Activation of Small Functionalized Molecules
(40 mgmLꢀ1 and chloramphenicol (20 mgmLꢀ1). Plasmids from
eight colonies were isolated, and the desired construct
(pBOU71106) was confirmed by sequencing of relevant regions.
)
quired inert atmosphere (nitrogen or argon) were carried out by
using standard Schlenk techniques.
Procedure for scaled-up biohydroxylation reactions with P450
mutants:
A disruption cassette flanked by short regions homologous to
dkgA was amplified from pBOU71106 by using primers Up-dkgA-
FRT-Kan (atg gct aat cca acc gtt att aag cta cag gat ggc aat gtc atg
ccc cag caa tta acc ctc act aaa ggg cgg ccg) and Down-GDH-dkgA
(tta gcc gcc gaa ctg gtc agg atc ggg acc gag acg ctt gcc ctg atc
gag ttt tgt tat tat ccg cgt cct gct tgg aat gat ggg tac). The homolo-
gous regions are underlined. E. coli BL21-Gold(DE3) strain harbor-
ing pRedETamp(R) (Gene Bridges) was transformed with the afore-
mentioned disruption cassette. Expression of reda, b, and g genes
of the l phage red recombinase with the recA gene under the con-
trol of an arabinose-inducible promoter facilitated recombination
among the homologous regions of the disruption cassette and the
dkgA locus of E. coli chromosomal DNA (Figure S2). Cells were
grown at 378C to eliminate pRedETamp(R) (due to its temperature-
sensitive replicon) with selection for kanamycin resistance. Four-
teen kan-resistant colonies were re-streaked on LB-agar plates con-
taining kan (20 mgmLꢀ1) and screened by colony PCR for the dis-
ruption of dkgA. A single colony, BOU72304, contained the desired
disruption. The expression of GDH under the T7 promoter was
confirmed by SDS-PAGE analysis of boiled cells induced with IPTG
(Figure S3). An aliquot of clear lysate from the same culture was
used to monitor the increment of absorbance at 340 nm due to
GDH-dependent formation of NADPH in presence of glucose/
NADP+. Uninduced BOU72304 cells were used as a negative con-
trol (Figure S3). The resulting strain, E. coli BL21-Gold(DE3) DdkgA::
FRT-KAN-FRT-T7-GDH, was rendered electrocompetent and trans-
formed with pCP20 (Coli Genetic Stock Center, CGSC) and/or with
707-FLPe plasmid (Gene Bridges). In our hands, transformation of
707-FLPe into BL21(DE3) proved inefficient. The kanamycin resist-
ance gene was removed by FLP-mediated site-specific recombina-
tion to leave a single FRT site, along with the T7 promoter–GDH
cassette, at the original dkgA locus. The elimination of the marker
in colony BOU73009 was confirmed by absence of resistance to
kanamycin and by colony PCR. The resulting strain, E. coli BL21-
Gold(DE3) DdkgA:: FRT-T7-gdh, was designated BOU730.
Preparation of (R)-methyl 3-hydroxycyclohex-1-enecarboxylate
(2): For scaling up the biohydroxylation reactions, an Erlenmeyer
flask (50 mL) containing LB (10 mL) and kan (50 mgmLꢀ1), was ino-
culated with a colony from BOU730 cells expressing (R)-P450
mutant (F87V/A328N), and incubated overnight at 378C with shak-
ing. An aliquot of this pre-culture was inoculated into TB (500 mL)
containing kan (50 mgmLꢀ1) (initial OD of 0.1 at 600 nm). Culture
was grown at 308C until an OD600 of 0.6–0.8 was reached, then
IPTG was added to a final concentration of 0.2 mm, and the culture
was grown at 308C over 12 h with agitation. Cells were pelleted by
centrifugation (4000 rpm, Fiberlite F10-6x500y, Sorvall, 15 min at
48C). The pellet [4.5–5 g wet mass (ca. 20 mm P450)[40] was resus-
pended in M9 minimal salts medium without a nitrogen source
[50 mL, pH 7.0 containing Na2HPO4 (12.8 gLꢀ1), KH2PO4 (3.0 gLꢀ1),
and NaCl (0.5 gLꢀ1)] containing glucose (100 mm) and NADP+
(0.5 mm). Resuspended cells were transferred to a 250 mL three-
necked, round-bottomed flask, and carboxy-methyl-cyclohexene
(1) was added via a Hamilton syringe [154 mL total volume, 14ꢃ
11 mL (0.08 mmol) every 45 min, 1.13 mmol in total]. The reaction
was carried out at 258C for 8 h at 150 rpm (Heidolph magnetic stir-
rer). A pH meter was also mounted to the installation in order for
the pH to be monitored continuously and maintained in the 7–7.5
range by the addition of NaOH (5m). During the reaction time, sev-
eral aliquots were withdrawn to evaluate conversion and (R)-2
ee%. After completion, the reaction mixture was extracted with
EtOAc (4ꢃ100 mL), the organic phase was dried with Na2SO4, the
solvent was evaporated, and the residue was subjected to column
chromatography to give (R)-methyl 3-hydroxycyclohex-1-enecar-
boxylate (2) as a colorless oil. GC analyses of the crude reaction
extract indicated final conversion ꢁ99% and, in addition to (R)-2
(155 mg, 88%), 7% of other oxidation products were detected:
(Rf =0.25 EtOAc/petroleum ether 1:4); 1H NMR (300 MHz, CDCl3):
d=6.87 (s, 1H), 4.35 (s, 1H), 3.75 (s, 3H), 2.25 (m, 2H), 1.98–
1.50 ppm (m, 5H); 13C NMR (75 MHz, CDCl3): d=167.84, 139.87,
132.58, 66.10, 51.93, 31.29, 24.36, 19.22 ppm; HRMS (APCI+) calcd
for C8H13O3 [M+H]+: 157.0859, found: 157.0857; 94–96% ee/R.
Chemistry
General remarks: Starting compounds 1 and 3 were purchased
from Sigma–Aldrich and Acros and were used without further pu-
rification. Racemic standards 2[35] and 4[36] were prepared by reduc-
tion of the corresponding six-membered[37] and five-membered[36]
ring ketones with NaBH4 in MeOH as described in the Supporting
Information. Compound 5[38] was prepared according to similar lit-
erature protocols.[39] All other reagents, including dry solvents,
were purchased from Acros, Sigma–Aldrich, and Alfa and were
used without further purification. NMR spectra were recorded on
a Bruker Avance 300 or DRX 400 (1H: 300 MHz or 400 MHz, 13C:
75 MHz or 101 MHz) spectrometer with TMS as internal standard
(d=0) unless otherwise noted. High-resolution EI mass spectra
were measured on a Finnigan MAT 95S spectrometer. High-resolu-
tion mass spectra recorded in ESI and APCI mode were performed
on a ThermoScientific LTQ-FT spectrometer. Conversion and enan-
tiomeric excess were determined by achiral and chiral gas chroma-
tography as described. Alternatively, the enantiomeric excess of
product 9 could be measured by HPLC. Optical rotation measure-
ments were performed on a Rudolph Research Analytical, Autopol
IV at 258C. Analytical thin layer chromatography was performed on
Merck silica gel 60 F254q, while Merck silica gel 60 (230–400 mesh
ASTM) was used for column chromatography. Reactions that re-
Preparation of (S)-methyl 3-hydroxycyclohex-1-enecarboxylate
(2): For scaling up the production of (S)-2, BOU730 cells expressing
(S)-P450 mutant (I263G/A328S) were grown in LB as described
above. Although we scaled up the production of (S)-2 using the
whole-cell strategy as described for (R)-2, the best results were ob-
served with lysed cells. Briefly, an aliquot of the LB pre-culture was
inoculated into TB (500 mL) containing kan (50 mgmLꢀ1; initial OD
of 0.1 at 600 nm). The culture was grown until an OD600 of 0.6–0.8
was reached, then IPTG was added to a final concentration of
0.2 mm, and the culture was grown at 308C for 20–24 h with agita-
tion. Cells were harvested by centrifugation (4000 rpm, Fiberlite
F10-6x500y, Sorvall, 15 min at 48C), and the pellet (4.5–5 g wet
mass, ca. 23 mm P450) was resuspended in lysis buffer (40 mL)
[phosphate buffer (pH 7.4, 100 mm), lysozyme (14 mgmLꢀ1), and
DNAse I (6 UmLꢀ1)]. The suspension was incubated for 30 min with
agitation in an ice bath, then sonicated for four cycles of 30 s, al-
ternating with four cycles of 30 s on ice. Cellular debris was pellet-
ed by centrifugation (5000 rpm, fixed rotor F34-6-38, Eppendorf,
20 min at 48C), and the supernatant was diluted to 50 mL with re-
action buffer [phosphate buffer (pH 7.4, 100 mm), glucose (100 mm
final concentration), and NADP+ (500 mm final concentration)].
Lysed cells were transferred to a 250 mL round bottomed flask
ChemBioChem 0000, 00, 1 – 10
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Agudo, Ruben
