 H 4.79803882 -4.27629267 1.11425272 H 3.26762224 -5.15665720 1.15179216 O 0.21006761 -0.02829675 -2.83744467 Cl 2.50837144 -0.23232951 -4.56179193 H -1.03555051 -0.01924441 -1.58031147 H 2.02053889 0.02263798 3.81863201 Cl 2.95610002 2.00444468 -2.74452974 H 3.43549763 -3.66730495 2.06979831 C 5.45887666 -2.98521675 -1.03885804 H 5.87180707 -3.06933699 -0.02963261 H 5.90020736 -2.08582717 -1.47311592 C 5.82946273 -4.20465464 -1.84557842 C 6.41157614 -4.24275576 -3.04950754 C 6.70278600 -5.56921162 -3.71045530 H 6.32598458 -6.39003932 -3.09007181 H 6.17103092 -5.62477062 -4.67195536 C 6.84551105 -3.02373432 -3.82623280 H 6.34314924 -2.10871761 -3.50518955 H 7.92774109 -2.86751478 -3.73681652 H 6.63559576 -3.16333129 -4.89337469 C 8.20461788 -5.79520997 -3.97170619 H 8.74632229 -5.69646880 -3.02571479 H 8.57727528 -4.99939321 -4.62961466 C 8.45816478 -7.12488420 -4.62232814 C 9.04012438 -8.20557070 -4.09218738 C 9.18932592 -9.47537060 -4.88968058 H 10.24520744 -9.76116606 -4.97352116 H 8.67623212 -10.30934891 -4.39466123 H 8.78134481 -9.37213778 -5.89836323 C 9.59357032 -8.28828110 -2.69401322 H 9.46554542 -7.36724808 -2.12365610 H 9.10443155 -9.09956282 -2.14101512 H 10.66371334 -8.52768612 -2.71997360 H 8.08623153 -7.20904201 -5.64523133 H 5.54500395 -5.15268492 -1.38565902 EM06-2X/6-31G(d) -2266.572065
ZPEM06-2X/6-31G(d) 0.511104 ΔEthermal,M06-2X/6-31G(d) 0.544206 ΔHthermal,M06-2X/6-31G(d) 0.545151 ΔGthermal,M06-2X/6-31G(d) 0.443838 EM06-2X/6-311+G(d,p) -2267.032326
56
5. Supplementary Figures
Supplementary Figure 1 ½ pH screen for optimization of the production of 10 by Mcl24. Reversed-
phase HPLC chromatograms (254 nm) of assays conducted using a range of pH conditions (6.5-8.0)
tested for maximization of production of 10 by Mcl24. A pH of 8.0 was determined to be required for
maximal production. Blue band indicates compound 10.
57
Supplementary Figure 2 ½ Experimental and calculated circular dichroism spectra of 10. The
spectrum of isolated 10 (expt) is indicative of an enantiopure chiral molecule. This implies the enzymatic
activity of Mcl24, in particular the α-hydroxyketone rearrangement, is enantiospecific. The calculated
spectrum (calc) was obtained using time-dependent density functional theory (TD-DFT). The vertical axis
has been scaled so that the maximum in the calculated spectrum matches that in the experimental
spectrum and the horizontal axis has been red-shifted by 32 nm. Most importantly, the relative positions
of the positive and negative lobes in the experimental spectrum are correctly predicted, confirming the
assigned absolute configuration of the molecule.
58
Supplementary Figure 3 ½ Incorporation of 18OH2 into 10. a, Mass spectrum of product 10 of the
Mcl24 reaction in unlabeled 16OH2. The observed m/z ([M-H]-) of 479.142 is consistent with the
calculated m/z ([M-H]-) of 479.147. The isotope distribution pattern is consistent with a dichlorinated
compound displaying an ([M-H]- + 2) peak in ~64% abundance to that of the [M-H]- peak. b, Mass
spectrum of product 10 of the Mcl24 reaction in labeled 18OH2 (~97% enriched). The major observed m/z
([M-H]-) of 481.142 is consistent with the calculated m/z ([M-H]-) of 481.142 if oxygen incorporation
originates from 18OH2. In addition, the compound still exhibits the dichlorinated isotope distribution as
previously described. To be certain 18O-incorporation was a result of enzymatic catalysis and not due to
the exchange of carbonyl oxygen atoms with water through hydration, 10 isolated from the unlabeled
reaction was incubated in the same reaction components, without enzyme, resuspended in 18OH2. After
incubation, reisolated 10 displayed no trace of 18O-incorporation, with a mass spectrum and isotope
distribution almost identical to that observed in panel a.
Mcl24H2O2
OH
OH
HO
HO
2
4
pre-merochlorin (9)
HO O
HOO
OH
ClCl
10
18OH2
a b
m/z m/z
Rel
ativ
e ab
unda
nce
Rel
ativ
e ab
unda
nce
59
Supplementary Figure 4 ½ UV/Visible and mass spectra of the substrate, product, and synthetic
standard in the NapT8 in vitro assay. The UV/Visible and mass spectra of the major product of the
NapT8 reaction are identical to those of the synthetic standard 33. a, racemic substrate 34 (trace I, Fig.
6a), observed m/z ([M-H]-): 377.120, calculated m/z ([M-H]-): 377.123. The isotope distribution is
consistent with monochlorination, with an ([M-H]- + 2) peak ~32% the intensity of the ([M-H]-) peak. b,
0
200
400
600
800
1000
200 250 300 350 400 450 500
0
500
1000
1500
2000
200 250 300 350 400 450 500
Wavelength (nm)
Wavelength (nm)
Abso
rban
ce (m
Au)
Abso
rban
ce (m
Au)
m/z
Rel
ativ
e ab
unda
nce
a
b
m/z
Rel
ativ
e ab
unda
nce
0
500
1000
1500
2000
200 250 300 350 400 450 500Wavelength (nm)
Abso
rban
ce (m
Au)
c
m/z
Rel
ativ
e ab
unda
nce
m/z ([M-H]-): 377.1204
m/z ([M-H]-): 445.1811
m/z ([M-H]-): 445.1796
60
NapT8 product 33 (trace II, Fig. 6a), observed m/z ([M-H]-): 445.181, calculated m/z ([M-H]-): 445.186.
The isotope distribution is consistent with monochlorination, with an ([M-H]- + 2) peak ~32% the
intensity of the ([M-H]-) peak. c, synthetic 33 (trace III, Fig. 6a), observed m/z ([M-H]-): 445.180,
calculated m/z ([M-H]-): 445.186. The isotope distribution is consistent with monochlorination, with an
([M-H]- + 2) peak ~32% the intensity of the ([M-H]-) peak.
61
Supplementary Figure 5 ½ UV/Visible and mass spectra of the product and synthetic
naphthomevalin (1) in the coupled NapH3 in vitro assay. The UV/Visible and mass spectra of the
major product of the NapH3 reaction are identical to those of synthetic naphthomevalin (1). a, NapH3
major product 1 (trace IV, Fig. 6a), observed m/z ([M-H]-): 445.180, calculated m/z ([M-H]-): 445.186.
The isotope distribution is consistent with monochlorination, with an ([M-H]- + 2) peak ~32% the
intensity of the ([M-H]-) peak. b, synthetic naphthomevalin (1) (trace V, Fig. 6a), observed m/z ([M-H]-):
445.180, calculated m/z ([M-H]-): 445.186. The isotope distribution is consistent with monochlorination,
with an ([M-H]- + 2) peak ~32% the intensity of the ([M-H]-) peak.
0
500
1000
1500
2000
200 250 300 350 400 450 500
0
500
1000
1500
2000
200 250 300 350 400 450 500
Wavelength (nm)
Wavelength (nm)
Abso
rban
ce (m
Au)
Abso
rban
ce (m
Au)
a
bm/z
Rel
ativ
e ab
unda
nce
m/z
Rel
ativ
e ab
unda
nce
m/z ([M-H]-): 445.1804
m/z ([M-H]-): 445.1804
62
Supplementary Figure 6 ½ UV/Visible and mass spectra of the product and napyradiomycin A1 (2)
standard in the coupled NapH1 in vitro assay. The UV/Visible and mass spectra of the major product
of the NapH3 reaction are identical to those of napyradiomycin A1 (2) a, NapH1 major product 2 (trace
VI, Fig. 6a), observed m/z ([M-H]-): 479.147, calculated m/z ([M-H]-): 479.147. The isotope distribution
is consistent with dichlorination, with an ([M-H]- + 2) peak ~64% the intensity of the ([M-H]-) peak. b,
isolated napyradiomycin A1 standard (2) (trace VII, Fig. 6a), observed m/z ([M-H]-): 479.146, calculated
m/z ([M-H]-): 479.147. The isotope distribution is consistent with dichlorination, with an ([M-H]- + 2)
peak ~64% the intensity of the ([M-H]-) peak.
0
500
1000
1500
2000
200 250 300 350 400 450 500
0
500
1000
1500
2000
200 250 300 350 400 450 500
Wavelength (nm)
Wavelength (nm)
Abso
rban
ce (m
Au)
Abso
rban
ce (m
Au)
a
bm/z
Rel
ativ
e ab
unda
nce
m/z
Rel
ativ
e ab
unda
nce
m/z ([M-H]-): 479.1472
m/z ([M-H]-): 479.1460
63
Supplementary Figure 7 ½ Reaction requirements for NapT8 activity. Overlaid reversed-phase HPLC
chromatograms (254 nm) comparing the production of 33 in the standard NapT8 assay with the substrate
34 and: no enzyme (control), MgCl2 omitted (using DMAPP as the isoprene substrate), or when the
isoprene/terpene was varied between DMAPP, IPP, or GPP as described in the Supplementary
Information. Activity was only observed when DMAPP was used as the isoprene substrate (red trace),
and activity is reproducibly diminished when omitting MgCl2 in the reaction (purple trace), alluding to a
possible Mg2+-dependence of the enzyme as frequently observed for ABBA prenyltransferase enzymes.
There was no activity observed when the enzyme was assayed with the substrate THN (8) and either
DMAPP or GPP as the isoprene/terpene (data not shown).
0
100
200
300
400
500
600
700
800
21 21.2 21.4 21.6 21.8 22
no MgCl2
IPPGPP
no enzyme
DMAPP
Time (min)
Abso
rban
ce (m
Au)
64
Supplementary Figure 8 ½ In vitro assays of NapH3 with synthetic 33. The capacity of NapH3 to
catalyzed an α-hydroxyketone rearrangement on synthetic 33 was analyzed. Reversed-phase HPLC
chromatograms (254 nm) of assays containing only compound 33 (21.8 min)(no enzyme), 33 and NapH3,
and naphthomevalin (1) standard (20.8 min). Clear production of naphthomevalin (1) from 33 is observed
with the addition of NapH3. The reaction went to completion within 2 h, and the remaining synthetic 33
is the non-utilized enantiomer (see Supplementary Figure 13). The naphthomevalin (1) present in the 33
control reaction is a result of the non-enzymatic reaction described in the manuscript over the course of
the 2 h incubation and the purification of compound 33 in aqueous conditions.
Time (min)18 19 20 21 22 23 24
33 control
NapH3 + 33
naphthomevalin (1)control
65
Supplementary Figure 9 ½ In vitro assays of NapH3 with other synthetic substrates. The capacity of
NapH3 to catalyzed an α-hydroxyketone rearrangement on various synthetic, C4-prenylated substrates
was analyzed. For each compound indicated, no NapH3 activity was detected further demonstrating the
necessity of prenylation at C2 for turnover. Reversed-phase HPLC chromatogram (254 nm) of an assay
containing: I) SI-7 control (14.0 min). II) SI-7 and NapH3. III) 34 control (15.0 min). IV) 34 and NapH3.
V) 35 control (18.4 min). VI) 35 and NapH3. The peak at 15.0 min in traces I and II is an uncharacterized,
non-chlorinated compound as determined by LC-MS. However, the peak at 15.0 min found in traces V
and VI is compound 34 formed through spontaneous dechlorination.
12 14 16 18 20 22 24Time (min)
I
II
III
VI
V
IV
34
OHHO
HO OOH
Cl
35
OHO
HO OOH
ClCl
SI-7
OHHO
HO OOH
66
Supplementary Figure 10 ½ Coupled in vitro assays of NapT8 and NapH1/Mcl24. The capacity of
NapH1 or Mcl24 to catalyze an α-hydroxyketone rearrangement on (33) was analyzed. No activity was
detected for either NapH1 or Mcl24 when assayed with 33 produced by NapT8. Reversed-phase HPLC
chromatogram (254 nm) of an assay containing: I) 34 control (~15.0 min); II) 34 and NapT8, product of
the reaction 33 elutes at ~21.8 min; III) 34 and NapT8, followed by NapH1; IV) 34 and NapT8, followed
by Mcl24. The compound eluting at ~18.7 min is an uncharacterized, dichlorinated compound formed
through a reaction of Mcl24 with remaining 34. A minor amount of naphthomevalin was present in traces
II and IV (~20.8 min), due to the slow, non-enzymatic α-hydroxyketone rearrangement described in the
manuscript, that was equal to the amount present in control reactions. In trace III, this was converted to a
minor amount of napyradiomycin A1 (2) found at ~23.0 min by NapH1.
12 14 16 18 20 22 24
IV
III
II
I
Time (min)
67
Supplementary Figure 11 ½ Comparison of the initial velocities for the NapH3 catalyzed and non-
enzymatic conversion of 33 to naphthomevalin (1). The conversion of 33 to the product
naphthomevalin (1) monitored over time for both the NapH3 catalyzed and the non-enzymatic reaction.
Under the conditions described in the methods, the rate of the NapH3 catalyzed reaction was 0.374 ±
0.021 µM/min, while the non-enzymatic rate was 0.041 ± 0.002 µM/min, with an approximate order of
magnitude enzymatic rate enhancement.
2
4
6
8
10
12
14
16
18
0 50 100 150 200 250Time (min)
Nap
htho
mev
alin
Pro
duct
(µM
)
non-enz.NapH3
68
Supplementary Figure 12 ½ Experimental and calculated circular dichroism spectra of 33. The
spectrum of 33 isolated from the enzymatic reaction of NapT8 and 34 (expt) is indicative of an
enantiopure chiral molecule. This implies the enzymatic activity of NapT8 is enantiospecific. The
calculated spectrum (calc) was obtained using time-dependent density functional theory (TD-DFT). The
vertical axis has been scaled so that the maximum in the calculated spectrum matches that in
the experimental spectrum and the horizontal axis has been red-shifted by 25 nm. Most importantly, the
relative positions of the positive and negative lobes in the experimental spectrum are correctly predicted,
confirming the assigned absolute configuration of the molecule. Furthermore, the CD spectrum of
unreacted 33 recovered from the reaction between racemic 33 and NapH3 (expt (recovered)) is the
inverse of the enzymatically produced 33.
33
OHO
HO O
Cl
OH
OHHO
HO O
Cl
OH
34
NapT8
prenylation
69
Supplementary Figure 13 ½ Experimental and calculated circular dichroism spectra of 1. The
spectrum of 1 isolated from the enzymatic reaction of NapH3 and 33 (expt) is indicative of an enantiopure
chiral molecule. This implies the enzymatic activity of NapH3 is enantiospecific. The calculated spectrum
(calc) was obtained using time-dependent density functional theory (TD-DFT). The vertical axis has been
scaled so that the maximum in the calculated spectrum matches that in the experimental spectrum and the
horizontal axis has been red-shifted by 35 nm. Most importantly, the relative positions of the positive and
negative lobes in the experimental spectrum are correctly predicted, confirming the assigned absolute
configuration of the molecule. Furthermore, the CD spectrum of unreacted 1 recovered from the reaction
between racemic 1 and NapH1 (expt (recovered)) is the inverse of the enzymatically produced 1.
naphthomevalin (1)33
OHO
HOO
OH
Cl
OHO
HO O
Cl
OH
NapH3
α-hydroxyketonerearrangement
70
Supplementary Figure 14 ½ Multiple sequence alignment of VHPO homologs. An alignment of the
three VHPO homologs from the napyradiomycin cluster (NapH1, 3, and 4) in Streptomyces sp. CNQ-525
and the two present in the merochlorin cluster (Mcl24 and 40) in Streptomyces sp. CNH-189. The serine
residue found to be critical for chlorination activity in NapH1 (highlighted in blue), is mutated to a
phenylalanine residue in NapH3. Sequence alignment was performed using ClustalX18.
71
napH3 sequence used in this study:
GTGACGACATCCGCCCCTGCCCAGCAGATTCCGTTCGACTTCGACAACGGCAACTTCATCCGGGACCTGATCACCACGCACGGTGGCGGAGGTTACCCGCCGGCGGATGCGATGGCTCCGGGGGATGTGTCCTCGTACACGTGGGTGACGCATCTGCTGCAGACGTCGTGGTTCGACGCGCTGGCCCCGTACCACCCGACCGCGGTCGGCGTGTACTCCCGGATCCCGCGCCGTCCCGCCGAGGAGTCGGCCACCAACCGGAACAAGAACATCGCGGGCCTTTACGCCATGTTCCAGGTGGTGAAGGCGGCGTTCACGGAGCGGGTGCCGGTCCTGCGGCAGGCGCTGGGGGCGCTGGGCCTGGACCCTGACGACGAGTCGCAGGACCTGTCCACCGCGGTCGGTATCGGCAACACGGCCGGCAAGGCCGTCGCCGCCGCCCGTATGGGGGACGGCATGAACGCCCTGGGCGGCAAGGACCGCACCCACAACGGCCAGCCCTACGAGGACTACACCGGCTACCGGCCGGTGAACACCGCCGACGAACTCGTCGACCCCTCGCGCTGGCAGCCCGCCGTCGAGCCGCACCGCCGCCGCACCGACGGCGGCCCGGGCGACAAGGGCATCTTCACCGCCCAGCGGTTCGCCACCCCGCAACTGGGCCTGGTCGCCCCCCAGACGTACCGGGACCCCGCCCGGTTCAAGCTCGCCGCGCCCGACCACCTCGACCACAACGACGCCGGCGCCTACCGGCAGGCGGTGGACGAGGTGCTCGCGGCGTCGGCCGGGCTGACCGACGAGCAGAAGGTCAAGGCGGAGTTCTTCGAGCACACCCCGCTGTCGGTCACGCTGTCGCCGCGCGCCGCGGCGATGGCGCACGACCTGGACCTGGACGGCTGGGCGCAGCTGTTCCTGGTGTGCTCGACCGCACGGTTCGACAGCCTGATCGCCGCCTGGCACCACAAGCGCGCCTACGACACCGTGCGGCCCTTCAGCGCCGTGCGGCACGTGTACGGCAGCAAGCCGGTCACCGCCTGGGGCGGGCCCGGCAAGGGCACCGTCGAGTCCATTCCCGCCGACGAGTGGACCGGCTACCTGCCCGTGGGCAACCACCCCGAGTACCCCTCCGGCTTCACCACCTTGATCGCGGCCCAGGCGCAGGCCGCCCGCAGCTTCCTCGGCGACGACGTCCTGAACTGGACCCATGCCTTCCCCGCCGGCTCCGGCCAGCGGGAGCCCGGCGCGGTCCCCGCCTCCGACCTCGAACTGACCTGGGCCACCTGGACCGACTTCGAGAACGACTGCGCCACCAGCCGCGTATGGGCCGGCGCCCACTTCACGAAGACCGCCGAAACCTCCCTGGCCTTCGGCACCCAGTTCGGCGACCTGGCCCACACCTTCGTCCAGCGGCACATCAACGGCGACGTCAAGGACTGA napT8 sequence used in this study:
ATGACTGACACAGGCATGGAAGGTCTTTACGCCGCCATCGAGGAGGCGTCCGGGTTGTTGGACGTAGCCCCCTCGCGTGACAAGGTGTGGCCGATCCTGTCCGCGTACGAGTTGGACAAGGTCGTTGTGGCCTTCCGCGTGACGACGCGCGGCAGCAAGGACCTCGACTGCCGTTTCACGGCGCTGCCGGCGGACGTCAACCCGTACCGCTACGCGGTGTCGAAGGGGATCGCCGAGGCCACGGACCATCCTGTCGGTACGCTCCTGGACGATGTCCAGGCGAACATCCCGGTGACCGCCCACGGTGTCGACTTCGGAGTCGTCGAGGGCTTCAGGAAGACCTGGACGTTCCTGCCGGGCAACGATCTGCAGAAGCTGTCGAAGGTCGCGGCGCTGCCGTCCATGCCGCCGAGCCTGGCCGAGAACCTCGACTTCTACGCCCGCTACGGCCTGGATGACAAGAACAGCATGATCGGGATCGACTACCCGAGCCGGACGGTGAACGTCTACTTCCTGCAGTTCCCCGACGAGACCCGCGAGCCGGAGACCGTCCGGGCCATGCTGCGGGATCTGGGGCTGCCGGAGCCGAGCGAACAGATGCTGACGCTCGCCAAGCAGGCCGTGGGCATCTACACCACTCTGACGTGGGACTCGCCGAAGATCCAGCGGATCACGTTCGCCACCCTGGTCCCCGACGCCGAGGCCCTGCCCGGCCGCATCGCGGTGGAGCCGAGCGTGGAGAAGTTCGCGAGGAACGTCCCGCACACCTACCCCGGTCCGGTCCAGGGCCTGTACAACGTGGCCTCGTACTCCGGCGGCGAGTACTTCAAGCTCCAGACCTACCACCAGCTTGCCGAGGGCTCGCTGGAGGCGCGGGTCCTGCTGGGCGCGGCGGGCGCCGGCAGCTGA Supplementary Figure 15 ½ Gene sequences used in this study as predicted by Genemark. Putative
open reading frames in the regions of the originally annotated napH3 and napT8 genes were predicted
using GeneMark4. The expressed open reading frames from 21833 bp to 23629 bp (encoding NapH3) and
19734 to 20642 bp (encoding NapT8) in the Streptomyces sp. CNQ-525 nap cluster resulted in soluble,
functional protein used in this study.
72
Supplementary Figure 16 ½ SDS-PAGE gels (12%) of purified NapH3 and NapT8. The bands are
consistent with the predicted molecular weights of His6-NapH3 (53.8 kDa), and His6-NapT8 (35.2 kDa).
Both proteins were judged to be >90% pure based on band intensities.
20015012010085
7060
50
40
30
25
20
15
NapT8
Std. 1 μg 5 μg 10 μg20015012010085
7060
50
40
30
25
20
15
Std. 1 μg 5 μg 10 μg
NapH3
(kDa) (kDa)
73
6. NMR and Compound Characterization
74
75
76
77
78
79
80
81
82
83
84
85
MeO
MeOCO2Me
O
SI-1600 MHz 1H
in CDCl3
MeO
MeOCO2Me
O
SI-1150 MHz 13C
in CDCl3
86
HO
HOCO2Me
O
26500 MHz 1H
in d6-acetone
HO
HOCO2Me
O
26125 MHz 13Cin d6-acetone
87
MOMO
MOMOCO2Me
O
SI-2500 MHz 1H
in CDCl3
MOMO
MOMOCO2Me
O
SI-2125 MHz 13C
in CDCl3
88
500 MHz 1Hin CDCl3
OHMOMO
MOMO OH27
125 MHz 13Cin CDCl3
OHMOMO
MOMO OH27
89
500 MHz 1Hin CDCl3
OHMOMO
MOMO OH
29
125 MHz 13Cin CDCl3
OHMOMO
MOMO OH
29
90
500 MHz COSYin CDCl3
OHMOMO
MOMO OH
29
500 MHz HSQCin CDCl3
OHMOMO
MOMO OH
29
91
500 MHz HMBCin CDCl3
OHMOMO
MOMO OH
29
OMOMO
MOMO OH
H
diagnostic HMBCcorrelations
92
500 MHz 1Hin CDCl3
30
OMOMO
MOMO O
Cl
OAc
Cl
125 MHz 13Cin CDCl3
30
OMOMO
MOMO O
Cl
OAc
Cl
93
500 MHz COSYin CDCl3
30
OMOMO
MOMO O
Cl
OAc
Cl
500 MHz HSQCin CDCl3
30
OMOMO
MOMO O
Cl
OAc
Cl
94
500 MHz HMBCin CDCl3
30
OMOMO
MOMO O
Cl
OAc
Cl
OMOMO
MOMO OOAc
ClCl
diagnostic HMBCcorrelations
95
500 MHz 1Hin CDCl3
OHMOMO
MOMO O
Cl
OH
31
125 MHz 13Cin CDCl3
OHMOMO
MOMO O
Cl
OH
31
96
500 MHz COSYin CDCl3
OHMOMO
MOMO O
Cl
OH
31
500 MHz HSQCin CDCl3
OHMOMO
MOMO O
Cl
OH
31
97
500 MHz HMBCin CDCl3
OHMOMO
MOMO O
Cl
OH
31
OMOMO
MOMO OO
Cl
H
H
diagnostic HMBCcorrelations
98
500 MHz 1Hin CDCl3
OMOMO
MOMO O
Cl
OH
32
125 MHz 13Cin CDCl3
OMOMO
MOMO O
Cl
OH
32
99
500 MHz 1Hin CDCl3
OHO
HO O
Cl
OH
33
125 MHz 13Cin CDCl3
OHO
HO O
Cl
OH
33
100
500 MHz COSYin CDCl3
OHO
HO O
Cl
OH
33
500 MHz HSQCin CDCl3
OHO
HO O
Cl
OH
33
101
500 MHz HMBCin CDCl3
OHO
HO O
Cl
OH
33
OHO
HO O
Cl
OH
diagnostic HMBCcorrelations
102
500 MHz 1Hin CDCl3
(±)-naphthomevalin (1)
OHO
HOO
OH
Cl
125 MHz 13Cin CDCl3
(±)-naphthomevalin (1)
OHO
HOO
OH
Cl
103
500 MHz COSYin CDCl3
(±)-naphthomevalin (1)
OHO
HOO
OH
Cl
500 MHz HSQCin CDCl3
(±)-naphthomevalin (1)
OHO
HOO
OH
Cl
104
500 MHz HMBCin CDCl3
(±)-naphthomevalin (1)
OHO
HOO
OH
Cl
500 MHz NOESYin CDCl3
(±)-naphthomevalin (1)
OHO
HOO
OH
Cl
OHO
HOO
OH
Cl
diagnostic HMBCcorrelations
105
500 MHz NOESYin CDCl3
(±)-naphthomevalin (1)
OHO
HOO
Cl
HH
OHHH
106
Supplementary Table 9. 1H NMR comparison of natural naphthomevalin (1)19, natural SF2415B1 (SI-9)20 and synthetic naphthomevalin (1) in CDCl3.
Assignment Natural naphthomevalin* Natural SF2415B1 Our sample 3-OH 4.15 s 4.14 s 4.18 s
5 7.03 s 7.04 s 7.06 d, J = 2.0 6-OH 7.16 s 6.66 s 6.81 s
7 6.69 s 6.72 d, J = 2.0 8-OH 11.94 s 12.28 s 11.96 s 11a 2.47 dd, J = 8.0, 14.5 2.51 dd, J = 8.0, 14.5 11b 3.00 dd, J = 8.0, 14.5 2.99 dd, J = 8.0, 14.5 12 4.93 t, J = 8.0 4.93 t, J = 8.0 14 1.58 s 1.59 s 15 1.28 s 1.29 s 16a 2.27 dd, J = 8.0, 14.5 2.31 dd, J = 8.0, 14.5 16b 2.96 dd, J = 8.0, 14.5 2.97 dd, J = 8.0, 14.5 17 4.82 t, J = 8.0 4.83 t, J = 8.0 19 1.89 m 1.90 m 20 1.95 m 1.96 m 21 5.02 t, J = 8.0 5.02 t, J = 8.0 23 1.70 s 1.70 s 24 1.57 s 1.56 s 25 1.30 s 1.30 s 26 N/A 2.23 s N/A
* Not all NMR peaks were reported in reference 19.
naphthomevalin (1)
OHO
HOO
OH
Cl
SF2415B1 (SI-9)
12
345
6
78
9
10
11 1213
1415
16 17
18
19
20
2122 23
24
25
OHO
HOO
OH
Cl12
345
6
7
89
10
11 1213
1415
16 1718
19
20
2122 23
24
25
26
107
Supplementary Table 10. 13C NMR comparison of natural naphthomevalin (1)19, natural SF2415B1 (SI-9)20 and synthetic naphthomevalin (1) in CDCl3.
Assignment Natural naphthomevalin* Natural SF2415B1 Our sample 1 195.6 195.5 195.4 2 82.3 83.2 83.0 3 84.4 84.3 84.5 4 196.6 196.8 196.6 5 107.3 106.3 107.3 6 163.6 161.4 163.4 7 109.2 119.6 109.2 8 164.7 162.5 164.8 9 110.5 109.7 110.5
10 134.3 130.7 134.3 11 38.4 38.3 12 116.4 116.5 13 137.9 138.2 14 25.7 25.8 15 17.7 17.7 16 37.3 37.3 17 115.4 115.4 18 141.3 141.5 19 39.7 39.8 20 26.3 26.3 21 123.8 123.8 22 131.7 131.8 23 25.6 25.7 24 17.6 17.7 25 16.1 16.1 26 N/A 8.3 N/A
* Not all NMR peaks were reported in reference 19.
naphthomevalin (1)
OHO
HOO
OH
Cl
SF2415B1 (SI-9)
12
345
6
78
9
10
11 1213
1415
16 17
18
19
20
2122 23
24
25
OHO
HOO
OH
Cl12
345
6
7
89
10
11 1213
1415
16 1718
19
20
2122 23
24
25
26
108
(±)-A80915G (SI-4)500 MHz 1H
in CDCl3
OHO
HOO
O
(±)-A80915G (SI-4)500 MHz 13C
in CDCl3
OHO
HOO
O
109
OHMOMO
MOMO OOAc
SI-5500 MHz 1H
in CDCl3
OHMOMO
MOMO OOAc
SI-5500 MHz 13C
in CDCl3
110
OHMOMO
MOMO OOH
SI-6500 MHz 1H
in CDCl31.6 : 1 mixture of
enol : keto tautomers
OHMOMO
MOMO OOH
SI-6
125 MHz 13Cin CDCl3
1.6 : 1 mixture ofenol : keto tautomers
111
OHHO
HO OOH
SI-7500 MHz 1H
in d6-acetone2.5 : 1 mixture of
enol : keto tautomers
OHHO
HO OOH
SI-7125 MHz 13Cin d6-acetone
2.5 : 1 mixture ofenol : keto tautomers
112
500 MHz 1Hin CDCl3
SI-8
OMOMO
MOMO OOH
ClCl
125 MHz 13Cin CDCl3
SI-8
OMOMO
MOMO OOH
ClCl
113
500 MHz 1Hin CDCl3
35
OHO
HO OOH
ClCl
125 MHz 13Cin CDCl3
35
OHO
HO OOH
ClCl
114
500 MHz 1Hin CDCl3
OHO
HOO
OH
36
ClCl
125 MHz 13Cin CDCl3
OHO
HOO
OH
36
ClCl
115
500 MHz 1Hin d6-acetone
34
OHHO
HO OOH
Cl
125 MHz 13Cin d6-acetone
34
OHHO
HO OOH
Cl
116
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