Expanding the chiral pool: Oxidation of meta-bromobenzoic acid by R. eutrophus B9 allows access to new reaction manifolds

Article abstract of DOI:10.1039/c1ob05131h

Metabolism of meta-bromobenzoic acid by the blocked mutant Ralstonia eutrophus B9 affords an enantiopure dearomatised halodiene-diol which we demonstrate is a versatile chiron for organic synthesis. The presence of the halogen leads to reactivity that is

Full text of DOI:10.1039/c1ob05131h

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PAPER
Expanding the chiral pool: oxidation of meta-bromobenzoic acid by
R. eutrophus B9 allows access to new reaction manifolds†
Julia A. Griffen,a Ame´lie M. Le Coz,b Gabriele Kociok-Ko¨hn,a Monika Ali Khan,a Alan J. W. Stewartc and
Simon E. Lewis*a
Received 24th January 2011, Accepted 7th March 2011
DOI: 10.1039/c1ob05131h
Metabolism of meta-bromobenzoic acid by the blocked mutant Ralstonia eutrophus B9 affords an
enantiopure dearomatised halodiene-diol which we demonstrate is a versatile chiron for organic
synthesis. The presence of the halogen leads to reactivity that is distinct to that observed for the
non-halogenated analogue and also serves as a synthetic handle for further functionalisation.
Introduction
The dearomatising dihydroxylation of an aromatic substrate by a
microorganism was first reported by Gibson over 40 years ago.1 It
was subsequently recognised that the resultant diene diols were
useful starting materials for synthesis owing to their densely-
packed, differentiated functionality. Indeed, their synthetic value
is enhanced further by the fact that substituted arenes give rise
to enantiopure diols in most instances. The production and
utilisation of these arene-derived diols has become established
methodology and the field has been the subject of several excellent
reviews.2
Thus far, over 400 arene cis-diol products have been reported.
The vast majority of these are produced by organisms expressing
toluene dioxygenase (TDO), naphthalene dioxygenase (NDO) and
biphenyl dioxygenase (BPDO) enzymes. These metabolise substi-
tuted arene substrates in a regio- and stereoselective fashion. A reli-
able predictive model has been reported for such transformations3
and the sense of enantioinduction is conserved across organisms
and substrates (Scheme 1a, ortho-meta oxygenation). In contrast,
organisms expressing benzoate dioxygenase (BZDO) enzymes
oxidise benzoic acids in a process that exhibits not only different
regioselectivity, but also the opposite sense of enantioinduction.
For example, R. eutrophus B9,4 P. putida U1035 and P. putida
KTSY01 (pSYM01)6 oxidise benzoic acid to benzoate 1,2-cis
dihydrodiol 4 (Scheme 1b, ipso-ortho oxygenation).
Scheme 1 Regio- and stereoselectivity of dioxygenases.
Diol acid 4 has seen several diverse applications in organic
synthesis to date. In 1995, Widdowson et al. reported the
production of 4 with P. putida U103.7 The absolute stereochemistry
of 4 was determined by means of X-ray crystallographic analysis
of a para-bromobenzoyl derivative. The ability of derivatives of 4
to participate in [4 + 2] cycloadditions with various dienophiles
was also demonstrated. In 2001, Myers et al. described multiple
approaches for elaborating 4, demonstrating that each position
on the ring could be functionalised in a selective fashion through
judicious choice of reaction sequence.8 They also described in
detail a large-scale preparation of 4. That the derivatives described
therein are of synthetic utility was first shown by the report in
2004 of the synthesis of carbocyclic analogues of topiramate.9
In this work, Parker et al. described a route to analogues of
this anticonvulsant agent, requiring between 3 and 4 steps from
one of Myers’ chirons; the authors also described the first use
of 4 to access a carbohydrate target, carba-b-L-fructopyranose.
Also in 2004, Mihovilovic et al. reported intramolecular Diels–
Alder reactions of derivatives of 4 bearing tethered dienophiles.10
In 2005, Myers et al. disclosed the total synthesis of natural
and unnatural tetracycline antibiotics11 via a synthetic sequence
commencing from a derivative of 4 they had described four years
previously.8 It is worth noting that whilst the first stereocentre in
the target tetracyclines was set in the arene dihydroxylation step, all
subsequent stereocentres were installed under substrate control; all
stereochemical information in the final products is thus of ultimate
enzymatic origin. In 2010, the Mihovilovic group published a
full paper12 on intramolecular Diels–Alder reactions with 4 and,
most recently, the chemistry of 4 has been augmented by our
aDoctoral Training Centre in Sustainable Chemical Technologies, University
of Bath, Bath, UK BA2 7AY. E-mail: S.E.Lewis@bath.ac.uk; Fax: +44
(0)1225 386231; Tel: +44 (0)1225 386568
bESCOM, 1 Alle´e du Re´seau Jean-Marie Buckmaster, 60200, Compie`gne,
France
cProsidion Limited, Windrush Court, Watlington Road, Oxford, UK OX4
6LT
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra for all novel compounds. 2D NMR spectra for selected compounds.
Crystallographic data for 13 and 21. CCDC reference numbers 808270
and 808269. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c1ob05131h
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report that simple derivatives of 4 are amenable to complexation
to form tricarbonyliron(0)diene complexes.13 We have been able
to demonstrate that such complexation permits the synthesis of
otherwise inaccessible moieties. Most notably a diene possessing
the ortho-meta pattern of oxygenation found in 2 but antipodal to 2
may be accessed from 4 by means of such organoiron complexes.14
The unique synthetic versatility of 4 has thus been demon-
strated. However, we sought to enhance further the utility of the
BZDO-mediated benzoate dihydroxylation by use of substituted
benzoate substrates. The viability of metabolising substituted
benzoates by this approach has been established previously.
Studies on Ralstonia eutrophus B9‡ have shown that a variety
of mono- and disubstituted benzoates are acceptable substrates.
It was noted that turnover rates decreased in accordance with the
steric demand of a substituent, with the ortho position being least
tolerant of substitution (only ortho-fluorobenzoate underwent
dioxygenation) and the meta position being the most tolerant.4,15
Many other organisms expressing BZDOs have been evaluated for
their ability to process substituted benzoates,16 as have organisms
expressing TADO17 (toluate dioxygenase), TERDOS18 (tereph-
thalate dioxygenase) and IPADO19 (isophthalate dioxygenase).
To our knowledge, however, there is just one example to date
of the use in synthesis of an arene dihydrodiol derived from a
substituted benzoate: Banwell’s use of a metabolite derived from
meta-ethyltoluene in an approach to vinblastine20 (Scheme 2).
product formation and found that 5-substituted diols analogous
to 10 were formed more rapidly than the corresponding 3-
substituted regioisomers analogous to 9.15a In both of the above
studies, however, only meta-chloro- and meta-methyl benzoate
were employed as substrates, not meta-bromobenzoate 8. Reineke
and co-workers’ subsequent study15c has been the only one thus
far to specifically address the metabolism of meta-bromobenzoate
8 by R. eutrophus B9. Whilst the production of both 9 and
10 is described, the product ratio was not determined. In this
same study, it is stated that when using meta-methylbenzoate,
the 5-methyl analogue of 10 is “accessible only with difficulty”,
whereas the 3-methyl analogue of 9 is “isolable in good yield”;
such statements seem to contradict the earlier study.15a Thus, it
was unclear at the outset what the regiochemical outcome of
metabolism of 8 by R. eutrophus B9 would be.
Results and discussion
We undertook the bio-oxidation of 8 in accordance with the
procedure of Myers et al.,8 but on a 15 L scale. R. eutrophus
B9 cells were induced with a small quantity of benzoate before
addition of sodium meta-bromobenzoate solution portionwise
over 48 h; disodium succinate solution was added as a sole carbon
source. The fermentation broth was then centrifuged to remove
cellular material and the supernatant was concentrated under
reduced pressure. The concentrate was acidified to pH 3.0 and
extracted numerous times with ethyl acetate. Organic washings
were dried, then concentrated under reduced pressure to give
a crude mixture of 8, 9 and 10. NMR analysis indicated that
unreacted 8 was the major constituent of the crude mixture.
meta-Bromobenzoate 8 had been introduced to the fermentation
vessel at a rate comparable to that used previously8 in the non-
halogenated case, but R. eutrophus B9 metabolised the brominated
substrate much more slowly, as expected, leading to accumulation
of unreacted 8. Formation of 3-bromo product 9 was found to
have predominated (>10 : 1) over the 5-bromo isomer 10.
Purification of this crude material was effected by repeated
trituration with dichloromethane; this served to remove both the
starting material 8 and also the traces of 5-bromo product 10.
After 7 triturations, NMR analysis showed the residual quantity
of 8 to be negligible. By this means, pure 3-bromo product 9
was obtained in a yield of 65 mg per litre of fermentation broth,
approximately two orders of magnitude lower than that reported
for the unsubstituted benzoate.8
Scheme 2 Banwell’s access to arene diol 7 via meta-ethylbenzoic acid.
In the present study, we opted to exploit arene diols derived from
the metabolism of meta-bromobenzoic acid by R. eutrophus B9
(Scheme 3). We anticipated a twofold effect due to incorporation of
a bromine in the products. Firstly, this halide would modulate the
electron density of the diene such that it would exhibit reactivities
distinct from those of the parent system 4. Secondly, a bromodiene
would be amenable to diverse cross-coupling reactions to permit
further functionalisation in a manner that would not be possible
for unsubstituted diene 4.
With access to 9 secured we sought to verify the absolute
stereochemistry through X-ray analysis of a crystalline derivative.
The configuration of 4 was originally determined by Wid-
dowson et al. through formation of the corresponding para-
bromobenzoylmethyl ester and subsequent X-ray analysis;7 the
same approach was adopted to determine the configuration of
7.20 However, as 9 already incorporates a heavy atom, we opted
instead to target a crystalline cycloadduct. Esterification of 9 by
means of (trimethylsilyl)diazomethane to give 11 was followed by
diol protection as acetonide 12. Upon treatment with 4-phenyl-
1,2,4-triazoline-3,5-dione, 12 underwent [4 + 2] cycloaddition to
afford crystalline adduct 13 (Scheme 4).21
Scheme 3 Arene diol metabolites of meta-bromobenzoic acid.
As is the case for any meta-substituted benzoate, metabolism of
8 may give rise to two regioisomeric diols, 9 and 10, reflecting
the possibility of the substrate being accommodated in two
possible orientations in the BZDO active site. In the specific
case of substrate 8, literature precedent was ambiguous. In their
original report on R. eutrophus B9,4 Reiner and Hegeman describe
the isolation of both 3- and 5-substituted arene diols from the
metabolism of meta-substituted benzoates, but product ratios were
not quantified. Subsequently, Knackmuss and Reineke quantified
Single crystals of 13 suitable for X-ray structure determination
were obtained from diffusion of petroleum ether into a solution
of 13 in ethyl acetate. The structure of 13 is depicted in Fig. 1 and
‡ Previously known as Alcaligenes eutrophus B9.
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has also been reported22,26). The behaviour of the bromodiene
acetonide 12 reported here is in marked contrast to the above cases,
in that we have observed no dimerisation of 12 upon prolonged
storage at room temperature or below. Dimerisation of the non-
brominated analogue of 12 has also not been reported. We ascribe
this inertness to dimerisation to the fact that such acetonides
with the ipso-ortho pattern of oxygenation (cf. 4) present sterically
demanding substituents on both sides of the diene, hence retarding
the formation of all possible isomeric dimers.
We next examined the susceptibility of the bromodiene to
oxidative elaboration. Neither 11 nor 12 gave tractable products
upon attempted epoxidation with mCPBA, in contrast to the cor-
responding non-halogenated dienes.8 However, osmium tetroxide-
mediated dihydroxylation of 12 proceeded smoothly to afford
diol 14 as the sole regio- and diastereoisomer (Scheme 5). Such
selectivity is also in contrast to the non-halogenated series, wherein
the corresponding diene 15 undergoes dihydroxylation to afford
a 5 : 1 mixture of regioisomers favouring the other olefin.8 The
installation of the free diol functionality in 14 on the b-face was
confirmed by NOESY NMR experiments.† Reductive elaboration
of bromodiene 12 also proceeded in a markedly different fashion
to the non-halogenated analogue 15. Whereas LiAlH4-mediated
reduction of the ester in 15 to give 16 is reportedly7,10,12 high-
yielding (Scheme 6a), the corresponding reduction of bromodiene
12 affords the primary alcohol 17 in only 11% yield. We ascribe
this low yield partly to the concomitant formation of appreciable
amounts of debrominated product 16, which we have isolated
(Scheme 6b).
Scheme 4 Formation of crystalline derivative.
Fig. 1 Solid state structure of 13. Ellipsoids are represented at 50%
probability. H atoms are shown as spheres of arbitrary radius.
confirms the absolute stereochemistry as (1S,2S). Thus, the sense
of enantioinduction in the formation of 9 is the same as in the
formation of 4 and 7, as expected.
That the sole cycloadduct formed (13) is that in which the
dienophile approaches anti to the acetonide is in keeping with
Widdowson’s precedent.7 Aside from that report, all literature
examples of heterodienophile cycloadditions with arene diol
derived acetonides involve substrates with the ortho-meta pattern
of oxygenation (cf. 2). Here also, every report describes dienophile
addition anti to the acetonide.22,23 In such substrates the original
arene substituent is attached to the diene. In contrast, in 12 (which
has the ipso-ortho pattern of oxygenation, cf. 4), the original
arene substituent is attached to an sp3 centre and will not be
coplanar with the diene. If the substituent is of sufficient steric
bulk, approach of the dienophile to both diene faces may be
hindered. Exclusive formation of 13 in this instance is indicative
of the steric bulk of the acetonide being the controlling factor in
determining regioselectivity of cycloaddition, not steric bulk of
the ester.
Scheme 5 Dihydroxylation on the b-face. Selected NOESY correlations
in 14 shown with double-headed arrows.
The above considerations are also relevant in the context of
Diels–Alder dimerisation. Arene diol derived acetonides with
the ortho-meta pattern of oxygenation (cf. 2) are known to
undergo spontaneous dimerisation in many instances; this has
been reported with halodienes, e.g. R = Br,23j,24a–c Cl23j,24b,c and
also with many other substituents on the diene (R = CF3,23b,j,24c
Scheme 6 Reductive transformations.
The complexation of arene diols of type 2 with a tricarbonyl-
iron fragment has been extensively studied.27 We have reported
previously13 on the complexation of 18 (the methyl ester of 4) to
afford 19 as the sole product, with iron complexed to the a-face
(Scheme 7a). A later attempt14 to reverse the facial selectivity in
this complexation by use of an acetonide 15 to block the a-face did
indeed result in complexation of iron to the diene b-face, but the
isolated product (20) was that in which acetonide migration had
occurred (Scheme 7b). We were therefore keen to determine the
R = C2H3,23j,24c,d R = CN,24e R = COOMe,24f R = COOEt,23w R =
COOCH2CCH,23w R = SiMe2H,24g,h R = SiMe2C2H3
and R =
24g,h
Me24i). In each instance, the dimerisation is reportedly totally
selective for formation of the adduct which is anti both with
respect to the diene and also the dienophile. (The only reported
exception to this trend is dimerisation of the acetonide of the
parent unsubstituted diene 2, R = H25 – a minor cycloadduct
deriving from syn diene addition and anti dienophile addition
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substituent by using transformations that would not be possible
for the unsubstituted case. A dienyl bromide is suggestive of
applications in cross-coupling chemistry and in the more common
ortho-meta arene dihydrodiol series, both 2 (R = Br)24a,28 and 2
(R = I)28a,h,n,29 have indeed been exploited in this context. To date,
such cross-couplings have not been reported for derivatives of 4.
We first examined a Suzuki-Miyaura coupling30 of protected
bromodiene 12 with para-tolylboronic acid. Union of these
fragments under palladium catalysis was indeed achieved, al-
though it was found that the reaction conditions also effected
ester hydrolysis. The free acid 23, recovered from the aque-
ous phase, was re-subjected to esterification with (trimethylsi-
lyl)diazomethane to afford the originally targeted methyl ester
24, albeit in moderate yield (Scheme 8). A much more straight-
forward cross-coupling was the Sonogashira31 reaction of 12 with
(triisopropylsilyl)acetylene, which afforded the dienyne 25 in near-
quantitative yield (Scheme 8).
Scheme 7 Iron complex formation for various related dienes.
outcome of complexation of bromodiene acetonide 12 as an iron
carbonyl in order to shed light on the mechanism of formation
of 20. In the event, treatment of 12 with nonacarbonyldiiron in
THF afforded isomeric complexes 21 and 22, in which 21 was the
major isomer (Scheme 7c). The structure of 21 was determined
unambiguously by X-ray crystallography (Fig. 2). (The minor
isomer 22, which was appreciably less stable than 21, could not be
crystallised successfully; its structure was assigned by 2D NMR
experiments† and by comparison with 21.) In this instance, the sole
products are those in which acetonide migration has not occurred.
This may be explained by the fact that in 12, the carbon to which an
acetonide oxygen would be bonded upon rearrangement already
bears the bromine substituent. We had previously concluded
that the formation of 20 arose via “clockwise” migration of the
acetonide, rather than “anticlockwise” migration of the ester.14
The absence of any such rearrangement in the case of bromodiene
12 is in keeping with this conclusion. The presence of the bromine
in 12 also retards the rate of complexation with respect to
unsubstituted analogue 15–identical reaction conditions result
in consumption of all of 15, but recovery of 60% unreacted 12
(Scheme 7).
Scheme 8 Cross-couplings of the bromodiene.
Dienyne 25 is a useful intermediate for further functionalisation.
Desilylation with TBAF affords terminal acetylene 26, which is
amenable to the Huisgen32 copper-catalysed azide-alkyne cycload-
dition protocol. We have demonstrated this through the reaction
of 26 with benzyl azide to afford triazole 27 (Scheme 9).
Scheme 9 Azide-alkyne “click” cycloaddition.
Fig. 2 Solid state structure of 21. Of two independent molecules in
the unit cell, only one is shown for clarity. Ellipsoids are represented at
50% probability. H atoms are shown as spheres of arbitrary radius.
Conclusions
The above transformations of bromodiene 12 represent cases
in which the presence of the halogen modulates the reactivity of
the system with respect to the unsubstituted diene case. However,
we wished to exploit further the value inherent in the bromine
We have demonstrated the versatility of a halobenzoate-derived
ipso,ortho-oxygenated arene dihydrodiol in various synthetic con-
texts and shown that the presence of the halogen fundamentally
alters the course of the reaction in many instances. The formation
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◦
R
of cross-coupling products 23–27 is especially significant, as
these structures are arene dihydrodiol derivatives that would not
be accessible by direct metabolism of the corresponding arene
precursors. Thus, for example, accessing 23 by biotransformation
of biaryl carboxylic acid 28 would not be expected to succeed as the
steric bulk of the tolyl substituent would likely preclude docking
of 28 in the R. eutrophus B9 BZDO active site. Compound 23 is
nevertheless accessible, by means of the indirect route described in
this work (Scheme 10).
was maintained at 30 C by means of immersed Nalgeneꢀ 380
food grade tubing, through which was passed heated water.
Subsequently, sodium benzoate solution (5 mL of a 1.0 M
solution) was added to initiate BZDO expression. After 3 h,
sodium meta-bromobenzoate (50 mL of a 1.0 M solution) and
sodium succinate (25 mL of a 1.5 M solution) were added. Over
the next 48 h, sodium meta-bromobenzoate solution (1.0 M)
and sodium succinate solution (1.5 M) were added portionwise.
In total 60.3 g (270 mmol) sodium meta-bromobenzoate and
48.6 g (300 mmol) sodium succinate were introduced. The culture
was incubated for a further 72 h, then the fermentation broth
was centrifuged at 6000 rpm for 25 min and the supernatant
liquid decanted. The supernatant was concentrated under reduced
◦
pressure (rotary evaporator bath temperature <40 C) to a total
volume of 1.5 L. The concentrated supernatant was then carefully
acidified to pH 3.0 with concentrated hydrochloric acid. The
acidified solution was then extracted with ethyl acetate (20 ¥
1 L). After each two extractions, the aqueous phase was re-
acidified to pH 3.0. Each organic extract was dried over MgSO4
and filtered. Combined filtrates were concentrated under reduced
pressure to give 51.4 g of crude material, of which unreacted meta-
bromobenzoic acid was the major constituent. This material was
repeatedly triturated with copious quantities of dichloromethane,
resulting in the dissolution of most of the material. (Toluene was
also determined to be a suitable triturant.) After 7 such triturations
anddryingunder vacuum, the residualsolid materialwas shown by
NMR to be pure (1S,6S)-5-bromo-1,6-dihydroxycyclohexa-2,4-
dienecarboxylic acid 9, a brown powder; 979 mg (4.17 mmol),
1.5%, 65 mg dm-3. [a]D25 -0.16 (c 0.1, CHCl3); dH (300 MHz,
CD3OD)15c 6.30 (1H, dd, J = 6.0, 1.0 Hz, CBr CH), 5.89 (1H,
dd, J = 9.5, 6.0 Hz, CBr CH–CH), 5.75 (1H, d, J = 9.5 Hz,
CBr CH–CH CH), 4.69 (1H, d, J = 1.0 Hz, HO–CH); dC
(75 MHz, CD3OD) 177.3 (C O), 130.6 (CBr), 128.5 (CBr CH–
CH CH), 127.0 (CBr CH–CH), 126.7 (CBr CH), 77.6 (C–
COOH), 74.6 (HO–CH); vmax(film) 3220, 1698, 1417, 1358,
1229, 1091, 1028, 992, 671 cm-1; HRMS (ESI-) m/z calcd for
(C7H8BrO4–CO2–H2O–H)-, 170.9446, 172.9425; found 170.9446,
172.9422.
Scheme 10
Current efforts in our laboratory are focused on optimising the
production process for 9 and incorporating this versatile chiron
in the synthesis of more complex targets, including appropriate
natural products. The results of these endeavours will be reported
subsequently.
Experimental
General
Reactions which required the use of anhydrous, inert atmosphere
techniques were carried out under an atmosphere of nitrogen.
Nonacarbonyldiiron was dispensed in a glovebox. Solvents were
dried and degassed by passing through anhydrous alumina
columns using an Innovative Technology Inc. PS-400-7 solvent
purification system. Petrol refers to petroleum ether, bp 40–
60 ◦C. TLCs were performed using aluminium-backed plates
precoated with AlugramꢀSIL G/UV and visualized by UV light
(254 nm) and/or KMnO4 followed by gentle warming. Flash
column chromatography was carried out using Davisil LC 60 A
silica gel (35–70 micron) purchased from Fisher Scientific. IR
spectra were recorded on Perkin–Elmer 1600 FT IR spectrometer
with absorbances quoted as n in cm-1. NMR spectra were run
in CDCl3 (unless otherwise specified) on Bru¨ker Avance 250,
300. 400 or 500 MHz instruments at 298 K. Mass spectra were
recorded with a micrOTOF electrospray time-of-flight (ESI-TOF)
mass spectrometer (Bru¨ker Daltonik).
R
˚
(1S,6S)-Methyl 5-bromo-1,6-dihydroxycyclohexa-2,4-diene-
carboxylate (11)
To a stirred solution of 9 (61 mg, 0.260 mmol, 1 eq.) in MeOH–
benzene (1 : 1, 32 mL) at room temperature was added dropwise
(trimethylsilyl)diazomethane (1.5 mL, 2.0 M in hexane) until the
yellow colour persisted and effervescence ceased. The solution
was stirred for 2 h then concentrated under reduced pressure to
give crude (1S,6S)-methyl 5-bromo-1,6 dihydroxycyclohexa-2,4-
diene carboxylate 11 (61 mg, 94%) as a brown oil, sufficiently pure
to be used without further purification: Rf 0.73 (50% EtOAc–
petrol); [a]2D5 -40 (c 0.175, CH2Cl2); dH (250 MHz) 6.38 (1H, ddd,
J = 6.0, 2.5, 0.5 Hz, CBr CH), 5.99 (1H, dd, J = 9.5, 6.0 Hz,
CBr CH–CH), 5.76 (1H, d, J = 9.5 Hz, CBr CH–CH CH),
4.79 (1H, brs, C(OH)H), 3.89 (3H, s, CH3); dC (75 MHz, CD3OD)
175.5 (C O), 130.3 (CBr), 127.9, 127.3, 126.8, 78.0 (C-COOMe),
74.6 (C(OH)H), 53.9 (O–CH3); vmax (film) 3451, 2953, 1734, 1643,
1572, 1255, 1105, 1034, 940, 809, 695 cm-1; HRMS (ESI+) m/z
calcd for (C8H9BrO4+Na)+, 270.9582, 272.9561; found 270.9576,
272.9559.
Biotransformation of meta-bromobenzoic acid
This was performed in accordance with a literature procedure,8
substituting meta-bromobenzoate for benzoate in the biotransfor-
mation step. A sterile pipette tip was streaked across the surface
of a frozen glycerol stock solution of R. eutrophus B9 cells to
produce small shards (approx. 10 mg). The frozen shards were
added to a sterile 250 mL Erlenmeyer flask containing 100 mL of
Hutner’s mineral base medium8,33 and aqueous sodium succinate
solution (500 mL of a 1.5 M solution). The flask was shaken at
250 rpm for 48 h at 30 ◦C. The culture was then transferred
to a 20 L plastic carboy and additional medium was added
to give a total volume of 15 L. Air was continuously sparged
through the culture. Sodium succinate solution (50 mL) was
added and the culture was incubated for 48 h. The temperature
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(3aS,7aS)-Methyl 7-bromo-2,2-dimethyl-3a,7a-dihydrobenzo-
[d][1,3]dioxole-3a-carboxylate (12)
EtOAc–petrol) to give (3aS,4R,5R,7aS)-methyl 7-bromo-4,5-
dihydroxy-2,2-dimethyl-3a,4,5,7a-tetrahydrobenzo[d][1,3]dio-
xole-3a-carboxylate 14 (11 mg, 80%) as a colourless oil: Rf 0.32
(40% EtOAc–petrol); [a]2D5 -5.5 (c 0.55, CH2Cl2); dH (500 MHz)
6.23 (1H, d, J = 2.5 Hz, CBr CH), 5.08 (1H, d, J = 0.5 Hz,
CBr–CH–O), 4.42 (1H, br s, CHCH(OH)CH(OH)), 4.32 (1H,
br s, CHCH(OH)CH(OH)), 3.87 (3H, s, –OCH3), 2.80 (1H, br s,
–OH), 2.76 (1H, br s, –OH), 1.46 (3H, s, C–CH3), 1.41 (3H, s,
C–CH3); dC (75 MHz) 170.8 (C O), 130.9 (CBr CH), 121.2
(CBr), 111.3 (–O–C–O–), 83.5 (C–COOCH3), 77.1 (CBr–CH–O–
C), 69.9 (C–OH), 66.2 (C–OH), 52.2 (O–CH3), 26.4 (C–CH3),
25.8 (C–CH3); vmax (film) 3405, 1734, 1647, 1373, 1236, 1098,
1062, 620 cm-1; HRMS (ESI+) m/z calcd for (C11H15BrO5+Na)+,
344.9950, 346.9929; found 344.9948, 346.9935.
To diol 11 (84 mg, 0.337 mmol, 1 eq.) and para-toluenesulfonic
acid (2.0 mg, 0.01 mmol, 0.03 eq.) in acetone (30 mL) was
added 2,2-dimethoxypropane (600 mL, 4.88 mmol, 14.5 eq.).
The reaction mixture was stirred at room temperature for 48 h,
transferred to a separating funnel, washed with saturated NaCl(aq)
then extracted with EtOAc. The organic phase was dried over
MgSO4 and filtered. The filtrate was concentrated under reduced
pressure to give (3aS,7aS)-methyl 7-bromo-2,2-dimethyl-3a,7a-
dihydrobenzo-[d][1,3]dioxole-3a-carboxylate 12 (68 mg, 70%) a
brown oil, sufficiently pure to be used without further purification:
Rf 0.82 (25% EtOAc–petrol); [a]2D5 -192 (c 0.65, CH2Cl2); dH
(250 MHz) 6.45 (1H, d, J = 6.0 Hz, CBr CH), 5.97 (1H, dd, J =
9.0, 6.0 Hz, CBr CH–CH), 5.90 (1H, d, J = 9.0 Hz, CBr CH–
CH CH), 5.08 (1H, s, CH–O–), 3.81 (3H, s, O–CH3), 1.47 (3H, s,
C–CH3), 1.43 (3H, s, C–CH3); dC (75 MHz) 171.3 (C O), 125.9
(CBr), 124.2, 124.1, 123.3, 108.5 (–O–C–O–), 81.8 (C–C–O), 78.5
(CH–O), 53.2 (–OCH3), 26.9 (–C–CH3), 25.5 (–C–CH3); vmax (film)
2971, 1775, 1706, 1602, 1358, 1240, 1211, 1159, 1009, 957, 758,
723, 687 cm-1; HRMS (ESI+) m/z calcd for (C11H13BrO4+Na)+,
310.9895, 312.9874; found 310.9886, 312.9873.
((3aR,7aS)-7-Bromo-2,2-dimethyl-3a,7a-dihydrobenzo-
[d][1,3]dioxol-3a-yl)methanol (17)
To ester 12 (68.9 mg, 0.235 mmol, 1 eq.) was added lithium
aluminium hydride (9.0 mg, 0.237 mmol, 1 eq.) as a solution
in diethyl ether (25 mL). The reaction mixture was stirred
at room temperature for 1 h, then cooled to 0 ◦C. Excess
EtOAc was added dropwise by syringe to quench unreacted
reductant, then the reaction mixture was cautiously added to
a saturated aqueous solution of sodium potassium tartrate
(Rochelle’s salt). The reaction mixture was stirred vigorously for
1 h, then the phases were separated. The organic phase was
dried over MgSO4 then filtered. The filtrate was concentrated
under reduced pressure and purified by chromatography (30%
EtOAc–petrol) to afford ((3aR,7aS)-7-bromo-2,2-dimethyl-3a,7a-
dihydrobenzo[d][1,3]dioxol-3a-yl)methanol (17) (7.0 mg 11%) as
a colourless oil. Debrominated product ((3aR,7aS)-2,2-dimethyl-
3a,7a-dihydrobenzo[d][1,3]dioxol-3a-yl)methanol (16) was also
isolated. Alcohol 17: Rf 0.45; (30% EtOAc–petrol); [a]2D5 -10 (c 0.2,
CH2Cl2); dH (300 MHz) 6.42 (1H, dd, J = 6.0, 0.5 Hz, BrC CH),
5.90 (1H, dd, J = 9.5, 6.0 Hz, BrC CH–CH), 5.78 (1H, dd,
J = 9.5, 0.5 Hz, BrC CH–CH CH), 4.91 (1H, s, CH–O–), 3.61
(1H, d, J = 11.5 Hz, –CHH–OH), 3.44 (1H, dd, J = 11.5, 7.0 Hz,
–CHH–OH), 1.85 (1H, br s, –OH), 1.49 (3H, s, CH3), 1.39 (3H, s,
CH3); dC (75 MHz) 127.9, 126.4, 123.4, 123.3, 107.9 (–O–C–O–),
82.9 (C–CH2OH), 77.7 (CH–O–C), 64.7 (–CH2OH), 27.0 (CH3),
26.9 (CH3); vmax (film) 3413, 2918, 2956, 2852, 1465, 1383, 1222,
1943, 750 cm-1; HRMS (ESI+) m/z calcd for (C10H11BrO3+Na)+,
282.9946: 284.9925; found 282.9932, 284.9922.
(3aS,4R,10R,10aS)-Methyl 10-bromo-2,2-dimethyl-6,8-dioxo-7-
phenyl-4,6,7,8,10,10a-hexahydro-3aH-4,10-etheno[1,3]-dioxolo-
[4,5-d][1,2,4]triazolo[1,2-a]pyridazine-3a-carboxylate (13)
To a solution of diene 12 (49 mg, 0.17 mmol, 1 eq.) in acetone
(4.0 mL) at room temperature was added in a dropwise fashion
a solution of 4-phenyl-1,2,4-triazoline-3,5-dione in acetone, until
a faint red colour of the dione persisted. After 1 h, the reac-
tion mixture was concentrated under reduced pressure to give
(3aS,4R,10R,10aS)-methyl 10-bromo-2,2-dimethyl-6,8-dioxo-7-
phenyl-4,6,7,8,10,10a-hexahydro-3aH-4,10-etheno[1,3]dioxolo-
[4,5-d][1,2,4]-triazolo[1,2-a]pyridazine-3a-carboxylate 13 (86 mg,
93% yield) as a white crystalline solid: mp 172–174 ◦C
(EtOAc : petrol); Rf 0.54 (50% EtOAc–petrol); [a]2D5 -11.5 (c 0.78,
CH2Cl2); dH (300 MHz) 7.44–7.37 (5H, m, Ar–H), 6.62 (1H,
dt, J = 10.0, 1.5 Hz, CBr–CH–CH), 6.37 (1H, dd, J = 10.0,
7.0 Hz, CBr–CH CH), 5.39 (1H, dd, J = 7.0, 1.5 Hz, N–CH–
C–COO–), 5.33 (1H, d, J = 1.5 Hz, CBr–CH–O), 3.93 (3H, s,
–OCH3), 1.42 (3H, s, C–CH3), 1.36 (3H, s, C–CH3); dC (75 MHz)
169.7 (O–C O), 154.3 (N–C O), 154.0 (N–C O), 135.4, 130.7,
129.2, 128.8, 127.6, 125.8, 114.3 (–O–C–O–), 84.2, 82.9, 67.7,
53.9, 53.8, 26.2, 26.1; vmax (film) 2993, 1786, 1721, 1559, 1501,
1457, 1400, 1260, 1214, 1149, 1088, 974, 880, 754 cm-1; HRMS
(ESI+) m/z calcd for (C19H18BrN3O6+H)+, 464.0457, 466.0437;
found 464.0449, 466.0429.
(4S)-Tricarbonyl(g4-(3aS,7aS)-methyl 7-bromo-2,2-dimethyl-
3a,7a-dihydrobenzo[d][1,3]dioxole-3a-carboxylate)iron(0) (21) and
(4R)-Tricarbonyl(g4-(3aS,7aS)-methyl 7-bromo-2,2-dimethyl-
3a,7a-dihydrobenzo[d][1,3]dioxole-3a-carboxylate)-iron(0) (22)
(3aS,4R,5R,7aS)-Methyl 7-bromo-4,5-dihydroxy-2,2-dimethyl-
3a,4,5,7a-tetrahydrobenzo[d][1,3]dioxole-3a-carboxylate (14)
To a flask containing 12 (185 mg, 0.59 mmol, 1 eq.) in a glovebox
was added nonacarbonyldiiron (440 mg, 1.21 mmol, 2 eq.). THF
(40 mL) was added and the reaction mixture was stirred at
room temperature for 7 d. The reaction mixture was then con-
centrated under reduced pressure (Care! Toxic pentacarbonyliron
distilled over at this point) and purified by column chromatogra-
phy (10% EtOAc–petrol) to give (4S)-tricarbonyl(h -(3aS,7aS)-
methyl 7-bromo-2,2-dimethyl-3a,7a-dihydrobenzo[d][1,3]dioxole-
3a-carboxylate)iron(0) 21 as fine brown needles (43 mg, 17%)
To a solution of 12 (14.0 mg, 0.048 mmol, 1 eq.) and NMO
(6.6 mg, 0.048 mmol, 1 eq.) in acetone/H2O 4 : 1 (3 mL) was
added OsO4 (10 mL, 2.5 wt% in tBuOH, 2 mol%). The reaction
mixture was stirred at room temperature for 24 h, then diluted with
Na2S2O3(aq) and extracted with EtOAc. The organic extracts were
dried over MgSO4 and filtered. The filtrate was concentrated under
reduced pressure and purified by column chromatography (40%
4
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Org. Biomol. Chem., 2011, 9, 3920–3928 | 3925
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4
and (4R)-tricarbonyl(h -(3aS,7aS)-methyl 7-bromo-2,2-dimethyl-
821 cm-1; HRMS (ESI+) m/z calcd for (C18H20O4+Na)+, 323.1259;
3a,7a-dihydrobenzo-[d][1,3]dioxole-3a-carboxylate)iron(0) 22 as a
brown oil (36 mg, 14%). Unreacted 12 (100 mg, 60%) was also
isolated. Complex 21: mp 95-97 ◦C (EtOAc : petrol); Rf 0.32 (10%
EtOAc–petrol); [a]D25 -10 (c 0.2, CH2Cl2); dH (300 MHz) 5.95
(1H, dt, J = 4.5, 1.5 Hz, CBr CH), 5.47 (1H, dd, J = 6.5,
4.5 Hz,CBr CH–CH ), 5.36 (1H, d, J = 1.5 Hz, CH–O–),
3.88 (3H, s, O–CH3), 3.06 (1H, dd, J = 6.5, 1.0 Hz, CBr CH–
CH CH), 1.45 (3H, s, C–CH3), 1.23 (3H, s, C–CH3); dC (75 MHz)
171.3 (–COOMe), 117.8 (–O–C–O–), 89.9 (CBr CH), 87.0 (C–
COOMe), 86.7 (CH–O–), 83.6 (CBr CH-CH ), 70.2 (CBr),
54.0 (CBr CH–CH CH), 53.3 (O–CH3), 28.1 (C–CH3), 27.3
(C–CH3); vmax (film) 2981, 2068, 2003, 1730, 1437, 1375, 1261,
1214, 1162, 1062, 1027, 978, 865, 752, 687, 636 cm-1; HRMS
(ESI+) m/z calcd for (C14H13BrFeO7+Na)+, 450.9092, 452.9071;
found 450.9106, 452.9164. Complex 22: Rf 0.26 (10% EtOAc–
petrol); [a]2D5 -90 (c 0.6, CH2Cl2); dH (500 MHz) 5.76 (1H, d,
J = 3.5 Hz, CBr CH), 5.15 (1H, dd, J = 6.0, 4.5 Hz, CBr CH–
CH ), 4.60 (1H, s, CH–O–), 3.75 (3H, s, O–CH3), 2.97 (1H, d, J =
6.5 Hz, CBr CH–CH CH), 1.71 (3H, s, C–CH3), 1.20 (3H, s,
C–CH3); dC (75 MHz) 207.1 (Fe C O), 173.3 (–COOMe), 109.7
(–O–C–O–), 89.3 (CBr CH), 85.4 (C–COOMe), 84.8 (CH–O–),
80.5 (CBr CH-CH ), 73.6 (CBr), 61.2 (CBr CH–CH CH),
53.1 (O–CH3), 25.0 (C–CH3), 24.2 (C–CH3); vmax (film) 2980, 2061,
1995, 1729, 1460, 1380, 1252, 1207, 1168, 1070, 1030 cm-1; HRMS
(ESI+) m/z calcd for (C14H13BrFeO7+Na)+, 450.9092, 452.9071;
found 450.9091, 452.9073.
found 323.1258.
(3aS,7aR)-Methyl 2,2-dimethyl-7-((triisopropylsilyl)ethynyl)-
3a,7a-dihydrobenzo[d][1,3]dioxole-3a-carboxylate (25)
To a solution of bromodiene 12 (81 mg, 0.28 mmol, 1
eq.), tetrakis(triphenylphosphine)palladium (16 mg, 0.014 mmol,
5 mol%), copper(I) iodide (3.7 mg, 0.0196 mmol, 7 mol%)
dissolved in THF (20 mL), was added by syringe n-butylamine
(110 mL, 1.12 mmol, 4 eq.) and (triiso-propyl)acetylene (100 mL,
0.45 mmol, 1.6 eq.). The reaction mixture was stirred at room
temperature for 24 h, then diluted with EtOAc and washed
with NH4Cl(aq) and NaCl(aq). The organic phase was dried over
MgSO4 and filtered. The filtrate was concentrated under reduced
pressure and purified by column chromatography (10% EtOAc–
petrol) to give (3aS,7aR)-methyl 2,2-dimethyl-7-((triisopropyl-
silyl)ethynyl)-3a,7a-dihydrobenzo[d][1,3]dioxole-3a-carboxylate
(25) (108 mg, 98%) as a yellow oil: Rf 0.45 (10% EtOAc–petrol);
[a]2D5 -176 (c 0.89, CH2Cl2); dH (250 MHz) 6.37 (1H, d, J = 6.0 Hz,
SiC C–C CH), 6.12 (1H, dd, J = 9.5, 6.0 Hz, SiC C–C CH–
CH), 5.85 (1H, dd, J = 9.5, 0.5 Hz, SiC C–C CH–CH CH),
4.91 (1H, d, J = 0.5 Hz, CH–O–), 3.78 (3H, s, O–CH3), 1.45 (3H, s,
C–CH3), 1.39 (3H, s, C–CH3), 1.08 (21H, br s, Si–CH and Si–CH–
CH3); dC (75 MHz) 171.7 (C O), 129.0 (alkene C), 125.0 (alkene
C), 124.4 (alkene C), 120.7 (alkene C), 108.2 (–O–C–O–), 105.5
(alkyne C), 96.7 (alkyne C), 80.0 (C–COOMe), 75.6 (CH–O–),
53.0 (O–CH3), 26.9 (C–CH3), 25.6 (C–CH3), 18.6 (Si–C–CH3),
11.3 (Si–C–CH3); vmax (film) 2943, 2865, 2158, 2032, 1741, 1462,
1381, 1243, 1039, 883, 677 cm-1; HRMS (ESI+) m/z calcd for
(C22H34O4Si+Na)+, 413.2124; found 413.2127.
(3aS,7aR)-Methyl 2,2-dimethyl-7-(para-tolyl)-3a,7a-dihydro-
benzo[d][1,3]dioxole-3a-carboxylate (24)
Bromodiene 12 (25.0 mg, 0.096 mmol, 1 eq.), tetrakis(triphenyl-
phosphine)palladium (2.0 mg, 0.002 mmol, 2 mol%), para-
tolylboronic acid (105 mg, 0.78 mmol, 8 eq.) and potassium
carbonate (238 mg, 1.73 mmol, 18 eq.) were dissolved in DMF–
H2O 5 : 1 (30 mL) and stirred at room temperature for 72 h. The
reaction mixture was diluted with EtOAc and washed with water.
The organic layer was devoid of product; thus, the aqueous layer
was concentrated under reduced pressure to afford crude free acid
cross-coupling product 23. The crude acid 23 was then dissolved
in MeOH–benzene 1 : 1 (35 mL) and (trimethylsilyl)diazomethane
(1.5 mL, 2.0 M in hexane) was added dropwise with stirring
until the yellow colour persisted and effervescence ceased. The
solution was stirred for 2 h then concentrated under reduced
pressure. Purification by column chromatography (10% EtOAc–
petrol) gave (3aS,7aR)-methyl 2,2-dimethyl-7-(para-tolyl)-3a,7a-
dihydrobenzo[d][1,3]dioxole-3a-carboxylate (24) (8 mg, 30% over
two steps) as a colourless oil: Rf 0.36 (5% EtOAc–petrol); [a]D25
-156 (c 0.3, CH2Cl2); dH (250 MHz) 7.50 (2H, d, J = 8.0 Hz,
Ar–H), 7.18 (2H, d, J = 8.0 Hz, Ar–H) 6.48 (1H, d, J = 6.0 Hz,
Ar–C CH), 6.23 (1H, dd, J = 9.5, 6.0 Hz, Ar–C CH–CH), 5.83
(1H, d, J = 9.5 Hz, Ar–C CH–CH CH), 5.26 (1H, s, CH–O–C),
3.77 (3H, s, O–CH3), 2.36 (3H, s, Ar–CH3), 1.53 (3H, s, C–CH3),
1.42 (3H, s, C–CH3); dC (75 MHz) 171.8 (C O), 138.2 (4◦), 135.3
(4◦), 134.6 (4◦), 129.4 (3◦ Ar), 125.8 (3◦ Ar), 124.8 (Ar–C CH–
CH), 124.5 (Ar–C CH–CH CH), 119.7 (Ar–C CH), 107.6
(–O–C–O–), 81.2 (C–COOMe), 74.6 (CH–O–), 53.1 (O–CH3),
27.1 (C–CH3), 25.4 (C–CH3), 21.4 (Ar–CH3); vmax (film) 2973,
2937, 2888, 1741, 1469, 1381, 1308, 1163, 1131, 1105, 951,
(3aS,7aR)-Methyl 7-ethynyl-2,2-dimethyl-3a,7a-dihydro-
benzo[d][1,3]dioxole-3a-carboxylate (26)
To a stirred solution of silylacetylene 25 (9.6 mg, 0.03 mmol,
1 eq.) in THF (30 mL) at room temperature was added
tetra-n-butylammonium fluoride (1.0
M solution in THF,
0.05 mL, 0.05 mmol, 1.1 equiv). The reaction mixture was
stirred for 24 h, then diluted with EtOAc and washed with
NaCl(aq). The organic layer was dried over MgSO4 and fil-
tered. The filtrate was concentrated under reduced pressure
and purified by column chromatography (15% EtOAc–petrol)
to give (3aS,7aR)-methyl 7-ethynyl-2,2-dimethyl-3a,7a-dihydro-
benzo[d][1,3]dioxole-3a-carboxylate (26) (4.4 mg, 0.015 mmol,
76%) as a pale white gum: Rf 0.24 (15% EtOAc–petrol); [a]D25
-184 (c 0.32, CH2Cl2); dH (400 MHz) 6.46 (1H, d, J = 6.0 Hz,
HC C–C CH), 6.14 (1H, dd, J = 9.5, 6.0 Hz, HC C–C CH–
CH), 5.90 (1H, d, J = 9.5 Hz, HC C–C CH–CH CH), 4.92
(1H, s, CH–O–), 3.80 (3H, s, O–CH3), 3.23 (1H, s, C CH),
1.48 (3H, s, C–CH3), 1.43 (3H, s, C–CH3); dC (75 MHz)
171.5 (C O), 130.6 (HC C–C CH), 126.1 (HC C–C CH–
CH CH), 123.8 (HC C–C CH–CH), 118.8 (HC C–C ),
108.1 (–O–C–O–), 82.6 (HC C), 81.8 (HC C), 80.1 (C–
COOMe), 75.0 (CH–O), 53.3 (O–CH3), 27.0 (C–CH3), 25.4 (C–
CH3); vmax (film) 2981, 2889, 1737, 1462, 1382, 1251, 1152, 954,
807 cm-1; HRMS (ESI+) m/z calcd for (C13H14O4+Na)+, 257.0784;
found 257.0751.
3926 | Org. Biomol. Chem., 2011, 9, 3920–3928
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(3aS,7aR)-Methyl 7-(1-benzyl-1H-1,2,3-triazol-4-yl)-2,2-
dimethyl-3a,7a-dihydrobenzo[d][1,3]dioxole-3a-carboxylate (27)
11 (a) M. G. Charest, C. D. Lerner, J. D. Brubaker, D. R. Siegel and A. G.
Myers, Science, 2005, 308, 395; (b) M. G. Charest, D. R. Siegel and
A. G. Myers, J. Am. Chem. Soc., 2005, 127, 8292.
12 T. C. M. Fischer, H. G. Leisch and M. D. Mihovilovic, Monatsh. Chem.,
2010, 141, 699.
13 M. Ali Khan, M. F. Mahon, A. J. W. Stewart and S. E. Lewis,
Organometallics, 2010, 29, 199.
14 M. Ali Khan, J. P. Lowe, A. L. Johnson, A. J. W. Stewart and S. E.
Lewis, Chem. Commun., 2011, 47, 215.
15 (a) H.-J. Knackmuss and W. Reineke, Chemosphere, 1973, 2, 225;
(b) W. Reineke and H.-J. Knackmuss, Biochim. Biophys. Acta, Gen.
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Knackmuss, Appl. Environ. Microbiol., 1980, 39, 68.
16 (a) J. T. Rossiter, S. R. Williams, A. E. G. Cass and D. W. Ribbons,
Tetrahedron Lett., 1987, 28, 5173; (b) K.-H. Engesser, R. B. Cain and
H.-J. Knackmuss, Arch. Microbiol., 1988, 149, 188; (c) K. A. Vora,
C. Singh and V. V. Modi, Curr. Microbiol., 1988, 17, 249; (d) K. A.
Vora and V. V. Modi, Indian J. Exp. Biol., 1989, 27, 967; (e) K.-H.
Engesser, G. Auling, J. Busse and H.-J. Knackmuss, Arch. Microbiol.,
1990, 153, 193; (f) F. G. H. Boersma, W. C. McRoberts, S. L. Cobb and
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17 (a) J. Zeyer, P. R. Lehrbach and K. N. Timmis, Appl. Environ. Microbiol.,
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To a stirred solution of terminal alkyne 26 (13.0 mg, 0.05 mmol,
1 eq.) in EtOH–H2O 5 : 1 (25 mL) were added benzyl azide
(7.9 mg, 0.06 mmol, 1.2 eq.), CuSO4 (1.1 mg, 1 mol%) and
ascorbic acid (5.9 mg, 10 mol%). The solution was stirred
at room temperature for 48 h, then diluted with NaCl(aq)
and extracted with EtOAc. The organic layer was dried over
MgSO4 and filtered. The filtrate was concentrated under
reduced pressure and purified by column chromatography (10%
to 50% EtOAc–petrol) to give unreacted 26 (7.8 mg, 66%)
and (3aS,7aR)-methyl 7-(1-benzyl-1H-1,2,3-triazol-4-yl)-2,2-
dimethyl-3a,7a-dihydrobenzo[d][1,3]dioxole-3a-carboxylate (27)
(6.3 mg, 34%) as a pale brown oil: Rf 0.48 (50% EtOAc–petrol);
[a]2D5 -40 (c 0.18, CH2Cl2); dH (300 MHz) 7.62 (1H, s, HetAr–H),
7.37–7.29 (5H, m, Ph–H) 6.94 (1H, d, J = 6.0 Hz, HetAr–C CH),
6.28 (1H, dd, J = 9.0, 6.0 Hz, HetAr–C CH–CH ), 5.92 (1H,
d, J = 9.0 Hz, HetAr–C CH–CH CH), 5.61 (1H, d, J =
15.0 Hz, Ph–CHH–), 5.49 (1H, d, J = 15.0 Hz, Ph–CHH–) 5.27
(1H, s, CH–O–), 3.78 (3H, s, O–CH3), 1.48 (3H, s, C–CH3), 1.34
(3H, s, C–CH3); dC (75 MHz) 172.0 (C O), 134.8, 129.3, 128.8,
128.1, 127.8, 126.1, 124.9, 124.5, 121.0 (3◦ HetAr), 119.7 (HetAr–
C
CH–), 108.7 (–O–C–O–), 80.6 (C–COOMe), 74.2 (CH–O–),
18 H. R. Schla¨fli, M. A. Weiss, T. Leisinger and A. M. Cook, J. Bacteriol.,
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Aust. J. Chem., 2005, 58, 14.
54.3 (Ph–CH2–), 53.2 (O–CH3), 27.1 (C-CH3), 25.8 (C-CH3);
vmax (film) 2995, 2917, 1857, 1739, 1496, 1457, 1258, 1066, 887,
799, 727 cm-1; HRMS (ESI+) m/z calcd for (C20H21N3O4+Na)+,
390.1429; found 390.1440.
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Acknowledgements
We thank EPSRC, CIKTN, Prosidion Limited and the Univer-
sity of Bath for funding (DTC studentship for J.A.G., CASE
studentship to M.A.K.). We also thank ESCOM for funding
(technician placement to A.M.L). We are grateful to Prof. Andrew
G. Myers (Harvard University) for a generous gift of R. eutrophus
B9 cells and we thank Dr Petra J. Cameron (Bath) for translation
and Prof. J. Grant Buchanan (Bath) for helpful discussions.
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