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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: [email protected] or

[email protected]

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2829

NATURE CHEMISTRY | www.nature.com/naturechemistry 1

http://dx.doi.org/10.1038/nchem.2829

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:


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:


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.


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.


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.


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.


(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.


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.


(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.


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.



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.





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:


445.1787 [M−H]−, found 445.1795.


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.





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:


445.1787 [M−H]−, found 445.1795.


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


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.


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,


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


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


ΔEgas = +11.8 kcal mol-1ΔG°gas = +12.0 kcal mol-1ΔG°soln = +11.4 kcal mol-1

36

37


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


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


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


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


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


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


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


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


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


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


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

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


30

OMOMO

MOMO O

Cl

OAc

Cl


30

OMOMO

MOMO O

Cl

OAc

Cl

94


30

OMOMO

MOMO O

Cl

OAc

Cl

OMOMO

MOMO OOAc

ClCl


95

500 MHz 1Hin CDCl3

OHMOMO

MOMO O

Cl

OH

31

125 MHz 13Cin CDCl3

OHMOMO

MOMO O

Cl

OH

31

96


OHMOMO

MOMO O

Cl

OH

31


OHMOMO

MOMO O

Cl

OH

31

97


OHMOMO

MOMO O

Cl

OH

31

OMOMO

MOMO OO

Cl

H

H


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


OHO

HO O

Cl

OH

33


OHO

HO O

Cl

OH

33

101


OHO

HO O

Cl

OH

33

OHO

HO O

Cl

OH


102

500 MHz 1Hin CDCl3


OHO

HOO

OH

Cl

125 MHz 13Cin CDCl3


OHO

HOO

OH

Cl

103



OHO

HOO

OH

Cl



OHO

HOO

OH

Cl

104



OHO

HOO

OH

Cl

500 MHz NOESYin CDCl3


OHO

HOO

OH

Cl

OHO

HOO

OH

Cl


105

500 MHz NOESYin CDCl3


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

7. References: 1. Teufel, R. et al. One-pot enzymatic synthesis of merochlorinA and B. Angew. Chem. Int. Ed. 53,

11019–11022 (2014). 2. Diethelm, S., Teufel, R., Kaysser, L. & Moore, B. S. A multitasking vanadium-dependent

chloroperoxidase as an inspiration for the chemical synthesis of the merochlorins. Angew. Chem. Int. Ed. 53, 11023–11026 (2014).

3. Bernhardt, P., Okino, T., Winter, J. M., Miyanaga, A. & Moore, B. S. A stereoselective vanadium-dependent chloroperoxidase in bacterial antibiotic biosynthesis. J. Am. Chem. Soc. 133, 4268–4270 (2011).

4. Besemer, J. & Borodovsky, M. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res. 33, W451–4 (2005).

5. Kaysser, L. et al. Merochlorins A–D, cyclic meroterpenoid antibiotics biosynthesized in divergent pathways with vanadium-dependent chloroperoxidases. J. Am. Chem. Soc. 134, 11988–11991 (2012).

6. Beekman, A. M., Castillo Martinez, E. & Barrow, R. A. First syntheses of the biologically active fungal metabolites pestalotiopsones A, B, C and F. Org. Biomol. Chem. 11, 1109–1115 (2013).

7. Ichinose, K., Ebizuka, Y. & Sankawa, U. Mechanistic studies on the biomimetic reduction of tetrahydroxynaphthalene, a key intermediate in melanin biosynthesis. Chem. Pharm. Bull. 49, 192–196 (2001).

8. Gaussian 09, Revision D.01, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, M. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian, Inc., Wallingford CT, (2009).

9 Cornell, W. D., et al. A second generation force field for the simulation of protiens, nucleic acids, and organic molecules. J. Am. Chem. Soc. 117, 5179-5197 (1995).

10. Allouche, A.-R. Gabedit-a graphical user interface for computational chemistry softwares. J. Comput. Chem. 32, 174-182 (2011).

11. Zhao, Y. & Truhlar, D. The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionals. Theor. Chem. Acc. 120, 215-241 (2008).

12. Gonzalez, C. & Schlegel, H. B. An improved algorithm for reaction path following J. Chem. Phys. 90, 2154-2161 (1989).

13. Gonzalez, C. & Schlegel, H. B. Reaction path following in mass-weighted internal coordinates. J. Phys. Chem. 94, 5523-5527 (1990).

14. Marenich, A. V., Cramer, C. J. & Truhlar, D. G. Universal solvation model based on solute electron density and on a continuum model of the solvent defined by the bulk dielectric constant and atomic surface tensions. J. Phys. Chem. B 113, 6378-6396 (2009).

15. Yanai, T., Tew, D. R. & Handy, N.C. A new hybrid exchange–correlation functional using the Coulomb-attenuating method (CAM-B3LYP). Chem. Phys. Lett. 393, 51-57 (2004).

16. Pescitelli, G., Di Bari, L. & Berova N. Conformational aspects in the studies of organic compounds

117

by electronic circular dichroism. Chem. Soc. Rev. 40, 4603-4625 (2011). 17. Tomasi, J., Mennucci, B. & Cammi, R. Quantum mechanical continuum solvation models.

Chem. Rev. 105, 2999-3093 (2005). 18. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007). 19. Henkel, T. & Zeeck, A. Secondary metabolites by chemical screening, 15. Structure and absolute

configuration of napthomevalin, a new dihydro-naphthoquinone antibiotic from Streptomyces sp. J. Antibiot. 44, 665-669 (1991).

20. Gomi, S., Ohuchi, S., Sasaki, T., Itoh, J. & Sezaki, M. Studies on new antibiotics SF2415 II. The structure elucidation. J. Antibiot. 40, 740-749 (1987).

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