Stereoselective Friedel-Crafts alkylation catalyzed by squalene hopene cyclases

Article abstract of DOI:10.1016/j.tet.2012.06.041

In organic synthesis the Friedel-Crafts alkylation is of eminent importance, as it is a key reaction in many synthetic routes. A general access to enzymatic Friedel-Crafts alkylations would be very beneficial due to the high selectivity of biocatalysts. We used designed polyprenyl phenyl ethers to specifically address this reaction by using squalene hopene cyclases as catalysts. Polycyclic products with aromatic rings constituting important biological active compounds were obtained. Our results demonstrate that squalene hopene cyclases can be utilized for Friedel-Crafts alkylations and reveal the potential of these enzymes for chiral Bronsted acid catalysis.

Full text of DOI:10.1016/j.tet.2012.06.041

Tetrahedron 68 (2012) 7624e7629
Contents lists available at SciVerse ScienceDirect
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journal homepage: www.elsevier.com/locate/tet
Stereoselective FriedeleCrafts alkylation catalyzed by squalene hopene cyclases
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*
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ꢀ
Stephan C. Hammer , Jorg M. Dominicus , Per-Olof Syren, Bettina M. Nestl, Bernhard Hauer
Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In organic synthesis the FriedeleCrafts alkylation is of eminent importance, as it is a key reaction in many
synthetic routes. A general access to enzymatic FriedeleCrafts alkylations would be very beneficial due to
the high selectivity of biocatalysts. We used designed polyprenyl phenyl ethers to specifically address
this reaction by using squalene hopene cyclases as catalysts. Polycyclic products with aromatic rings
constituting important biological active compounds were obtained. Our results demonstrate that
squalene hopene cyclases can be utilized for FriedeleCrafts alkylations and reveal the potential of these
enzymes for chiral Brønsted acid catalysis.
Received 5 March 2012
Received in revised form 23 May 2012
Accepted 8 June 2012
Available online 15 June 2012
Keywords:
Biocatalysis
Squalene hopene cyclase
FriedeleCrafts alkylation
Substrate promiscuity
Polyprenyl phenyl ether
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
alkylations are prenyltransferases.9 Yet, their alkyl group activation
isalsochallengingsince itisbasedondiphosphateasa leavinggroup.
The formation of CeC bonds to generate carbon scaffolds of or-
ganic molecules is often the key step in synthesis. Due to their fre-
quently high chemo-, regio-, and stereoselectivity, enzymes are
promising catalysts for CeC bond formations.1 However, the number
of CeC bond forming reactions applied in biocatalysis is limited. This
is mainly due to the poor availability of enzymes for such reactions.
Beside the well-established aldol reaction (aldolases), benzoin-like
condensation (thiamine diphosphate-dependent enzymes), and
stereoselective HCN addition to carbonyls (hydroxynitrile lyases),
several novel CeC bond formations have recently been applied in
biocatalysis.2 These include the catalysis of oxidative CeC bond
formation,3 PicteteSpengler4 as well as Stetter5 reactions, the
enantioselective ring opening of epoxides,6 and the alkylation of
enolates with crotonases.7 The FriedeleCrafts alkylation is an im-
portant reaction in organic chemistry, however, a general approach
to address this reaction in biocatalysis is somewhat unexplored.
FriedeleCrafts alkylation involves the generation of an electrophile,
In contrast to these approaches, a simple way of activation is the
protonation of different functional groups like alkenes, carbonyls or
epoxides. Since there exist enzymes, which use protonation for ca-
talysis, we hypothesized that they can be utilized for FriedeleCrafts
alkylation. Such an approach is highly interesting, due to the chiral
environment of the active site, which leads to selective CeC bond
formation between the electrophile and the aromatic moiety.
One of the most prominent examples of enzymes, which use
a relative strong Brønsted acid, are squalene hopene cyclases
(SHCs).10e17 SHCs catalyze the regio- and stereoselective poly-
cyclization of squalene 1 to form a 6/6/6/6/5-fused pentacyclic ring
system with nine stereocenters (Fig. 1).
The reaction is initiated by protonation of the terminal, non-
activated double bond of squalene, proceeds through carboca-
tionic intermediates and is terminated by deprotonation or reaction
with a water molecule. Initial studies using a SHC from Alicyclo-
bacillus acidocaldarius (AacSHC) revealed a high flexibility con-
cerning their substrate scope. Besides truncated C15eC25 squalene
analogs,18 elongated C35 polyprenoids19,20 could also be converted
in a selective manner. Furthermore, it has been shown that alter-
native nucleophiles, like hydroxyl groups, are accepted by these
enzymes. Representative examples include the conversion of
homofarnesol 5 to the important fragrance ambroxan 6, as well as
the conversion of 7 to products, which contain the scaffold of a class
of cyclic meroterpenoids 8 (Fig. 2).21,22
which is then attacked by an aromatic p-system. The recently pub-
lished enzymatic approach utilizing methyltransferases for meth-
ylation, allylation, propargylation, and benzylation of aromatic
compounds is a magnificent progress.8 However, these transferases
depend ona challenging syntheticcofactor foralkyl groupactivation.
Another class of enzymes, which is known to catalyze FriedeleCrafts
In this context, we have recently published a series of reactions
using a novel SHC from Zymomonas mobilis (ZmoSHC1).23,24 Re-
markably, this cyclase exhibits a higher activity for the truncated
* Corresponding author. Tel.: þ49 (0)711 685 63193; e-mail addresses: bern-
hard.hauer@itb.uni-stuttgart.de, itbbha@itb.uni-stuttgart.de (B. Hauer).
y
Both authors contributed equally to this work.
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.06.041

S.C. Hammer et al. / Tetrahedron 68 (2012) 7624e7629
7625
Fig. 1. Enzymatic conversion of squalene. The reaction is initiated via a Brønsted acid, which protonates the non-activated terminal C]C bond of squalene 1 whereupon a highly
reactive carbocation is formed. The chiral environment of the active site enables the stereospecific attack of an adjacent C]C bond. The conversion proceeds via the final carbocation
intermediate 2. Deprotonation or reaction with water leads to products hopene 3 and hopanol 4.
phenyl ethers, namely prenyl phenyl ether 11, geranyl phenyl ether
12, farnesyl phenyl ether 13, and geranylgeranyl phenyl ether 14
were used as model substrates. These compounds were synthesized
via Williamson ether synthesis to specificallyaddress FriedeleCrafts
alkylation. Biotransformations of these compounds with semi-
purified ZmoSHC1 followed by GC analysis revealed the formation
of compounds corresponding to cyclization products for most of the
tested substrates. Control experiments performed in buffer alone
and preparations obtained from Escherichia coli (E. coli) cells
expressing empty pET-22b(þ) vector did not exhibit any new peaks.
To determine the structure of these products, we conducted con-
versions in the range of 150e300 mg. After extraction, detergent
molecules were removed by passing through a small silica column
and the different products were separated using preparative HPLC.
The structures of the isolated compounds were solved by 1H and 13
C
NMR spectroscopy, including DEPT, 1He1H COSY, HSQC, HMBC, and
NOESYas well as IR spectra (see Supplementary data for details). The
molecular formulas were determined by high-resolution EIMS
(HREIMS). For the sake of clarity, herein, we only discuss the
structures of the main products. Structures of side compounds can
be found in Supplementary data. Compounds 15 and 17 were
identified as main products for the conversion of 12 and 13 (Figs 3
and 4).
These are promising results, since they demonstrate that the
Friedel–Crafts alkylation is catalyzed by SHCs. Moreover, the chiral
environment of the active site directed the cyclization, where up to
four stereocenters were formed. Such polycyclic compounds con-
taining aromatic moieties are scaffolds of a variety of important
biological active compounds like (þ)-nimbidiol, (þ)-totarol, inca-
none, and others.25e29 The determined yields were between 1.3
and 5.4%, respectively, after 20 h reaction time (Fig. 3). In-
terestingly, these low conversions are in the same range as for the
natural substrate squalene under these in vitro conditions (data not
shown).
The smallest ether 11 was not transformed into the corre-
sponding polycyclic product. However, this is in accordance with
other studies, which suggested that SHCs are not, or just barely,
active toward molecules smaller than C15 atoms.18,21 The reaction
of ZmoSHC1 with 12 led to the formation of a single product, while
the transformation of 13 generated three (two main and one side
product) and the reaction of 14 afforded six products (two main and
four side products). The generation of several products can be at-
tributed to the fact that the carbocation intermediates are highly
reactive species. Even for the natural substrate squalene 1 several
side products have been identified.30 A high amount of a single
product implies exclusion of alternative reaction pathways.31
Hence, the number of side products should correspond to the
number of cyclization steps. Therefore, it is not surprising that we
see multiple products formed from a single non-natural substrate,
Fig. 2. Conversions of alternative substrates with SHCs.
substrate homofarnesol 5 compared to the well-known AacSHC.
Furthermore, ZmoSHC1 shows a significant acceleration of the se-
lective conversion of citronellal 9 to compound 10, compared to its
buffer catalyzed background reaction. These results indicate that in
addition to C]C bond protonation, the catalytic machinery of SHCs
can be further utilized to activate C]O bonds. This catalytic flexi-
bility is in agreement with the much higher basicity of C]O
compared to C]C bonds. As a consequence, we assume that SHCs
can be used in a general approach for Brønsted acid catalysis in
a chiral environment. To further verify this hypothesis, we ratio-
nally designed and synthesized different substrates to address the
FriedeleCrafts alkylation. We used truncated squalene analogs,
which were linked with sterically challenging benzene moieties.
These compounds were chosen, due to their similarity with the
natural substrate squalene. We show that these compounds were
accepted by ZmoSHC1 and demonstrate that the stereoselective
FriedeleCrafts alkylation is part of the cyclization reaction. Impor-
tant scaffolds of a variety of biological active compounds were
formed for which a classical chemical synthesis is challenging.
2. Results and discussion
In the present paper, we extend the reaction diversity of SHCs to
the FriedeleCrafts alkylation via cyclization of the different poly-
prenyl phenyl ethers 11e14. Fig. 3 summarizes the results of these
alkylation reactions with ZmoSHC1. Four different polyprenyl

7626
S.C. Hammer et al. / Tetrahedron 68 (2012) 7624e7629
Fig. 3. Conversions of polyprenyl phenyl ethers catalyzed by ZmoSHC1 after 20 h reaction time. The values in brackets describe the percentage of the products formed. Differences
to 100% correspond to the amount of side products.
especially for the larger compounds. While the conversion of 12
yielded a single product, several compounds were generated for 13
and 14. The cyclization of 13 resulted in an alkene 16 as second
major compound along with the FriedeleCrafts alkylation product
17. This alkene 16 most likely originated from the carbocationic
intermediate 13a. This indicates that the attack of the aromatic
moiety to 13a is partially hindered. Since the aromatic moiety
cannot attack within a specific time, a nearby base (e.g., a water
molecule) deprotonates 13a in a regioselective manner. This final
attack (the FriedeleCrafts alkylation) was not observed for sub-
strate 14. The main products 18 and 19 most likely originated from
the deprotonation of 14a. We further isolated three additional side
products for this reaction. However, we could not identify the
FriedeleCrafts alkylation product for this substrate. The relative
high number of cyclization products for 14 reveals the existence of
a variety of alternative reaction pathways, which finally compete
with the FriedeleCrafts alkylation.
Recent studies by Tanaka et al. demonstrated that AacSHC was
not applicable for the CeC bond formation of substrates comprising
aromatic moieties.32,33 However, in contrast to our study, only large
substrates with four isoprene units similar to 14 were used. We used
AacSHC to determine the ability of a second SHC for FriedeleCrafts
alkylation with our set of substrates. Interestingly, the same sub-
strateacceptance andsimilar product patternswere found. The most
obvious difference was the presence of one further product for the
cyclization of 12 and 13 with AacSHC (see Supplementary data for
Fig. 4. Important NOE and HMBC signals for 15 and 17.

S.C. Hammer et al. / Tetrahedron 68 (2012) 7624e7629
7627
details). A related oxidosqualene cyclase from Arabidopsis thaliana
has been shown to catalyze FriedeleCrafts alkylation as well.34 The
aromatic alkylation product of this reaction has been observed as
a minor product. These results, together with our findings, demon-
strate that this class of enzymes can be in general harnessed for
FriedeleCrafts alkylations. This is of great interest, especially in
combination with Brønsted acid activation of different functional
groups. Although the CeC bond formation of substrates comprising
an aromatic moiety was not described for SHCs before, this activity is
not surprising due to the intrinsic high chemical reactivity of car-
bocations. The surprise lies in the fact that substrates like 12 can bind
to the active site without putting water molecules into a reactive
position in order to quench carbocationic intermediates. Hence, the
enzyme active site excludes water molecules mimicking an inert
solvent even for such sterically challenging substrates.
In conclusion, we extended the reaction diversity of SHCs to-
ward the FriedeleCrafts alkylation using challenging aromatic
substrates. Several polyprenyl phenyl ethers were synthesized and
studied in biotransformations using our set of SHCs. Interestingly,
both ZmoSHC1 and AacSHC showed a similar product profile. For
two of four substrates the corresponding FriedeleCrafts alkylation
product was obtained and characterized. This demonstrates that
the polycyclization machinery of these enzymes can be utilized for
‘novel’ CeC bond forming reactions. These results are of great in-
terest, particularly given that the number of enzymes for CeC bond
formation is limited.
3 mL THF at 0 ꢁC and stirred for 1 h. Saturated NaHCO3 (5 mL) was
added and the mixture was extracted with n-hexane (3ꢀ15 mL).
The combined organic layers were dried over Na2SO4 and con-
centrated in vacuo. The geranylgeranyl bromide was used without
further purification or characterization.
3.3. General procedure for the preparation of polyprenyl aryl
ethers 11e14
Polyprenyl aryl ethers were prepared by Williamson ether
synthesis altering a procedure previously described.36 Under a ni-
trogen atmosphere in dry THF dissolved phenol (1.0 equiv) was
added dropwise to a suspension of sodium hydride (1.1 equiv) in
dry THF and stirred for 2 h. Polyprenyl halide (1.0 equiv, for 11e13
chloride, for 14 bromide) dissolved in DMSO (ca. 15% solution) was
added and the mixture was stirred for 22 h. The mixture was
poured onto ice-water and extracted with diethyl ether. The com-
bined organic layers were dried over Na2SO4, concentrated in vacuo
and purified by flash column chromatography on silica gel 60
(0.040e0.063 mm) to give polyprenyl aryl ethers 11e14 (45e75%).
Analytical data of the ethers are given below.
3.3.1. Prenyl phenyl ether 11. Pale yellow oil. 1H NMR (CDCl3,
250 MHz)
d
1.74 (s, 3H, CH3), 1.80 (s, 3H, CH3), 4.51 (d, J¼7 Hz, 2H,
OeCH2), 5.46e5.55 (m, 1H, ]CH), 6.90e6.97 (m, 3H, Aro/peH),
7.23e7.33 (m, 2H, ArmeH); 13C NMR (CDCl3, 62.5 MHz)
d 18.2, 25.9,
3. Experiments
64.6, 114.6 (2C), 119.7, 120.6, 129.4 (2C), 138.2, 158.9.37
3.1. General procedures
3.3.2. Geranyl phenyl ether 12. Pale yellow oil. 1H NMR (CDCl3,
250 MHz) 1.60 (s, 3H, CH3), 1.68 (s, 3H, CH3), 1.73 (s, 3H, CH3),
d
All chemicals and solvents were purchased from SigmaeAldrich
(Steinheim, Germany), Alfa Aesar (Karlsruhe, Germany) or Carl-
Roth (Karlsruhe, Germany). The 1H and 13C nuclear magnetic res-
onance (NMR) spectra were recorded on a Bruker Avance 500
spectrometer operating at 500.15 and 125.76 MHz, respectively. All
spectra were recorded at room temperature in CDCl3. Chemical
2.02e2.20 (m, 4H, 2ꢀCH2), 4.54 (d, J¼6.6 Hz, 2H, OeCH2),
5.04e5.15 (m, 1H, ]CH), 5.44e5.55 (m, 1H, ]CH); 6.87e6.99 (m,
3H, Aro/peH), 7.22e7.34 (m, 2H, ArmeH); 13C NMR (CDCl3,
62.5 MHz)
d 16.7, 17.7, 25.7, 26.3, 39.6, 64.8, 114.7 (2C), 119.6, 120.6,
123.8, 129.4 (2C), 131.8, 141.1, 158.9.36
shifts are expressed in parts per million (ppm,
d) and referenced to
3.3.3. Farnesyl phenyl ether 13. Pale yellow oil. 1H NMR (CDCl3,
tetramethylsilane (TMS,
d
¼0 ppm). The correct assignment of the
250 MHz)
d
1.53 (s, 6H, 2ꢀCH3), 1.61 (s, 3H, CH3), 1.66 (s, 3H, CH3),
chemical shifts was confirmed by application of two-dimensional
correlation measurements, including correlation spectroscopy
(COSY), heteronuclear single quantum coherence (HSQC), hetero-
nuclear multiple bond coherence (HMBC), and nuclear Overhauser
enhancement spectroscopy (NOESY). IR spectra were measured on
a Bruker Vector 22 FT-IR spectrometer in an ATR mode. Mass
spectra were measured using electron impact ionization on a Fin-
nigan MAT 95. GC analyses were performed on a Shimadzu GC-
2010 equipped with a flame ionization detector using a DB-5 cap-
illary column (Agilent, 0.25 mmꢀ30 m) and H2 as carrier gas (linear
velocity 30 cm/s). Product yields were quantified using 1-decanol
as internal standard. The purified main products were used for
external calibration via GC (triplicates). The products of the enzy-
matic cyclizations were dissolved in acetonitrile and separated via
reversed phase HPLC on an Agilent HPLC 1200 Series equipped with
1.83e2.14 (m, 8H, 4ꢀCH2), 4.46 (d, J¼6.5 Hz, 2H, OeCH2), 4.95e5.09
(m, 2H, 2ꢀ]CH), 5.37e5.48 (m, 1H, ]CH), 6.79e6.91 (m, 3H, Aro/
peH), 7.14e7.26 (m, 2H, ArmeH); 13C NMR (CDCl3, 62.5 MHz)
d 16.0,
16.7, 17.7, 25.7, 26.2, 26.7, 39.6, 39.7, 64.7, 114.7 (2C), 119.6, 120.6,
123.7, 124.3, 129.4, 131.4 (2C), 135.4, 141.2, 158.9.38
3.3.4. Geranylgeranyl phenyl ether 14. Pale yellow oil. 1H NMR
(CDCl3, 500 MHz)
d
1.58e1.62 (m, 9H, 3ꢀCH3), 1.68 (s, 3H, CH3), 1.74
(s, 3H, CH3), 1.94e2.01 (m, 4H, 2ꢀCH2), 2.02e2.18 (m, 8H, 4ꢀCH2),
4.53 (d, J¼6.5 Hz, 2H, OeCH2), 5.06e5.14 (m, 3H, 3ꢀ]CH),
5.47e5.52 (m, 1H, ]CH), 6.88e6.96 (m, 3H, Aro/peH), 7.24e7.30 (m,
2H, ArmeH); 13C NMR (CDCl3, 125 MHz)
d 16.02, 16.04, 16.7, 17.7,
25.7, 26.2, 26.6, 26.8, 39.6, 39.70, 39.73, 39.74, 114.7 (2C),
119.6, 120.6, 123.7, 124.2, 124.4, 129.4 (2C), 131.3, 135.0, 135.5, 141.1,
158.9.
a diode array detector using a Reprosil 100-5 C18 column (5 mm,
250ꢀ20 mm column, Trentec Analysentechnik, Rutesheim, Ger-
many, acetonitrile/water gradient). Fractions were manually col-
lected according to absorption at 200 nm and were concentrated in
vacuo.
3.4. Vector construction
Genes encoding for cyclases SHC1 from Z. mobilis (ZmoSHC1,
NCBI no. YP_163283.1) and SHC from A. acidocaldarius (AacSHC,
NCBI no. BAA25185.1) were gratefully obtained from Michael Bre-
uer (BASF SE, Ludwigshafen, Germany). The genes were amplified
via PCR and cloned into a pET-22b(þ)-vector system (Merck,
Darmstadt, Germany). The plasmids were transformed into
3.2. Preparation of geranylgeranyl bromide
The preparation of geranylgeranyl bromide followed a pro-
cedure previously described.35 Under a nitrogen atmosphere,
phosphorus tribromide (40
mL, 0.43 mmol, 0.65 equiv) was added
Escherichia coli DH5a and successful cloning was verified by se-
to a solution of geranylgeraniol (192 mg, 0.66 mmol, 1.0 equiv) in
quencing (GATC Biotech, Konstanz, Germany).

7628
S.C. Hammer et al. / Tetrahedron 68 (2012) 7624e7629
3.5. Expression conditions
3.8. General procedure for preparative enzymatic cyclization
For protein expression each vector construct was transformed
For product isolation and characterization larger bio-
transformations (150e300 mg substrate) were performed in
round-bottom flasks equipped with a magnetic stirrer. To afford
a greater amount of cyclic products, the reaction mixtures were
stirred for 3e6 days. The extract was filtered through silica and
concentrated in vacuo. The products were separated via reversed
phase HPLC. For detailed information regarding IR and NMR see
Supplementary data.
€
in E. coli BL21(DE3) (Agilent, Boblingen, Germany) and 100
mg/mL
ampicillin was added to all growth media. A glycerol stock was
used to inoculate a 5 mL LB medium preculture, which was in-
cubated for approximately 8 h at 37 ꢁC and 180 rpm. This pre-
culture was used to inoculate 50 mL LB medium cultures in
250 mL Erlenmeyer flasks, which were incubated at same condi-
tions (180 rpm, 37 ꢁC) over night. These cultures were used to
inoculate 500 mL TB medium in 2 L Erlenmeyer flasks (with
baffles) with an optical density of OD600¼0.05 and were incubated
as described above (180 rpm, 37 ꢁC). When an optical density of
OD600¼0.5e0.7 was reached, the SHC expression was induced
3.8.1. Product 15. Isolated as a colorless oil. IR (KBr): 2925, 2866,
2360, 1605, 1579, 1488, 1447 cmꢂ1. 1H NMR (CDCl3, 500 MHz)
d 0.93
(s, 3H, CH3-16), 1.01 (s, 3H, CH3-17), 1.28 (s, 3H, CH3-15), 1.23e1.30
(m, 1H, H-3), 1.41 (dt, J¼3.8, 13.1 Hz, 1H, H-1), 1.46e1.51 (m, 1H, H-
3), 1.62e1.68 (m, 1H, H-2), 1.68 (dd, J¼3.7, 12.1 Hz, 1H, H-5),
1.70e1.81 (m, 1H, H-2), 2.21e2.27 (m, 1H, H-1), 4.19 (dd, J¼10.6,
11.9 Hz, 1H, H-6), 4.38 (dd, J¼3.6, 10.5 Hz, 1H, H-6), 6.73e6.76 (m,
1H, H-14), 6.81e6.85 (m, 1H, H-12), 7.04e7.08 (m, 1H, H-13), 7.13
with isopropyl
b-D-1-thiogalactopyranoside (IPTG) with a final
concentration of 1 mM. Expression was carried out for 4 h at 30 ꢁC
and 180 rpm. The cells were harvested by centrifugation
(16,900ꢀg, 14 min, 4 ꢁC), frozen in liquid nitrogen and stored at
ꢂ80 ꢁC.
(dd, J¼1.4, 7.7 Hz, 1H, H-11); 13C NMR (CDCl3, 125 MHz)
d 18.9 (C-2),
21.9 (C-16), 24.5 (C-15), 32.0 (C-4), 33.0 (C-17), 34.7 (C-10), 37.2 (C-
1) 41.9 (C-3), 47.8 (C-5), 64.0 (C-6), 116.3 (C-14), 119.8 (C-12), 124.3
(C-11), 127.0 (C-13), 135.7 (C-9), 152.6 (C-8); HREIMS m/z 230.1670
(calcd for C16H22O 230.1671).
3.6. Enzyme purification
All enzyme purification steps were carried out on ice or at 4 ꢁC.
For enzyme isolation, the frozen cells were thawed and resus-
pended in lysis buffer (3 mL per gram cell pellet, 200 mM citrate
buffer, pH 6.0, 1 mM EDTA), phenylmethanesulfonylfluoride
(PMSF) with a final concentration of 1 mM and a pinch of DNaseI
were added. The cells were disrupted by passing the suspension
3.8.2. Product 16. Isolated as a colorless oil. IR (KBr): 2921, 2848,
1709, 1599, 1587, 1497, 1474, 1456 cmꢂ1. 1H NMR (CDCl3, 500 MHz)
d
0.88 (s, 3H, CH3-11), 0.89 (s, 3H, CH3-13), 0.90 (s, 3H, CH3-12),
1.10e1.23 (m, 2H, H-2 & H-6), 1.23e1.29 (m, 1H, H-4), 1.40e1.49 (m,
2H, H-1 & H-2), 1.51e1.62 (m, 1H, H-1), 1.71 (s, 3H, CH3-22),
1.85e1.95 (m, 1H, H-7), 1.95e2.06 (m, 2H, H-6 and H-7), 2.19 (br s,
1H, H-10), 3.91 (dd, J¼9.6, 6.2 Hz, 1H, H-14), 4.11 (dd, J¼9.5, 3.0 Hz,
1H, H-14), 5.49e5.54 (m,1H, H-8), 6.87e6.95 (m, 3H, H-17 and H-19
and H-21), 7.23e7.30 (m, 2H, H-18 and H-20); 13C NMR (CDCl3,
twice through
a high-pressure homogenizer (EmulsiFlex C5,
Avestin, 100 MPa). The broken cells were centrifuged (38,700ꢀg,
45 min, 4 ꢁC) and the pellet was resuspended in solubilization
buffer containing 1% CHAPS as detergent (1 mL per gram pellet,
60 mM citrate buffer, pH 6.0). The mixture was incubated for 1 h
at 4 ꢁC and centrifuged again (38,700ꢀg, 45 min, 4 ꢁC). The
AacSHC containing supernatant was heated for 15 min to 60 ꢁC to
precipitate unstable E. coli proteins. The ZmoSHC1 containing
supernatant was incubated at 30 ꢁC to precipitate unstable E. coli
proteins. The solutions were centrifuged (38,700ꢀg, 45 min, 4 ꢁC)
again and the supernatant was used for biotransformations. SDS-
PAGE was used for controlling successful expression and enzyme
isolation (see Supplementary data). The protein concentration
was determined according to the Bradford Ultra (Expedeon)
method using bovine serum albumin (BSA)/CHAPS solution as
standard.
125 MHz) d 14.7 (C-13), 18.8 (C-1), 21.9 (C-22), 22.0 (C-12), 23.7 (C-
7), 33.0 (C-3), 33.3 (C-11), 36.0 (C-5), 39.7 (C-6), 42.1 (C-2), 49.9 (C-
4), 54.1 (C-10), 66.3 (C-14), 114.6 (C-17 & C-21), 120.4 (C-19), 123.2
(C-8), 129.4 (C-18 and C-20), 133.3 (C-9), 158.9 (C-16); HREIMS m/z
298.2295 (calcd for C21H30O 298.2297).
3.8.3. Product 17. Isolated as a white solid. IR (KBr): 2923, 2867,
2360, 1734, 1606, 1581, 1488, 1446 cmꢂ1. 1H NMR (CDCl3, 500 MHz)
d
0.86 (s, 3H, CH3-19), 0.88 (s, 3H, CH3-20), 0.91e0.95 (m, 1H, H-4),
0.94 (s, 3H, CH3-21), 1.05 (dt, J¼12.9, 4.0 Hz, 1H, H-10), 1.17 (dt,
J¼13.2, 4.1 Hz, 1H, H-2), 1.29 (s, 3H, CH3-22), 1.38e1.43 (m, 1H, H-2),
1.43e1.50 (m, 1H, H-1), 1.50e1.57 (m, 2H, H-5 and H-6), 1.62 (dd,
J¼11.7, 3.5 Hz,1H, H-8),1.62e1.67 (m, 1H, H-1), 1.70e1.78 (m, 2H, H-
5 and H-10) 2.31e2.36 (m, 1H, H-6), 4.18 (dd, J¼10.6, 11.6 Hz, 1H, H-
11), 4.35 (dd, J¼10.5, 3.5 Hz,1H, H-11), 6.73 (dd, J¼8.2, 0.9 Hz,1H, H-
18), 6.81e6.85 (m, 1H, H-16), 7.02e7.07 (m, 1H, H-17), 7.13 (dd,
3.7. General procedure for the enzymatic cyclization of
substrates 11e14
For the biotransformations an emulsion of substrate 11e14
(10
citrate buffer, pH 6.0) was prepared. Protein solution was added
(250 L, 2.4 mg/mL AacSHC protein solution, 13.6 mg/mL
m
L, 200 mM DMSO stock) in reaction buffer (740
m
L, 60 mM
J¼7.7, 1.2 Hz, 1H, H-15); 13C NMR (CDCl3, 125 MHz)
d 16.8 (C-21),
18.4 (C-1), 18.9 (C-5), 21.4 (C-19), 25.9 (C-22), 33.2 (C-3), 33.3 (C-
20), 35.0 (C-7), 36.6 (C-9), 39.2 (C-6), 39.7 (C-10), 41.9 (C-2), 52.2 (C-
8), 56.3 (C-4), 63.3 (C-11), 116.2 (C-18), 119.9 (C-16), 214.5 (C-15),
126.9 (C-17), 135.9 (C-14), 152.4 (C-13); HREIMS m/z 298.2297
(calcd for C21H30O 298.2297).
m
ZmoSHC1 protein solution) resulting in a total volume of 1 mL
(0.25% CHAPS final conc.). The biotransformations were per-
formed in glass tubes and were shaken in a thermomixer
(Eppendorf) at 1000 rpm and 60 ꢁC (AacSHC) and 30 ꢁC
(ZmoSHC1), respectively. After 20 h of incubation, 1-decanol
3.8.4. Product 18. Isolated as a white solid. IR (KBr): 2921, 2849,
(10
The reaction was terminated by extraction with ethyl acetate
L). The organic phases were combined, dried over
m
L, 200 mM DMSO stock) was added as internal standard.
2361, 1736, 1599, 1586, 1497, 1474 cmꢂ1. 1H NMR (CDCl3, 500 MHz)
d
0.82 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.90 (s, 3H,
(3ꢀ500
m
CH3), 1.09e1.17 (m, 1H), 1.20 (dd, J¼11.4, 5.4 Hz, 1H), 1.24e1.27 (m,
2H), 1.29 (dd, J¼12.8, 3.7 Hz, 1H), 1.32e1.42 (m, 3H), 1.56e1.61 (m,
2H), 1.61e1.67 (m, 1H), 1.71 (s, 3H, CH3), 1.87e2.01 (m, 2H),
2.04e2.09 (m, 1H), 2.18e2.23 (m, 1H), 3.91 (dd, J¼9.5, 6.4 Hz, 1H),
4.10 (dd, 9.5, 2.9 Hz, 1H), 5.46e5.50 (m, 1H), 6.87e6.95 (m, 3H),
Na2SO4, and analyzed by GC-FID. The biotransformations were
performed in triplicates. As negative control substrate in re-
action buffer, substrate in reaction buffer containing 0.25%
CHAPS and preparations from cells harboring empty pET-
22b(þ)-vector were used.
7.24e7.30 (m, 2H); 13C NMR (CDCl3, 125 MHz)
d 15.65, 15.7, 18.5,

S.C. Hammer et al. / Tetrahedron 68 (2012) 7624e7629
7629
ꢀ
7. Hamed, R. B.; Henry, L.; Gomez-Castellanos, J. R.; Mecinovic, J.; Ducho, C.;
Sorensen, J. L.; Claridge, T. D. W.; Schofield, C. J. J. Am. Chem. Soc. 2012, 134,
471e479.
18.8, 21.7, 21.7, 22.6, 33.1, 33.4, 36.1, 37.3, 39.9, 41.3, 41.9, 54.6, 54.9,
56.2, 66.2, 114.6 (2C), 120.4, 123.0, 129.4 (2C), 133.1, 158.9; HREIMS
m/z 366.2930 (calcd for C26H38O 366.2923).
8. Stecher, H.; Tengg, M.; Ueberbacher, B. J.; Remler, P.; Schwab, H.; Griengl, H.;
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€
€
3.8.5. Product 19. Isolated as a white solid. IR (KBr): 2928, 2866,
1645, 1599, 1586, 1496, 1474 cmꢂ1. 1H NMR (CDCl3, 500 MHz)
d 0.81
(s, 3H, CH3), 0.82 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.86 (s, 3H, CH3),
1.11e1.19 (m, 2H), 1.32e1.37 (m, 2H), 1.37e1.45 (m, 4H), 1.51 (s, 1H),
1.57e1.63 (m, 2H), 1.66e1.74 (m, 2H), 1.85e1.89 (m, 1H), 2.09 (td,
J¼13.1, 4.7 Hz, 1H), 2.19e2.24 (m, 1H), 2.39e2.45 (m, 1H), 4.07e4.12
(m, 1H), 4.16 (dt, J¼9.5, 3.7 Hz, 1H), 4.56 (d, J¼1 Hz, 1H), 4.87 (d,
J¼1 Hz, 1H), 6.89e6.95 (m, 3H), 7.26e7.29 (m, 2H); HREIMS m/z
366.2925 (calcd for C26H38O 366.2923).
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Acknowledgements
Fundings by a scholarship of the Fonds der Chemischen Indus-
trie (to S.C.H.) and from the Royal Swedish Academy of Sciences (to
P.-O.S.) are gratefully acknowledged. We thank Miriam Seitz for
discussion and Dr. Birgit Claasen (Universitaet Stuttgart) for helpful
advice regarding NMR.
Supplementary data
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