Enzymatic oxidation of para-substituted arenes: Access to new non-racemic chiral metabolites for synthesis

Article abstract of DOI:10.1016/j.tetasy.2013.01.002

A series of para-substituted benzene derivatives were subjected to whole-cell fermentation with Escherichia coli JM109 (pDTG601), an organism expressing toluene dioxygenase (TDO). Several compounds proved to be excellent substrates for TDO, including 4-bromo-phenylacetylene, 4-bromobenzaldehyde, 4-bromobenzyl alcohol and 4-bromo-allylbenzene. Some of the first para-functionalized diene diols produced using TDO, are useful substrates for further synthetic manipulations, including their use in the potential synthesis of complex natural products.

Full text of DOI:10.1016/j.tetasy.2013.01.002

Tetrahedron: Asymmetry 24 (2013) 184–190
Contents lists available at SciVerse ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Enzymatic oxidation of para-substituted arenes: access to new
non-racemic chiral metabolites for synthesis
⇑
John F. Trant, Jordan Froese, Tomas Hudlicky
Department of Chemistry and Centre for Biotechnology, Brock University, 500 Glenridge Avenue, St Catharines, Ontario, Canada L2S 3A1
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 November 2012
Accepted 4 January 2013
A series of para-substituted benzene derivatives were subjected to whole-cell fermentation with Esche-
richia coli JM109 (pDTG601), an organism expressing toluene dioxygenase (TDO). Several compounds
proved to be excellent substrates for TDO, including 4-bromo-phenylacetylene, 4-bromobenzaldehyde,
4-bromobenzyl alcohol and 4-bromo-allylbenzene. Some of the first para-functionalized diene diols pro-
duced using TDO, are useful substrates for further synthetic manipulations, including their use in the
potential synthesis of complex natural products.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
not been exploited for synthetic purposes, possibly because of low
enantiotopic differentiation by the enzyme. One exception is the
Chiral cyclohexadiene-cis-diols derived from aromatic precur-
sors through chemoenzymatic oxidation by mutants of the soil
bacterium Pseudomonas putida and recombinant clones of Esche-
richia coli have proven to be very useful additions to the chiral
pool.1–6 In wild-type organisms, this family of enzymes allow the
use of benzene, naphthalene and biphenyl compounds to be
metabolized as an energy source. Gibson et al. reported the first
bio-oxidative degradation of benzene and its simple derivatives
in 19687,8 and then in 1970 reported the first example of a mutant,
Pp39D, capable of blocking the further degradation of diols of type
1 (Scheme 1).9 This result was first exploited by Ley who used cis-
1,2-dihydroxycyclohexa-3,5-diene for the synthesis of pinitol.10
Since this report, the bio-oxidation of arenes has provided access
to many useful chiral building blocks with an accompanying inter-
est regarding the substrate-tolerance of the enzyme. Known
metabolites were comprehensively reviewed up to 2004 by John-
son et al.,6,11–17 while recent reviews describe the range of syn-
thetic targets that have been attained from cis-diene-diols.1–6,18
The published library of compatible mono-substituted and
ortho-substituted substrates is considerable, presumably because
their enzymatic dihydroxylation is almost completely enantiose-
lective.11,12 However, there have been very few para-substituted
substrates published to date, and the majority of the metabolites
do not contain useful functional groups at the 1- and 4-positions
that can be easily manipulated. Notable exceptions include several
dihalogenated metabolites, which have been used to access the
ent-series of diene diols after downstream reduction of an iodine
atom, as reported by Boyd et al. (Fig. 1).19,20 These compounds have
use of 4 by Hudlicky et al. in order to access the ent-series for
7-deoxypancratastatin synthesis;21 however, the 1,4-substitution
arising from the original functionality of the arene derivative was
not conserved in the final product. Consequently, there remains a
need to investigate the enzymatic tolerance for para-substituted
benzenes. Herein we report the isolation of several new metabo-
lites derived from their bio-oxidation.
2. Results and discussion
Diene diol metabolites provide ideal chiral starting materials for
an approach towards tetrodotoxin (Fig. 2), a marine toxin first iso-
lated in 1950,22 which has gathered significant synthetic atten-
tion23–34 since its structural elucidation in 1965.35 We envisioned
that 1,4-functionalized cyclohexadiene diols will allow access to
the target in a very efficient manner. The greatest challenge in this
approach towards the desired alkaloid was thought to be the
installation of the two syn-carbon chains at opposite poles of the
core cyclohexane (Fig. 2). If both carbon chains could be introduced
prior to the enzymatic dihydroxylation, the target molecule could
be readily accessed through simple elaboration of the two dienes.
To this end, a series of 1,4-difunctionalized substrates were exam-
ined as potential substrates for the toluene dioxygenase enzyme
expressed by E. coli JM109 (pDTG601A) a recombinant organism
developed by Gibson (Table 1).36
Initial results supported the hypothesis that para-substituted
benzene derivatives were poorer substrates than their better-stud-
ied ortho- or mono-substituted counterparts. The majority of the
compounds were not converted by the endogenous enzymes into
the desired diene diols. All three carboxylic acid derivatives 6, 7
and 8 failed to provide the desired compound when fed to the cells
⇑
Corresponding author. Tel.: +1 (905) 688 5550x3406; fax: +1 (905) 682 9020.
E-mail address: thudlicky@brocku.ca (T. Hudlicky).
0957-4166/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2013.01.002

J. F. Trant et al. / Tetrahedron: Asymmetry 24 (2013) 184–190
185
knocked-out in
JM109 (pDTG601)
R
R
R
toluene
catechol
OH
OH
OH
OH
dioxygenase
dehydrogenase
acetate
P. putida
wild strain
2
1
Scheme 1. Comparison of the degradation pathways of aromatics by wild type P. putida and the bioengineered strain.
determined for 4 was identical to that previously reported by
Boyd.19 This, however, is the first reported isolation of meso diene
diol 28. The diol had been previously prepared by Hudlicky et al. in
2006 in low yield during the study dealing with the dihydroxyla-
tion of all three isomeric dibromobenzenes and the synthesis of
(ꢀ)-conduritol E and consequently was not fully characterized at
the time.40
I
I
I
OH
OH
OH
OH
OH
OH
F
Cl
Br
The ideal substrate for our current purposes, 16, was investi-
gated along with a series of related methyl esters, 17, 18, 19 and
20. All of these esters failed to provide any detectable metabolites,
and the starting material was recovered from the fermentation
broth. Esters have proven to be less than ideal substrates in the past.
Previous reports from our laboratory have examined the substrate
scope of this enzyme using a variety of esters. Methyl benzoate pro-
vides its analogous diene diol in 1.3 g/L (on 9-L scale), considerably
less than the 20–22 g/L routinely achieved with bromobenzene. As
the steric bulk of the ester increases, the yield rapidly decreases;
thus butyl benzoate is not a suitable substrate for this enzyme.41
The introduction of an ortho halogen group has been shown to be
slightly tolerated: 2-bromomethyl benzoate provides the diene diol
in 200 mg/L;42 however it appears that the introduction of a para
substituent completely destroys the enzyme’s tolerance of the sub-
strate, as opposed to the better tolerated ortho substitution. Conse-
quently, it appears that adding the steric concerns of para
substitution to the poor enzyme tolerance of esters produces a com-
pound incompatible with the TDO enzymatic pocket. To circumvent
this problem, a series of para-bromo- and iodophenylacetylenes
were prepared, as small alkyl substituents are well precedented
as good substrates. Symmetrical bis-trimethylsilylprotected diacet-
ylene 21 was not metabolized and hence was quantitatively recov-
ered from the fermentation. However, both the iodo-TMS acetylene
derivative 22 and the bromo analogue 23 produced small amounts
of metabolites along with the recovered starting material. para-
Iodophenyl acetylene 24, provided only trace amounts of the prod-
uct, while bromo-substituted 25 furnished a respectable 640 g/L
(30% yield) of diene diol 29 (in a 95:5 enantiomeric ratio) after
extraction of the fermentation mixture. Due to the successful con-
version of the acetylene compound, the allyl derivative 26 was also
prepared. It furnished, upon fermentation, diene diol 30 in 850 g/L
(40% yield, 82:18 enantiomeric ratio).
3 94:6 er
5
58:42 er
4 61:39 er
Figure 1. Previously generated 1,4-‘difunctionalized’ diene diols.
O-
OH
O
OH
+
H
NH2
O
N
HO
N
H
HO
OH
tetrodotoxin
6
Figure 2. Tetrodotoxin with syn 1,4 carbon chains highlighted.
as either the free acid (dissolved in DMSO prior to addition to
broth) or as the sodium salt; in all cases the substrates were recov-
ered in high yields. This was somewhat surprising since 6 had been
previously reported by Ribbons et al. in 1987 using P. putida JT107
to be an excellent substrate, with complete enantiotopic discrimi-
nation.37,38 Even simpler structures provided unsatisfactory re-
sults. Benzyl bromide derivative 9 proved not to be a substrate
and was recovered unchanged. Similar results were obtained for
dioxolane 10 and acetate 11. These substrates were chosen to serve
as protected derivatives of the benzyl alcohol (a known toxin to the
bacteria) and benzaldehyde derivatives, respectively, but neither
provided any diene diol products; interestingly, dithiane 14 was
completely digested in the fermentation, presumably through
oxidation of the sulfur atoms as this is a generally recognized phe-
nomenon.39 However, when benzaldehyde derivative 12 or benzyl
alcohol derivative 13 was examined, both provided the same diol,
27, with similar yields, approximately 700 mg/L. This result was
particularly satisfying as the protected derivatives, hypothesized
to be more stable to the enzymatic conditions, failed to provide
the product; the aldehyde was presumably reduced to the alcohol
during the reaction through another process. However, as the
enantiomeric ratios of the 27 differ based on whether the source
material was the aldehyde 12 (60:40) or the benzyl alcohol 13
(78:22), it appears that this competing reduction occurs on the
same time-scale as the dihydroxylation.
In all cases, the enantiomeric ratios were determined through
chiral HPLC of the diene diols. Their conversion to known com-
pounds demonstrated their absolute stereochemistry. The derivati-
zations of the diene diols were carried out for the determination of
the absolute stereochemistry and are shown in Scheme 2.
Alkynyl diol 29 was protected as acetonide 31 and then reduced
with Adam’s catalyst to the fully saturated cyclohexane 32.
Although this compound has been previously reported by Gibson
et al.,43 they provided no characterization data for it at the time;
consequently, enantiopure acetonide diol 33, derived from ethyl
benzene, was treated in a similar fashion to provide an enantiopure
sample of 32.44,45
As a control of the fermentation methodology and bacterial
strain, 4-bromo-iodobenzene 4 and 1,4-dibromo benzene 15 were
treated under the fermentation conditions. Both provided their
diene diols, 730 mg/L (65% yield, 60:40 er) and 920 mg/L (80%
yield, meso), respectively. It is important to note that these were
prepared only on a 1-L scale with 1 g of substrate; consequently
these are excellent yields and conversions. The enantiomeric ratio
Allyl diene diol 30 was dehalogenated under standard radical
conditions to provide diol 34, whose properties were compared

186
J. F. Trant et al. / Tetrahedron: Asymmetry 24 (2013) 184–190
Table 1
Results of the whole-cell fermentation of potential metabolites using E. coli JM 109 (pDTG601)
Product
Product
Product
Substrate
R
Substrate
Br
Substrate
HO
(g/L; % yield, er)
(g/L; % yield, er)
(g/L; % yield, er)
O
d
a
a,c
6
14
21
22
R
Br
S
S
TMS
R=
CO2H
Br
I
TMS
Br
Br
a,c
OH
7
8
15
16
28
OH
Br
1.5 g/L, 80 %, meso
OH
OH
Br
CO2H
OH
TMS
Br
I
a,c
traceb
a
TMS
CO2Me
CO2Me
23
Br
OH
a
a
17
18
9
OH
Br
TMS
I
Br
O
Br
traceb
I
O
a
a
10
24
CO2Me
CO2Me
Br
AcO
AcO
traceb
OH
OH
a
11
12
19
OH
Br
Br
O
traceb
29
25
OH
HO
CO2Me
Br
Br
OH
OH
0.64 g/L, 30 %, 95:5
a
27
20
Br
Br
Br
0.68 g/L, 34 %, 60:40
OH
TMS
26
30
HO
HO
OH
Br
OH
OH
13
27
Br
0.85 g/L, 40 %, 81.5:18.5
Br
0.70 g/L, 35 %, 78:22
a
b
c
Unreacted arene recovered.
Trace diene diol observed by crude NMR of extract, deemed insufficient for isolation. Stereochemistry assigned by analogy to similar compound.
Substrate tested as both carboxylic acid and sodium salt.
No unreacted arene or other product observed in crude NMR of extract.
d
to those of a previously published standard.45 Diene diol 27
derived from benzyl alcohol could not be successfully dehalogenat-
ed but its acetonide derivative was successfully dehalogenated in
low yield to provide 36, whose enantiomer is known.46 Alterna-
tively, Adams’ reduction provided cyclohexane 37, which was tosy-
lated to the activated 38, which could be chromatographed. This

J. F. Trant et al. / Tetrahedron: Asymmetry 24 (2013) 184–190
187
HO
HO
O
O
O
O
O
O
DMP, TsOH
PtO2, H2
NEt3
PtO, H2
NEt3
Br
29
Br
31
32
33
OH
OH
OH
OH
Bu3SnH
AIBN, PhH
Br
30
34
TsO
HO
HO
HO
O
O
O
O
OH
OH
O
O
pTsOH
DMP
TsCl
Pyr.
PtO2, H2
38
37
Br
Br
35
1) stand overnight
2) LAH
27
Bu3SnH
AIBN, PhH
OH
OH
HO
39
O
O
36
Scheme 2. Derivatization of metabolites to known compounds for determination of absolute stereochemistry.
compound decomposed readily (in under 12 h) when left under ar-
gon at 4 °C, presumably through elimination of the tosylate and
then acid-catalysed loss of the acetonide. Treatment of the decom-
position residue with lithium aluminum hydride provided the
known diol 39 as the major product.47
3. Conclusion
We have identified several para-substituted arenes suitable as
substrates for toluene dioxygenase. The flexibility in the choice
of the substituents carries the potential for further elaboration to
readily incorporate carbon chains at the 1- and 4-positions of the
resulting cyclohexadiene ring. Although the enantioselectivity of
these transformations has been found to not be extremely high
in most cases, it could be significantly improved upon by investi-
gating substrates containing different halogen atoms, as based on
recent precedents.19,42 Studies to this effect are currently under-
way. Consequently, second-generation substrates of this family
could provide useful chiral starting materials to access complex
natural products containing this chiral cis-diol motif, such as tetro-
dotoxin, (+)-tutin,50 forskolin D,51 picrotoxin and picrotoxinin52
and nimbin,53 among many others. The formal synthesis of tetro-
dotoxin based on this technology will be reported in due course.
None of the three new diols obtained in good yield could be
isolated with complete selectivity, although alkynyl derivative
29 was obtained in a 95:5 enantiomeric ratio, which is consistent
with the size difference between the larger alkyne and smaller
bromine atom as predicted by Boyd.19 Allyl derivative 30 is not
generated with higher selectivity, although the allyl group is still
considered ‘larger’ by the enzyme than the bromine substituent,
and the enantiomeric ratio drops to 82:18. This discrepancy could
be related to the differences in the geometry of the linear alkyne
and the more flexible allyl chain. As aforementioned, benzyl alco-
hol has not been reported on as a substrate, possibly because of
its inherent toxicity towards E. coli (when benzyl alcohol was
subjected to fermentation conditions, only trace conversion was
noted).48,49 However, the theoretical product of this fermentation,
diene-diol 36, can be readily generated from either para-bromob-
enzyl alcohol or para-bromobenzaldehyde in acceptable yields.
Both substrates are better tolerated by the enzyme than methyl
benzoate, an alternative source of 36. The enantioselectivity of
the two processes varies greatly. The benzyl alcohol derivative
provides sufficient bias to allow for the formation of the product
in a 78:22 enantiomeric ratio. However, the benzaldehyde deriv-
ative provides almost no enantiotopic bias, as diol 27 is generated
in a near-racemic 60:40 enantiomeric ratio. The size difference
between the two substrates is not large, the difference in hydro-
gen-bonding properties is maybe responsible for the observed dif-
ference in enantioselectivity.
4. Experimental
4.1. General
All non-hydrogenation reactions were carried out under an ar-
gon atmosphere, hydrogenation reactions were carried out under
a hydrogen balloon. Glassware used for moisture-sensitive reac-
tions was flame-dried under vacuum and subsequently purged
with argon. THF, DME and toluene were distilled from potas-
sium/benzophenone. Methylene chloride and acetonitrile were
distilled from calcium hydride. Flash column chromatography
was performed using Kieselgel 60 (230–400 mesh) pre-neutralized
with 2% triethylamine. Analytical thin-layer chromatography was

188
J. F. Trant et al. / Tetrahedron: Asymmetry 24 (2013) 184–190
performed using silica gel 60-F254 plates. Melting points are re-
ported uncorrected. IR spectra were recorded as neat samples or
in KBr pellets. 1H and 13C NMR spectra were obtained on either a
300 or 600 MHz instrument. Data are reported as (s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet, br = broad; cou-
pling constants(s) in Hz, integration. Specific rotation measure-
ments are given in deg cm3 gꢀ1 dmꢀ1. Mass spectra and high
resolution mass spectra were performed by the analytical division
at Brock University. Chiral HPLC was carried out on an Agilent 1100
series instrument equipped with a UV detector monitoring at
254 nm and an ODH chiral column. HPLC flow-rate was 0.5 mL/
min using a gradient from 95:5 hexane/iso-propanol to 5:95 hex-
ane/iso-propanol over 20 min (Condition A); or 90:10 hexane:iso-
propanol to 60:40 hexanes/iso-propanol over 20 min (Condition B).
of crude material which was determined to be 75% pure diol. The
crude was purified by column chromatography (3:1 hexanes:ethyl
acetate) to provide 5.75 g of pure product as an off-white solid
(0.64 g/L). Off-white solid. Rf = 0.31 (3:1 hexanes:ethyl acetate);
mp = 118–120 °C (MeOH/Et2O); ½a D20
ꢁ
¼ þ34:6 (c 0.5, MeOH); 1H
NMR (300 MHz, CDCl3): dppm 6.28 (d, J = 6.2 Hz, 1H), 6.05 (d,
J = 6.1 Hz, 1H), 4.22 (d, J = 5.9 Hz, 1H), 4.15 (d, J = 6.0 Hz, 1H),
3.24 (s, 1H); 13C NMR (75 MHz, CDCl3): dppm 129.8, 127.8, 126.4,
122.1, 82.7, 81.6, 71.8, 66.8; IR
mmax: (KBr) 3734, 3288, 3200,
2839, 1557, 1414, 1304, 1102, 1085, 1014, 844 cmꢀ1; HRMS (EI)
Calcd for C8H7BrO2 (M+): 213.9629. Found: 213.9627; MS (EI)
216 (49.6), 214 (53.9), 198 (42.9), 196 (43.9), 145 (17.8), 143
(18.4), 135 (52.2), 117 (22.6), 118 (16.7), 89 (83.9), 77 (100); HPLC
Condition A as described above (minor enantiomer rt = 5.4 min,
major enantiomer rt = 10.2 min).
4.2. Small scale fermentation
4.3.3. (1R,2R)-3-Allyl-6-bromocyclohexa-3,5-diene-1,2-diol 30
In two 1 L fermentation cultures of grown E. coli JM109
(pDTG601) cells was added 500 mg of substrate [for the detailed
procedure see the literature42,54]. The fermentation cultures were
shaken at 37 °C for the reaction period. After this time, the cells were
separated from the broth by centrifugation at 7000 rpm for 20 min.
The cell-free broth was extracted three times with a total of 2.4 L of
base-washed ethyl acetate. Evaporation of the ethyl acetate afforded
the diene diol that could be further purified using column
chromatography.
OH
OH
Br
4.3. Large scale fermentation
p-Bromoallylbenzene was fermented according to standard pro-
cedures in an 18 L fermenter. Following standard work-up the
supernatant was extracted three times with ethyl acetate and con-
centrated and purified by column chromatography (3:1 hex-
anes:ethyl acetate) to provide 7.66 g of pure product as an off-
white solid (0.85 g/L). Off-white amorphous powder. Rf = 0.37
(1:1 hexanes:ethyl acetate); mp = 128–130 °C (MeOH/Et2O);
Protocol carried out as previously described in
fermenter.42,54
a 9 L
4.3.1. 3,6-Dibromocyclohexa-3,5-diene-1,2-diol 28
Br
½
a 2D0
ꢁ
¼ þ13:6 (c 0.86, MeOH); 1H NMR (300 MHz, CDCl3): dppm
OH
6.33 (d, J = 6.07 Hz, 1H), 5.85 (tdd, J = 16.89, 10.10, 6.71, 6.71 Hz,
1H), 5.61 (td, J = 5.97, 1.39, 1.39 Hz, 1H), 5.18–5.09 (m, 2H), 4.32
(br s, 2H), 2.97 (ddd, J = 6.47, 2.62, 1.49 Hz, 2H), 2.51 (s, 1H), 2.31
(s, 1H); 13C NMR (75 MHz, CDCl3): dppm 140.0, 135.0, 127.1,
OH
Br
123.9, 119.7, 117.3, 72.8, 71.0, 37.3. IR mmax: (MeOH) 3429, 2095,
1642, 1424, 1286, 1097, 1079, 1049, 1018, 913, 838 cmꢀ1. HRMS
(EI) Calcd for C9H11BrO2 (M+): 229.9942. Found: 229.9945. HPLC
Condition A as described above (minor enantiomer rt = 4.1 min,
major enantiomer rt = 7.0 min).
This compound was previously prepared by Hudlicky et al. in
2006.40 1,4-Dibromobenzene was shaken overnight with mature
cells of E. coli JM109 (pDTG601A) under the general protocol (small
scale) described above. The crude white solid was recrystallized
from ether to provide 1.6 g of white needles (1.6 g/L). White nee-
dles. Rf = 0.3 (4:1 ethyl acetate:hexanes); mp = 131–133 °C
(CDCl3); 1H NMR (300 MHz, CDCl3/MeOD): dppm 6.12 (s, 2H), 4.30
(s, 2H), 3.52 (s, 2H); 13C NMR (75 MHz, CDCl3/MeOD): dppm
4.3.4. (1R,2R)-3-Bromo-6-ethynyl-[1,2]-isopropylidenedioxycyc-
lohexa-3,5-diene 31
129.2, 125.7, 72.6; IR
C
m
max: (KBr) cmꢀ1. HRMS (EI) Calcd for
15H20O2 (M+): 232.1463. Found 232.1465.
4.3.2. (1R,2R)-3-Bromo-6-ethynylcyclohexa-3,5-diene-1,2-diol
29
O
O
Br
Diol 29 (300 mg, 1.4 mmol) was dissolved in acetone (10 mL)
and 2,2-dimethoxypropane (1 mL) along with catalytic pTsOH
(10 mg). The reaction mixture was stirred for 4 h, neutralized with
triethylamine (1 mL) and concentrated under reduced pressure.
The crude mixture was directly purified by flash chromatography
(4:1 hexanes:ethyl acetate, 0.5% triethylamine) to provide
278 mg of the acetonide as a white solid. White powder. Rf = 0.32
(9:1 hexanes:ethyl acetate); mp = 78–81 °C; 1H NMR (300 MHz,
CDCl3): dppm 6.39 (d, J = 6.5 Hz, 1H), 6.21 (d, J = 6.5 Hz, 1H), 4.77
(d, J = 8.4 Hz, 1H), 4.67 (d, J = 8.4 Hz, 1H), 3.28 (s, 1H), 1.45 (s,
3H), 1.45 (s, 3H); 13C NMR (75 MHz, CDCl3): dppm 135.8, 125.5,
OH
OH
Br
p-Bromoethynylbenzene55 was fermented according to stan-
dard procedures in an 18 L fermenter. Following standard work-
up and recovery of the unreacted material, the supernatant was
extracted three times with ethyl acetate. The combined organics
were then washed with brine and concentrated to provide 15.5 g

J. F. Trant et al. / Tetrahedron: Asymmetry 24 (2013) 184–190
189
122.9, 119.0, 106.6, 76.1, 72.9, 63.2, 26.5, 24.9; IR
m
max: (CHCl3)
4.3.7. (1S,2R,3S)-3-Hydroxymethyl-[1,2]-isopropylidenedioxy-
cyclohexane 37
3288, 2934, 1626, 1557, 1415, 1305, 1085, 1014, 896, 844,
778 cmꢀ1. HRMS (EI) Calcd for C11H11BrO2 (M+): 253.9942. Found:
253.9938.
OH
4.3.5. (1R,2R)-3-Bromo-6-(hydroxymethyl)cyclohexa-3,5-diene-
1,2-diol 27
O
O
OH
OH
To a degassed solution of acetonide-protected diene diol 35
(800 mg, 3.07 mmol) in methanol (16.0 mL) and triethylamine
(3.2 mL) was added Adams’ catalyst (PtO2, 80 mg) and the atmo-
sphere was evacuated and replaced with hydrogen. The reaction
mixture was stirred for 12 h at ambient temperature under a
hydrogen atmosphere, and then filtered through Celite along with
methanol. The solvent was removed under reduced pressure and
the residue was purified by flash chromatography to provide
320 mg of the product as a clear oil in 57% yield. Clear oil.
OH
Br
p-Bromobenzyl alcohol was fermented according to standard
procedures in an 18 L fermenter. Following standard work-up the
supernatant was extracted three times with ethyl acetate and con-
centrated and purified by recrystallization from diethyl ether to
provide 6.12 g of pure product as a white solid (0.68 g/L). White
Rf = 0.48 (1:1 hexanes:ethyl acetate); ½a D20
¼ ꢀ6:1 (c 2.0, CHCl3);
ꢁ
1H NMR (300 MHz, CDCl3): dppm 4.33 (dd, J = 5.3, 3.5 Hz, 1H), 4.16
(td, J = 9.0, 5.5, 5.5 Hz, 1H), 3.83 (dd, J = 11.0, 4.3 Hz, 1H), 3.73
(dd, J = 11.0, 5.8 Hz, 1H), 1.89–1.72 (m, 3H), 1.54 (s, 3H), 1.58–
1.45 (m, 3H), 1.39 (s, 3H) 1.35–1.27 (m, 1H); 13C NMR (75 MHz,
CDCl3): dppm 108.0, 74.7, 74.5, 60.2, 39.3, 28.4, 27.9, 25.9, 21.5,
powder. ½a 2D0
ꢁ
¼ ꢀ8:3 (c 1.0, MeOH, 7:3 er); 1H NMR (300 MHz,
CDCl3): dppm 6.38 (d, J = 6.0 Hz, 1H), 5.80 (ddd, J = 6.1, 3.0, 1.6 Hz,
1H), 4.38 (dd, J = 6.0, 0.8 Hz, 1H), 4.24–4.14 (m, 3H); 13C NMR
(75 MHz, CDCl3): dppm 142.3, 127.9, 126.5, 119.2, 74.1, 71.2, 63.0;
20.2; IR mmax: (CHCl3) 3691, 3626, 3011, 2940, 2870, 1732, 1451,
IR mmax: (CHCl3) 3337, 2920, 2861, 1648, 1579, 1388, 1333, 1096,
1382, 1372, 1235, 1039, 863 cmꢀ1; HRMS (EI) Calcd for C9H15O3
(M+-CH3): 171.1021. Found: 171.1036.
1014, 842 cmꢀ1; HRMS (EI) Calcd for C7H9BrO3 (M+): 219.9735.
Found: 219.9731; HPLC Condition B as described above (minor
enantiomer rt = 11.4 min, major enantiomer rt = 10.0 min).
Acknowledgments
4.3.6. (1S,2R,3R)-3-Ethyl-[1,2]-isopropylidenedioxycyclohexane
32
The authors are grateful to the following agencies for financial
support of this work: Natural Sciences and Engineering Research
Council of Canada (NSERC) (Idea to Innovation and Discovery
Grants), Canada Research Chair Program, Canada Foundation for
Innovation (CFI), TDC Research, Inc., TDC Research Foundation,
Brock University (Fellowship to J.F.), and the Ontario Partnership
for Innovation and Commercialization (OPIC). We are also grateful
to Dr. Costa Metallinos for the use of his chiral HPLC, and Joshni
John for her technical assistance with the HPLC.
O
O
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