Sequential enzymatic and electrochemical functionalization of bromocyclohexadienediols: Application to the synthesis of (?)-conduritol C

Article abstract of DOI:10.1016/j.tet.2020.130924

cis-Diene diol obtained from the microbial oxidation of bromobenzene was used as a substrate for the chemoenzymatic acetylation and epoxidation with lipases. The model studies showed that the regiochemistry of the acetylation is solvent-dependent. The chemoenzymatic epoxidation followed the expected regiochemistry when compared to the chemical epoxidation with m-CPBA, but with the unexpected formation of bromoconduritol-C, an important intermediate whose electrochemical reduction led to the synthesis of (?)-conduritol-C. Experimental and spectral data are provided for all new compounds.

Full text of DOI:10.1016/j.tet.2020.130924

Journal Pre-proof
Sequential enzymatic and electrochemical functionalization of
bromocyclohexadienediols: Application to the synthesis of (−)-conduritol C
Juana Goulart Stollmaier, Tomáš Hudlický
PII:
S0040-4020(20)30004-1
https://doi.org/10.1016/j.tet.2020.130924
TET 130924
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 12 November 2019
Revised Date: 23 December 2019
Accepted Date: 4 January 2020
Please cite this article as: Stollmaier JG, Hudlický Tomáš, Sequential enzymatic and electrochemical
functionalization of bromocyclohexadienediols: Application to the synthesis of (−)-conduritol C,
Tetrahedron (2020), doi: https://doi.org/10.1016/j.tet.2020.130924.
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Sequential enzymatic and electrochemical functionalization of
bromocyclohexadienediols: Application to the synthesis of (-)-conduritol C
Juana Goulart Stollmaier and Tomáš Hudlický*
Department of Chemistry and Centre for Biotechnology, Brock University, 1812 Sir Isaac Brock Way, St. Catharines,
Ontario, L2S 3A1, Canada
thudlicky@brocku.ca
1. Abstract
cis-Diene diol obtained from the microbial oxidation of bromobenzene was used as a substrate
for the chemoenzymatic acetylation and epoxidation with lipases. The model studies showed that the
regiochemistry of the acetylation is solvent-dependent. The chemoenzymatic epoxidation followed
the expected regiochemistry when compared to the chemical epoxidation with m-CPBA, but with the
unexpected formation of bromoconduritol-C, an important intermediate whose electrochemical
reduction led to the synthesis of (-)-conduritol-C. Experimental and spectral data are provided for all
new compounds.
Keywords: enzymatic dihydroxylation, lipases, acetylation, epoxidation, narciclasine, conduritol C
2. Introduction
For more than 30 years, arene cis-dihydrodiols obtained from the whole-cell fermentation of
aromatics by E. coli JM109 (pDTG601A)1 have been used as building blocks in natural product
synthesis. The enantiopure diols have been converted to morphine derivatives, cyclitols,
sesquiterpenes, the cytotoxic Amaryllidaceae alkaloids, and other complex natural products.2 This
biooxidation process has proven to be scalable and efficient within green chemistry metrics, and it
represents a good example of how biocatalysis can be utilized as a potentially sustainable tool in
organic synthesis.
Biocatalysis relies on the use of whole microorganisms or isolated enzymes and the tendency of
enzymes to control the environment of the reaction rather than the reaction itself to provide
outstanding enantio-, stereo- and regioselectivities in fewer steps than traditional chemical
strategies.3 By investigating and adding unconventional methods (e.g. biocatalysis, preparative
electrochemistry)2a to previously known synthetic routes unexpected results can be attained and
shorter synthetic routes can be achieved.
With this reasoning we considered sequential biotransformations applied to one of the
ubiquitously used cis-diols, namely bromocyclohexadienediol 2, which is obtained in high yields (>9
1

g/L) from the dihydroxylation of bromobenzene 1,4 Scheme 1. This compound was submitted as a
substrate for investigation of lipase-catalyzed acetylation under mild conditions using Amano PS
lipase immobilized in diatomite and for chemoenzymatic epoxidation using Candida antarctica
lipase (CAL). (Scheme 1) Each of these studies provided valuable results and added new options to
the repertoire of reactions that could be applied to the homochiral diols such as 2.
Scheme 1. Chemoenzymatic reactions investigated in this work.
In this work we report the results of chemoenzymatic acetylation of diols in organic media and a
short synthesis of (-)-conduritol C by a chemoenzymatic cascade followed by electrochemical
reduction of a vinyl bromide. The synthesis of conduritol was subjected to efficiency metric
evaluation5,6 in comparison to previous chemical syntheses. The lipase-catalyzed acetylation was
also extended to acetylation of the cytotoxic natural product narciclasine.
3. Results and discussion
a) Lipase–catalyzed acetylation of 2
Scheme 2. Acetylation of bromodiol (2) catalyzed by AmanoPS immobilized in diatomite.
Screening of lipases. Initial conditions similar to a previously reported acetylation methodology7
were used. Three out of nine lipases showed promising activity toward the acetylation of 2: Lipases
from Pseudomonas fluorescens (AmanoPF, 20,000 U/g); Burkholderia cepacia (AmanoIM, 500 U/g)
2

and immobilized in diatomite (AmanoPS-d, 500 U/g). The other lipases that were tested are listed in
Supporting Information Section I, Part A.
Effect of mass of catalyst and equivalents of acyl donor. The influence of the amount of lipase was
evaluated with an initial fixed value at 10 mg/mL with variation in the number of equivalents of acyl
donor. The best results are shown in Table 1 (For details and complete results see Supporting
Information Section I, Part B). AmanoIM and AmanoPS-d have similar activities with the same fixed
load of active enzyme, while the activity for AmanoPF is approximately 40-fold higher. For the
tested lipases, there was an increase in the overall conversion when two equivalents (1.0 mmol) of
vinyl acetate were used, but the selectivity between 3a and 3b was still low.
Table 1. Analysis of varying amount of acyl donor in chemoenzymatic acetylation. Reaction
conditions: 2 (0.5 mmol), vinyl acetate (0.5 – 1.0 mmol), lipase (100 mg), Methyl tert-butyl ether
(MTBE) (10 mL), 24h, 35 °C, 150 rpm. Conversion values calculated with 1H-NMR
Vinyl acetate
Overall
Conversion
22%
Lipase
AmanoIM
AmanoPF
AmanoPS-d
3a
3b 3c
Entry
(mmol)
0.5
64% 36%
49% 46% 5%
69% 31%
75% 25%
58% 42%
-
1
2
3
4
5
6
1.0
0.5
1.0
0.5
90%
38%
51%
32%
-
-
-
1.0
90%
49% 46% 5%
A uniform trend among the results presented in Table 1 was not observed. For the second
round of experiments the volume of the reaction mixture was lowered from 10 mL of tert-butyl
methyl ether (MTBE) to 5 mL with variable equivalents of vinyl acetate. The main results are shown
in Table 2. (For details and complete results see Supporting Information Section I, Part B)
Table 2. Analysis of varying amount of acyl donor in chemoenzymatic acetylation. Reaction
conditions: 2 (0.5 mmol), vinyl acetate (0.5 – 1.5 mmol), lipase (100 mg), Methyl tert-butyl ether
(MTBE) (5 mL), 24h, 35 °C, 150 rpm. Conversion values calculated with 1H-NMR
Vinyl acetate
Entry
Lipase
AmanoIM
AmanoPF
AmanoPS-d
Overall conversion 3a
3b
3c
(mmol)
1.0
>95%
>95%
76%
48% 32% 20%
49% 33% 18%
73% 27%
74% 26%
7
8
9
10
11
12
1.5
1.0
1.5
1.0
-
-
78%
>95%
>95%
39% 21% 40%
49% 32% 19%
1.5
When the volume of the reaction mixture is decreased while keeping the same relative
concentration of the substrate, acyl donor and lipases, AmanoIM and AmanoPS-d showed full
overall conversion. AmanoIM is less selective and less sensitive to variation of the concentration of
3

vinyl acetate, while AmanoPF had the best selectivity towards formation of 2, but without total
consumption of starting material. AmanoPS-d was selected for further studies.
By using more concentrated organic media we observed the formation of diacetate 3c and
complete consumption of starting material. To investigate the source of formation of diacetate 3c, the
concentrated crude product from entry 12 was purified by column chromatography and two mixtures
of 3a and 3b (1:1) were resubmitted in the shaker with conditions from entry 9. The first flask was
removed from the shaker after 24 h, and a mixture of 3a (55%), 3b (14%) and 3c (30%) was
observed through 1H-NMR. The second flask was removed after 48 h, and the mixture contained 3a
(41%), 3b (12%) and 3c (46%). The monoacetate 3b appears to be the main substrate for the
formation of diacetate 3c.
Effect of solvent. To better understand the effect of biocompatibility of a few organic solvents in the
acetylation reaction, two solvents and a mixture of solvents were selected with different logarithm of
the partition coefficient (log P).8 Acetonitrile (MeCN) (log P = -0.33), MTBE (log P = 1.24), and a
4:1 mixture of n-hexane (log P = 3.5) tetrahydrofuran (THF) (log P = 0.46) were selected. An
increase in selectivity was observed toward the formation of 3a when MTBE was switched for
MeCN, but with a decrease in the overall conversion. The solvent system n-hexane:THF 4:1
presented low selectivity in the acetylation of 2 and is highly dependent on the concentration of vinyl
acetate. Because of the observed trend, both MeCN and MTBE were selected for further
investigations. (For details and complete results see Supporting Information Section I, Part C)
Because of the intramolecular acyl migration from C-2 to C-3, it is not possible to purify 3a
and 3b by column chromatography. The crude product containing a mixture of starting material and
products 3a and 3b was treated with TBSCl and compound 6 was isolated as the major product.
Scheme 3. TBS protection of a mixture of 2, 3a and 3b.
An acyl migration study was performed by filtering and concentrating the crude product
obtained from the chemoenzymatic acetylation in MeCN and submitting it with a fresh load of
solvent. After a period of 24h, acyl migration was observed as there was a 11% conversion from 3a
4

to 3b. However, further acyl migration studies using imidazole or other suitable bases were not
performed.
Effect of temperature. Temperature can often be a key factor in reactions catalyzed by enzymes.9 To
evaluate the effect of temperature in the acetylation of bromodiene diol 2, reactions were performed
using T = 20 °C and T = 45 °C. (For details and complete results see Supporting Information Section
I, Part D)
Effect of co-solvents. For the subsequent application of narciclasine as a substrate for the
chemoenzymatic acetylation, DMSO (log P = -1.3) and dimethylformamide (DMF) (log P = -0.829)
were evaluated as co-solvents in the model studies, since these solvents are capable of solubilizing
narciclasine. Using the two standard MTBE/MeCN conditions, no conversion to any desired product
was obtained with DMSO as a co-solvent. However, overall conversions of 31% (3a (71%) and 3b
(29%)) and 37% (3a (78%) and 3b (22%)) with MTBE:DMF (4:1) and MeCN:DMF (4:1)
respectively were observed.
b) Lipase-catalyzed acetylation of narciclasine.
The reaction conditions for the acetylation of narciclasine were kept similar to the previous
study, but with longer reaction time of 72h. After purification, we observed different selectivity than
that in the model study regarding the solvents. The reaction in MTBE:DMF was selective in
acylating the C-2 position of narciclasine and trace amounts of acylated C-3 and C-4 products were
also detected. (Scheme 4) The reaction with MeCN:DMF is less selective towards acetylation of the
C-2 position because it was observed the formation of C-3/C-4 monoacetate derivatives of
narciclasine. The observed selectivity could be related to the allylic nature of the C-2 position,
similar to both allylic alcohols present in the model study with bromodiene diol. No acetylation
occurred at the phenolic alcohol at C-7 position, presumably because of the strong H-bond with the
neighboring amide.
Scheme 4. Chemoenzymatic acetylation of narciclasine. Reaction conditions: 7 (0.16 mmol), vinyl
acetate (1.0 - 1.5 mmol), AmanoPS-d (100 mg), MTBE:DMF (4:1) / MeCN:DMF (4:1) (5 mL), 72h,
35 °C, 150 rpm.
5

c) Epoxidation of bromodienediol 2
Diol 2 was tested as a potential substrate for a chemoenzymatic epoxidation.
Chemoenzymatic epoxidations catalyzed by lipases are known to promote the generation of a peracid
in situ through a cascade reaction, minimizing hazardous risks of handling peracids in stoichiometric
ratios.10 Following a previously reported epoxidation methodology,11 a few parameters were tested in
a similar way to the acetylation study.
Candida antarctica lipase (CAL) and lipase-B (CALB) were selected for the epoxidations
and the initial tests showed that the organic solvent and temperature are key parameters for
improving the conversion values. (For details and complete results see Supporting Information
1
Section II, Part A). Surprisingly, the syn-epoxide 9 was not observed in the crude product (via H-
NMR) after full consumption of starting material, and upon purification of the crude product,
compound 4 was obtained in 13% yield, the remaining mass balance being unidentified
decomposition products. (Scheme 5)
Scheme 5. Formation of tetraol 4 during chemoenzymatic epoxidation of 2.
Protection of diol 2 yielded acetonide 10, which was further used as substrate for the
chemoenzymatic epoxidation using the previously reported conditions. A 35% conversion was
observed for the anti-epoxide 11, formed as a result of the steric hindrance of the acetonide moiety.
(Scheme 6) Epoxide 11 is stable and was purified by column chromatography without the opening
observed in the syn-isomer.
Scheme 6. Chemoenzymatic epoxidation of 10 and formation of anti-epoxide 11
The diacetate 3c obtained from the chemoenzymatic acetylation of 1 was used in two
different approaches to the synthesis of epoxides. By using standard methods with m-CPBA, a 2:1
mixture of anti- and syn-epoxides 12a and 12b was obtained, with compound 12b quickly degrading.
6

When the chemoenzymatic epoxidation of 3c was performed, 19% conversion was observed for 12a,
while no conversion was observed for 12b. The rest of the mass balance was recovered starting
material. (Scheme 7). (For details and complete results see Supporting Information Section II, Part
A).
Scheme 7. Comparison between chemical and chemoenzymatic epoxidation of 3c
d) Electrochemical reduction of 4.
A methodology for preparative electrochemistry was adapted from a procedure previously
published,12 replacing the mercury pool with a glassy carbon electrode in a “greener” manner and
alternating the polarity of the electrodes to reduce the adsorption on the surface of the electrodes.
The constant voltage electrolysis of bromo-conduritol C 4 resulted in a 54% yield of (-)-conduritol C
13. (Scheme 8)
Scheme 8. Preparative electrochemical reduction of 4.
4. Conclusions
The chemoenzymatic acetylation and epoxidation were performed under mild conditions. The
organic medium had the greatest influence on conversions and selectivity. For the acetylation
reactions the regioselectivity was initially considered to be influenced only by the lipase, but it was
later shown that some migration of the acetyl groups occurred in solution and/or during purification
by chromatography. For the epoxidation of the free diol, the release of water in the catalytic cascade
reaction performed by the lipase is sufficient to promote opening of the epoxide and formation of the
bromo conduritol, but in low yield. The same effect is not observed when the diol moiety is
7

protected, and the reaction follows the stereochemistry expected from standard chemical
epoxidations. The overall yield starting from bromodiene diol is 7%, but there is opportunity to
improve the yield of the chemoenzymatic epoxidation, as the standard conditions can be reevaluated
and enhanced. Regardless, an old methodology for preparative electrochemistry of conduritol
derivatives was adapted for bromo-conduritol C and updated for greener standards. We believe that
this route represents a potentially efficient synthesis of a valuable building block. The E-factor for
this preparation cannot be contextualized as there are no alternate routes from the same starting
material.
5. Experimental section
5.1. General
Analytical thin-layer chromatography was performed using silica gel 60-F254 plates. IR
1
spectra were recorded as neat samples. All H-NMR spectra were recorded at ambient temperature
using Bruker Avance AV 300 (300 MHz) or Bruker Avance AV 600 (600 MHz) spectrometer. All
13C-NMR spectra were recorded at ambient temperature using Bruker Avance AV 300 (75 MHz) or
Bruker Avance AV 600 (150 MHz) spectrometer. Data are reported as (s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet, br = broad) coupling constants in Hz, integration. Specific rotation
measurements are given in deg.cm3.g-1.dm-1. Mass spectra and high-resolution mass spectra were
performed by the analytical division at Brock University. Electrochemical reactions and cyclic
voltammetry experiments were carried out in an IKA ElectraSyn 2.0. CV curves were recorded using
a three-electrode scheme: The working electrode was a CV glassy carbon electrode and a platinum
electrode served as counter electrode. Ag/AgCl was used as the reference electrode with a 3 M NaCl
as reference electrolyte. The preparative electrochemistry was performed using a three-electrode
scheme: The working electrode was a glassy carbon electrode (d = 3 mm) and a platinum electrode
served as counter electrode. Ag/AgCl was used as the reference electrode with a 3 M NaCl as
reference electrolyte.
5.2. Chemoenzymatic acetylation
In a typical chemoenzymatic acetylation reaction, a mixture of bromodiene diol 2 (95 mg, 0.5
mmol), vinyl acetate (0.15 mL, 1.5 mmol) and AmanoPS-d (100 mg) in methyl tert-butyl ether (5
mL), or acetonitrile (5 mL) was stirred at 150 rpm, at 35 °C, for 24 h. The crude mixture was filtered
to remove the catalyst and concentrated in vacuo to afford a mixture of 3a and 3b (and 3c when
specified) as a yellow oil.
5.2.1. (1S,6S)-2-bromo-6-hydroxycyclohexa-2,4-dien-1-yl acetate (3a)
8

1H NMR (300 MHz, CDCl3): δppm 6.51 (dd, J = 5.5, 1.1 Hz, 1H), 5.93 – 5.86 (m, 2H), 5.59
(d, J = 6.7 Hz, 1H), 4.68 (dt, J = 6.7, 2.0 Hz, 1H) , 2.16 (s, 3H).
5.2.2. (1S,6S)-5-bromo-6-hydroxycyclohexa-2,4-dien-1-yl acetate (3b)
1H NMR (300 MHz, CDCl3): δppm 6.39 (dd, J = 5.2, 0.9 Hz, 1H), 5.94 – 5.80 (m, 2H), 5.48
(ddd, J = 6.2, 3.0, 0.9 Hz, 1H), 4.43 (d, J = 6.2 Hz, 1H), 2.11 (s, 3H).
5.2.3. (1S,2S)-3-bromocyclohexa-3,5-diene-1,2-diyl diacetate (3c)
Diene diol 2 (2.0 g, 0.01 mol) was solubilized in dichloromethane (15 mL) and cooled to 0
°C. Triethylamine (4 mL, 0.03 mmol), 4-dimethylaminopyridine (DMAP, cat. amount, 3 crystals)
was added to the solution, followed by dropwise addition of acetic anhydride (3 mL, 0.03 mmol).
After 1 h, the reaction mixture was quenched with NaHCO3 (15 mL) and extracted with
dichloromethane (3x15 mL). The organic phases were combined and dried over MgSO4, filtered and
concentrated in vacuo. The residue was purified by column chromatography on deactivated silica gel
(10%) (hexanes:EtOAc (6:1)) to yield diacetate 3c as a yellow oil (1.8 g, 65 %).
1
ꢀꢁ: [α]21=-71.78 (c 1.0, MeCN); H NMR (300 MHz, CDCl3) δppm 6.55 (dd, J = 5.7, 0.6 Hz, 1H),
D
5.97 (ddd, J = 9.5, 5.8, 1.6 Hz, 1H), 5.84 (ddt, J = 9.6, 2.4, 0.7 Hz, 1H), 5.73 – 5.70 (m, 2H), 2.11 (s,
3H), 2.06 (s, 3H); 13C NMR (75 MHz, CDCl3) δppm 170.21, 170.12, 129.56, 125.63, 124.98, 120.32,
70.03, 69.04, 20.89, 20.80; IR νmax: 1739, 1369, 1208, 1068, 1015, 734, 596.
5.3. TBS-protection
5.3.1. (1S,6S)-2-bromo-6-((tert-butyldimethylsilyl)oxy)cyclohexa-2,4-dien-1-ol (5)13
Imidazole (200 mg, 2.9 mmol) and TBSCl (450 mg, 3.0 mmol) were added to a magnetically stirred
cold (0 °C) solution of diol 2 (500 mg, 2.6 mmol) in DMF (10 mL) maintained under an atmosphere
of nitrogen. After 16 h the reaction mixture was diluted with Et2O (20 mL) and washed with brine
(3x15 mL). The combined organic extracts were then dried with MgSO4, filtered and concentrated in
vacuo to give a pale-yellow oil. The crude product was purified by flash column chromatography on
deactivated silica gel (10%) (hexane:EtOAc (6:1 → 4:1)) to yield 5 (363 mg, 45%).
1
ꢂ: [α]21=44.60 (c 0.5, MeCN); H NMR (300 MHz, CDCl3) δppm 6.37 (dd, J = 4.1, 2.5 Hz, 1H), 5.78
D
(d, J = 3.7 Hz, 2H), 4.57 (d, J = 6.3 Hz, 1H), 4.14 (dd, J = 6.2, 4.6 Hz, 1H), 2.77 (d, J = 4.7 Hz, 1H),
13
0.92 (s, 9H), 0.13 (s, 6H); C NMR (75 MHz, CDCl3) δppm 129.65, 127.10, 125.64, 123.57, 77.16,
72.80, 70.75, 26.04, 25.90, 18.30, -4.40, -4.71; IR νmax: 3548, 2952, 2856, 1571, 1470, 1389, 1253,
1059, 834, 776, 676, 647 cm-1; HRMS (EI) Calcd for C12H21BrO2Si(M+): 304.0494, found 304.0492;
LRMS (ESI) found 322 [M+NH4]+, 327 [M+Na]+, 342.9 [M+K]+.
9

5.3.2. (1S,6S)-2-bromo-6-((tert-butyldimethylsilyl)oxy)cyclohexa-2,4-dien-1-yl acetate (6)
A similar procedure was repeated using a crude mixture of 2, 3a and 3b (285 mg) from a
chemoenzymatic acetylation solubilized in DMF (10 mL). The solution was cooled to 0 °C and
imidazole (200 mg, 2.9 mmol) and TBSCl (400 mg, 2.6 mmol) were added. After 16 h the reaction
mixture was diluted with Et2O (20 mL) and washed with brine (3x15 mL). The combined organic
extracts were then dried with MgSO4, filtered and concentrated in vacuo to give a pale-yellow oil.
The crude product was purified by flash column chromatography on deactivated silica gel (10%)
(hexane:EtOAc (10:1 → 4:1)) to yield 6 (125 mg).
1
ꢃ: [α]21=-78.16 (c 1.0, MeCN); H NMR (300 MHz, CDCl3) δppm 6.49 (dd, J = 4.1, 2.5 Hz, 1H),
D
5.82 (d, J = 3.3 Hz, 2H), 5.56 (d, J = 6.8 Hz, 1H), 4.68 (d, J = 6.8 Hz, 1H), 2.10 (s, 3H), 0.89 (s, 9H),
0.12 (s, 3H), 0.09 (s, 3H); 13C NMR (75 MHz, CDCl3) δppm 170.17, 131.78, 129.89, 122.46, 120.31,
72.17, 70.01, 25.84, 20.96, 18.29, -4.88, -5.02; IR νmax: 2927, 2855, 1736, 1740, 1222, 1119, 1097,
827, 784, 667 cm-1; HRMS (EI) Calcd for C14H23BrO3Si(M+): 346.0600, found 346.0594; LRMS
(ESI) found 364 [M+NH4]+, 369 [M+Na]+, 385 [M+K]+
5.4. (2S,3S,4S,4aR)-3,4,7-trihydroxy-6-oxo-2,3,4,4a,5,6-hexahydro-[1,3]dioxolo[4,5-
j]phenanthridin-2-yl acetate (8) 14
Two reaction mixtures were prepared with 7 (50 mg, 0.16 mmol) each, the first with 0.10 mL
(1.0 mmol) of vinyl acetate and 5 mL of MTBE:DMF (4:1) and the second with 0.15 mL of vinyl
acetate and 5 mL of MeCN:DMF (4:1). Both had 100 mg of lipase as the biocatalyst. Both reactions
were stirred at 150 rpm, at 35 °C, for 72 h. The reaction mixtures were combined and concentrated
by rotary evaporation and its crude product was purified by flash column chromatography on
deactivated silica gel (10%) (DCM:MeOH (50:1 → 30:1)) to obtain the title compound 8 (20 mg,
17%) as a pale-yellow solid.
1
8: Rf = 0.21 (DCM:MeOH 10:1); H NMR (300 MHz, DMSO-d6) δppm 13.17 (s, 1H), 8.01 (s, 1H),
6.86 (s, 1H), 6.10 (dd, J = 4.2, 2.5 Hz, 1H), 6.06 (d, J = 1.2 Hz, 2H), 5.48 (d, J = 4.1 Hz, 1H), 5.33
(d, J = 5.4 Hz, 1H), 5.17 (ddd, J = 4.4, 2.9, 1.3 Hz, 1H), 4.22 (d, J = 8.2 Hz, 1H), 3.78 – 3.65 (m,
2H), 2.00 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δppm 169.68, 168.92, 152.43, 144.88, 133.97,
133.32, 131.25, 119.32, 105.78, 102.27, 96.19, 71.00, 69.25, 69.14, 52.76, 20.83. The spectral data
were matched and are in agreement with the literature data.14
5.5. Epoxidation
10

In a typical chemoenzymatic epoxidation reaction, a mixture of substrate (2, 3c or 10 (0.5
mmol)), urea-hydrogen peroxide (UHP) (94 mg, 1.0 mmol), octanoic acid (80 µL, 0.5 mmol) and
Candida antarctica lipase (CAL) (10 mg) in methyl tert-butyl ether (5 mL) was stirred at 150 rpm, at
35 °C, for 24 h. The crude mixture was filtered for removal of the catalyst and concentrated in vacuo
and further analyzed by 1H-NMR.
5.5.1. (1R,2S,3S,4S)-5-bromocyclohex-5-ene-1,2,3,4-tetraol (4)15
The crude mixture of 6 batches was combined and filtered for removal of the catalyst, and the
flasks were washed with MeOH to remove product residues and combined with the MTBE filtrate
and concentrated in vacuo. The obtained white crude product was adsorbed in 24% deactivated silica
gel and purified by flash column chromatography (EtOAc:Hex:MeOH 8:1:1) to yield tetrol as a
white solid 4 (88 mg, 13%).
4: [α]2D2=-64.53 (c 0.5, MeOH); lit.15 [α]2D0=-50 (c 0.01, MeOH); 1H NMR (600 MHz, MeOD) δ 6.09
– 6.06 (m, 1H), 4.24 (dt, J = 6.5, 2.4 Hz, 1H), 4.20 – 4.17 (m, 1H), 4.06 (dd, J = 3.7, 2.0 Hz, 1H),
3.61 (dd, J = 6.8, 1.9 Hz, 1H).; 13C NMR (151 MHz, MeOD) δ 133.04, 127.66, 75.47, 73.10, 72.84,
71.53.; LRMS (ESI+/-) Calcd for C6H9BrO4[M]+: 223.9684, found 242 [M+NH4]+, 246.9 [M+Na]+,
262.9 [M+K]+. The spectral data were matched and are in agreement with the literature data.15
5.5.2. (3aS,7aS)-4-bromo-2,2-dimethyl-3a,7a-dihydrobenzo[d][1,3]dioxole (10)16
Diol 1 (2 g, 0.01 mol) was suspended in DCM (10 mL), and 2,2-DMP (5 mL, 0.04 mol) was
added followed by the addition of a catalytic amount of p-TSA. The reaction mixture was left stirring
for 4 h at room temperature and the consumption of starting material was checked with TLC
(hexane:EtOAc (4:1)). The reaction was quenched with NaHCO3 and extracted with DCM. The
combined organic phases were dried with MgSO4 and concentrated on the rotary evaporator. The
residue was adsorbed on deactivated silica gel (10%), and purified by column chromatography
(hexane:EtOAc (8:1)) to afford 10 as a colorless oil (1.43 g, 59%). This compound quickly dimerizes
and must be used immediately. Otherwise, it is necessary to keep it stored in solution in the freezer (-
13 °C).
10: [α]21= 77.08 (c 1.5, MeCN); 1H NMR (300 MHz, CDCl3) δ 6.34 (d, J = 6.1 Hz, 1H), 5.97 (d, J =
D
9.5 Hz, 1H), 5.87 (dd, J = 9.6, 6.0 Hz, 1H), 4.72 (d, J = 1.7 Hz, 2H), 1.44 (s, 2H), 1.43 (s, 3H). 13C
NMR (75 MHz, CDCl3) δ 125.90, 124.77, 124.53, 124.22, 106.34, 76.01, 72.60, 26.84, 25.08. The
spectral data were matched and are in agreement with the literature data.16
11

5.5.3. (3aS,5aR,6aR,6bS)-4-bromo-2,2-dimethyl-3a,5a,6a,6b
tetrahydrooxireno[2',3':3,4]benzo[1,2-d][1,3]dioxole (11)17
75 mg (0.43 mmol) of recrystallized m-CPBA was added to a cold solution of 10 (100 mg,
0.43 mmol) in DCM (5 mL) and left stirring overnight. TLC showed the presence of starting
material, so another 35 mg of m-CPBA was added to the cold reaction mixture. After 3 h, the
reaction was quenched with NaHCO3, extracted with DCM and the combined organic phases were
dried with MgSO4 and concentrated. The residue was purified by flash column chromatography with
deactivated silica gel (10%) (hexane:EtOAc (8:1)) to yield 11 as a colorless oil (80 mg, 75%).
1
11: [α]21= 95.6 (c 0.7, MeCN); H NMR (300 MHz, CDCl3) δ 6.47 (dd, J = 4.4, 1.1 Hz, 1H), 4.86
D
(ddd, J = 6.8, 1.8, 1.1 Hz, 1H), 4.41 (dd, J = 6.8, 0.9 Hz, 1H), 3.58 (dd, J = 3.7, 1.9 Hz, 1H), 3.34 –
3.30 (m, 1H), 1.45 (s, 3H), 1.43 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 130.01, 126.61, 111.54,
74.24, 72.74, 49.59, 48.42, 27.63, 26.12. The spectral data were matched and are in agreement with
the literature data.17
5.5.4. (1R,2S,3S,6R)-4-bromo-7-oxabicyclo[4.1.0]hept-4-ene-2,3-diyl diacetate (12a)
80 mg (0.46 mmol) of recrystallized m-CPBA was added to a cold solution of diacetate 3c
(114 mg, 0.46 mmol) in DCM (5 mL) and left stirring overnight. The reaction was quenched with
NaHCO3, extracted with DCM and the combined organic phases were dried with MgSO4 and
concentrated. The residue was purified by flash column chromatography with deactivated silica gel
(10%) (hexane:EtOAc (8:1)) to yield 12a as an opaque oil (28 mg, 21%) and 12b as a yellow solid
(14 mg, 10%). Compound 12b is unstable and quickly decomposes even when kept in the freezer (-
15 °C).
12a: [α]21= 112.8 (c 0.6, MeCN); 1H NMR (300 MHz, CDCl3) δ 6.55 (dd, J = 4.3, 2.5 Hz, 1H), 5.79
D
(ddd, J = 4.9, 3.0, 0.5 Hz, 1H), 5.49 (dd, J = 4.9, 2.5 Hz, 1H), 3.48 (t, J = 3.5 Hz, 1H), 3.37 (t, J = 3.8
Hz, 1H), 2.07 (s, 3H), 2.06 (s, 3H).13C NMR (75 MHz, CDCl3) δ 170.14, 169.31, 127.34, 125.82,
66.86, 66.27, 50.87, 48.33, 20.83, 20.64; IR νmax: 1742, 1371, 1210, 1069, 1047, 896, 597, 552, 471
cm-1.
5.5.5. (1S,2S,3S,6S)-4-bromo-7-oxabicyclo[4.1.0]hept-4-ene-2,3-diyl diacetate (12b)
12b: [α]21=-230.4 (c 0.6, MeCN); 1H NMR (300 MHz, CDCl3) δ 6.65 (d, J = 4.4 Hz, 1H), 5.82 (dd,
D
J = 5.5, 2.0 Hz, 1H), 5.42 (dd, J = 5.5, 1.2 Hz, 1H), 3.59 – 3.54 (m, 1H), 3.46 (t, J = 4.4 Hz, 1H),
2.15 (s, 3H), 2.08 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 170.75, 169.97, 131.45, 123.59, 70.30,
68.90, 53.34, 48.20, 20.88, 20.71. IR νmax: 1743, 1368, 1229, 1206, 1051, 897, 728, 552, 603 cm-1.
12

5.6. Preparative electrochemistry
5.6.1. (1R,2R,3S,4R)-cyclohex-5-ene-1,2,3,4-tetrol (13) [(-)-conduritol C]15,18
A 0.1 M solution of the charge carrier nBu4NBF4 (TBATFB) in 10mL of THF was added to
the IKA ElectraSyn vial. The solution was purged with an argon stream for 10min. Compound 4 (40
mg, 0.17 mmol) was added in the vial and the vial was closed with the three-electrode scheme. The
CV was done to obtain the reduction potential (-2.9 V) and the CV electrode was changed to the
glassy carbon electrode. The mixture was electrolyzed with constant voltage for 4 h with the polarity
being alternated every 5 min. Upon completion of the reaction, the solvent was evaporated, and the
resulting green solid was adsorbed in 10% deactivated silica gel and purified by flash column
chromatography (EtOAc to remove most of the charge carrier and EtOAc:MeOH 8:1) to yield (-)-
conduritol C 13 (14 mg, 54%) as a white solid.
1
13: [α]2D1=-141.13 (c 0.2, MeOH)*; lit.15 [α]2D0=-205 (c 0.2, MeOH); H NMR (300 MHz, MeOD) δ
5.65 (dt, J = 10.3, 2.0 Hz, 1H), 5.56 (ddd, J = 10.2, 3.5, 1.8 Hz, 1H), 4.27 (dd, J = 4.5, 2.3 Hz, 1H),
13
4.26 – 4.20 (m, 1H), 4.02 (dt, J = 3.5, 1.8 Hz, 1H), 3.53 (dd, J = 7.4, 2.0 Hz, 1H); C NMR (75
MHz, MeOD) δ 130.84, 130.11, 76.20, 74.05, 70.65, 69.57 *Small amounts of charge carrier still
.
present in the sample. The spectral data were matched and are in agreement with the literature data.18
6. Acknowledgements
We are grateful to the following agencies for financial support of this work: the Natural Sciences
and Engineering Research Council of Canada (NSERC; Idea to Innovation and Discovery Grants),
the Canada Research Chair Program, the Canada Foundation for Innovation (CFI), TDC Research,
Inc., the TDC Research Foundation, the Ontario Partnership for Innovation and Commercialization
(OPIC), and The Advanced Biomanufacturing Centre (Brock University). We thank Dr. Liqun Qui
for her help with mass spectrometry.
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14

Highlights:
•
arene cis-dihydrodiols obtained from whole-cell fermentation were submitted to lipase-
catalyzed acetylations;
•
•
The acetylation model was applied to the cytotoxic natural product narciclasine;
(-)-conduritol C was obtained via a chemoenzymatic epoxidation cascade followed by
electrochemical reduction.

Declaration of Interest Statement
The authors declare no conflict of interest.