Communications
Table 2: Product ee values for the biohydroxylation of 7 to 8 with different biocatalysts established by LC/
MS-based assay using (R)-9 and (S)-9 as substrates.
several distinctive features. 1) The
ee value can be determined with
satisfactory accuracy, independent
of the type of hydroxylation and the
nature of the biocatalyst. 2) The
analysis method is very sensitive. It
can analyze samples with product
concentration as low as 0.005 mm,
thus being suitable for the screening
of enantioselective enzymes for
biohydroxylation, for which the
product concentration is often low.
3) The analysis is based on MS and
does not require separation. It takes
only 1.0 minute for LC/MS analysis,
Entry Biocatalyst[a] t [h] Product conc.[b] [mm]
a
b
y
ee[c] [%] ee[d] [%]
1
Sphingomonas
1.0 0.023
0.080 0.017 3.25 53 (S) 54 (S)
sp. HXN-200
2
3
E. coli BL21(DE3) 1AF4 0.5 0.005
P. oleovorans GPo1 12 0.009
0.049 0.179 0.372 46 (R) 42 (R)
0.014 0.061 0.324 51 (R) 57 (R)
[a] Enantioselective hydroxylations of 2–5 mm (R)-9 and (S)-9 were performed in cell suspensions (5 mL,
5–30 gcdwLꢁ1) of different biocatalysts at 308C and 300 rpm. [b] Concentration refers to the analytical
sample with 20 times dilution of the original aqueous sample. [c] Determined and calculated based on
LC/MS analysis using the new method. [d] Determined by chiral HPLC analysis of the product from
biohydroxylation of 7 with different biocatalysts under the same biotransformation conditions and using
a concentrated sample.
the biotransformation mixtures. It also provides high sensi-
tivity, with no problem of using a 20 times diluted sample
containing 0.023 mm product. In comparison, chiral HPLC
analysis required the extraction of the product from aqueous
medium (5 mL) with ethyl acetate (5 mL) followed by
concentration of the sample by evaporation to 30 mL. In
fact, chiral HPLC analysis failed with a nonconcentrated
sample because of the sensitivity limitation. In addition, LC/
MS analysis took only 1.0 minute, which provided a theoret-
ical analytical throughput of 1440 samples per day. In
comparison, chiral HPLC analysis required about 30 minutes
for each sample.
thus providing high-throughput analysis of 1440 samples per
day. 4) The analysis method is simple. It allows direct analysis
of aqueous samples from biotransformation and does not
require further reactions of the bioproduct, which is often
necessary for many other assays. 5) The deuterated substrate
can be easily prepared from the corresponding alcohol, and
does not need to be enantiopure. Currently we are applying
this method to the discovery of enantioselective enzymes for
biohydroxylations through direct evolution of monooxyge-
nases.
The method was further examined with other available
biocatalysts for this hydroxylation. Recombinant Escherichia
coli BL21 expressing the 1AF4 mutant of the P450 mono-
oxygenase of Sphingomonas sp. HXN-200 catalyzed the
biohydroxylation of 7 to give the corresponding (R)-8 in
42% ee (Table 2, entry 2). This is the opposite enantioselec-
tivity to that obtained with Sphingomonas sp. HXN-200. To
obtain the product ee value by our new method, biotransfor-
mation of (S)-9 and (R)-9 with BL21 (DE3) 1AF4 was
performed under the same conditions, and samples from
aqueous buffer were analyzed by LC/MS without column
separation. As shown in Table 2, entry 2, the product ee value
was established as 46% (R) by this method, which has an
error of only 4% ee. Also in this case, the ee determination is
sensitive, with a product concentration of 0.005 mm, and fast,
with an analysis time of 1.0 minute.
Experimental Section
General procedure for determining the ee value of bioproduct 2 from
biohydroxylation of 1: In two parallel experiments, enantiomerically
enriched (R)-3 and (S)-3 were added to
a cell suspension
(10 gcdwLꢁ1) of P. monteilii TA-5 in 100 mm potassium phosphate
buffer (1 mL, pH 7.0) to a substrate concentration of 10 mm. The
mixture was shaken at 1000 rpm at 308C for 15 min. The cells were
removed by centrifugation, and the aqueous solution (0.4 mL) was
mixed with ethyl acetate (0.4 mL). The organic phase was separated
and analyzed by GC/MS. The ratio of the signals at M and M + 1 in
the mass spectra was determined as 3.050 for a and 0.093 for b,
respectively, and the values were used to calculate the ee value as
82% (R) for product 2 from the biohydroxylation of 1 with the same
enzyme. Upon comparison of the product ee value, 83% (R), from the
real biohydroxylation of 1 with the same enzyme and determined by
chiral HPLC analysis, the GC/MS-based method gave an accurate ee
value with an error of 1% ee. The analysis took 2.2 min for samples
with a product concentration from 0.034 to 0.170 mm.
P. oleovorans GPo1 contains a well-known membrane-
bound alkB hydroxylase system and catalyzes the hydroxyl-
ation of a number of aliphatic compounds.[21] Biohydroxyla-
tion of 7 with this strain gave (R)-8 in 57% ee (Table 2,
entry 3). Similarly, the ee value was also established by using
the new method through the separate biotransformation of
(S)-9 and (R)-9 with P. oleovorans GPo1 and LC/MS analysis
of the aqueous samples. As shown in Table 2, entry 3, the
product ee value was established as 51% (R) by this method
with an error of 6% ee. Once again, the analysis is sensitive
and fast, being able to determine samples containing
0.009 mm product within 1.0 minute.
General procedure for determining the ee value of bioproduct 8
from biohydroxylation of 7: In two parallel experiments, (S)-9 or (R)-
9 was added, to a final concentration of 5.0 mm, to a suspension of
cells of Sphingomonas sp. HXN-200 (5.5 gcdwLꢁ1) in 50 mm potas-
sium phosphate buffer (5 mL, pH 7.5) containing 2% (w/v) glucose.
The mixture was shaken at 300 rpm at 308C for 1 h, and the cells were
removed by centrifugation. The supernatant (50 mL) was diluted
20 times with methanol and used as the sample for LC/MS analysis.
From the mass spectra, a and b values were obtained as 0.080 and
0.017, respectively, thus establishing the ee value of product 8 as 53%
(S) from the biohydroxylation of 7 with Sphingomonas sp. HXN-200.
As biohydroxylation of 7 with the same strain gave product 8 in 54%
ee (S) determined by chiral HPLC analysis, the LC/MS based method
is accurate, with an error of only 1%. The analysis took 1.0 min for
samples with a product concentration of 0.023 mm.
In summary, we have developed a new method for
measuring the product ee value for enantioselective hydrox-
ylation based on the use of enantiopure or -enriched
deuterated substrates and MS detection. Our method has
Received: March 25, 2010
Published online: June 22, 2010
5282
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2010, 49, 5278 –5283