Microbial deracemisation of N-(1-hydroxy-1-phenylethyl)benzamide

Article abstract of DOI:10.1016/j.tetasy.2003.11.010

Microbial deracemisation of racemic N-(1-hydroxy-1-phenylethyl)benzamide to give the (R)-enantiomer is described using whole cells of Cunninghamella echinulata NRRL 1384. The deracemisation involves fast highly (S)-selective oxidation followed by slower p

Full text of DOI:10.1016/j.tetasy.2003.11.010

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 239–243
Microbial deracemisation of N-(1-hydroxy-
1-phenylethyl)benzamide
Gareth J. Cardus,a Andrew J. Carnell,a,* Harald Trauthweinb and Thomas Riermeirb
aDepartment of Chemistry, Robert Robinson Laboratories, University of Liverpool, Liverpool L69 7ZD, UK
bDegussa AG––Project House Catalysis, Industriepark H€ochst st. G 830, Frankfurt am Main D-65926, Germany
Received 18 September 2003; revised 11 November 2003; accepted 12 November 2003
Abstract—Microbial deracemisation of racemic N-(1-hydroxy-1-phenylethyl)benzamide to give the (R)-enantiomer is described
using whole cells of Cunninghamella echinulata NRRL 1384. The deracemisation involves fast highly (S)-selective oxidation followed
by slower partially (S)-selective reduction of the intermediate ketone. The yield and ee of (R)-N-(1-hydroxy-1-phenylethyl)benz-
amide were optimised (82%, 98% ee) by removal of a competing extracellular amidase/protease activity by using static cells at pH 5.
Use of the protease inhibitor PMSF with growing cells leads to a slower deracemisation (82% ee) but a very high (98%) overall
recovery of alcohol and ketone.
ꢀ 2003 Elsevier Ltd. All rights reserved.
1. Introduction
the racemic or enriched amino alcohol or its synthetic
precursor would be advantageous. We and others have
previously reported the use of whole cell microorgan-
isms14–17 to catalyse the deracemisation of secondary
alcohols and diols. The cells contain redox (dehydro-
genase) enzymes that carry out the oxidation and
reduction steps and the cofactors are recycled internally.
It can be speculated that the driving force for such
deracemisations derives from the cellular compartmen-
talisation of the enzymes catalysing each step. In addi-
tion only one of these steps needs to be enantioselective
for a successful deracemisation. It is also possible to
carry out dynamic kinetic resolutions of secondary
alcohols using a lipase in combination with either a
transition metal catalyst18 or a racemase.19
Chiral amino alcohols are widely used in synthesis as
ligands for asymmetric catalysis and as chiral auxiliaries
and are important structural elements in many phar-
maceuticals such as b-blockers and HIV inhibitors.1–3
Homochiral 2-amino-1-alkanols are easily obtained by
the reduction of the corresponding amino acids.4
However, the regioisomeric 1-amino-2-alkanols are not
as easy to access in enantiomerically pure form. A
number of approaches to their precursors have been
developed including a catalytic asymmetric nitro-aldol
reaction using rare earth Binol complexes5 and chiral
hydrocyanation6;7 of aldehydes leading to optically
active cyanohydrins. Chiral amino alcohols can be
synthesised by ring opening of cyclic ester derivatives of
chiral diols derived from Sharpless ADH reactions and
more directly using the aminohydroxylation,8–10 asym-
metric hydrogenation of amino ketones using chiral
ruthenium complexes11 and the highly regio- and
enantioselective ring opening of terminal epoxides using
TMSN3 and a (salen)Cr complex.12;13
2. Results and discussion
In order to extend our whole cell methodology to the
more synthetically useful amino alcohols, we screened a
number of microorganisms for the ability to deracemise
the model substrate amidoalcohol 1. This substrate was
chosen in preference to the free amino alcohol since we
envisaged that the solubility of the latter in aqueous
buffer would prohibit efficient extraction from the bio-
transformation media. From 12 strains of filamentous
fungi initially screened, one strain, Cunninghamella
echinulata NRRL 1384 gave a promising deracemisation
In cases where the above methods produce materials of
low ee or where the chiral reagent or catalyst ligands are
prohibitively expensive, a method for deracemisation of
* Corresponding author. Tel.: +44-151-794-3531; fax: +44-151-794-
3588; e-mail: acarnell@liv.ac.uk
0957-4166/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2003.11.010

240
G. J. Cardus et al. / Tetrahedron: Asymmetry 15 (2004) 239–243
OH
OH
O
C. echinulata
Ph
Ph
Ph
+
HN
O
HN
O
HN
O
NRRL 1384
6 days
Ph
Ph
Ph
2
4%
(R)-1
57%, 92% e.e.
( )-1
Scheme 1.
Table 1. Biotransformation of ketone 2
affording (R)-amidoalcohol 1 in 57% isolated yield and
92% ee (Scheme 1).
Reaction time (days)
Amidoalcohol 1
% Ee (conf.) % Yield
Ketone 2
% Yield
A small amount of the ketone 2 (ca. 4%) was also
detected in the reaction mixture, indicating that the
deracemisation is catalysed by redox enzymes. The
absolute configuration of the product was determined
by comparison (chiral HPLC) of a sample of the
(R)-configured product prepared from commercially
available amino alcohol (R)-2-amino-1-phenylethanol.
When the (R)-amidoalcohol 1 was incubated as the
substrate it showed no conversion to the ketone 2 or
the (S)-amidoalcohol 1 after 6 days, suggesting that the
deracemisation involves (S)-enantioselective oxidation
of the racemic substrate. In this case, the recovered yield
(72%) of (R)-1 was substantially higher than when
racemic-1 was used (57%). This suggests that (S)-1 or the
ketone 2 are being preferentially depleted by another
competing process, either spontaneous or enzymatic.
Incubation of the ketone 2 showed an unexpected
reaction profile (Table 1).
2
4
6
26 (S)
58 (R)
72 (R)
17
17
27
8
9
7
Microorganisms were grown in 50 cm3 of liquid nutrient media (2%
glucose, 2% corn steep liquors) in 250 cm3 flasks at 28 ꢁC in an orbital
incubator shaker at 200 rpm. Ketone 2 was added at a concentration of
0.5 mg/cm3 in 0.5 cm3 of DMF, after 2 days of growth. Chiral HPLC,
Chiralcel OJ column, eluent IPA–n-hexane 0.8:9.2, flow rate 1 cm3/
min, wavelength 210 nm, retention time for each enantiomer of ami-
doalcohol 1 13.2 and 15.1 min.
selective the overall result, after several cycles, is a de-
racemisation (Scheme 2).
In order to optimise the yield and ee of the alcohol from
this biotransformation and gain further insight into the
mechanism, we examined the reaction in greater detail.
A time-course study (Fig. 1) revealed that mass balance
was indeed poor, with only ca. 40% material remaining
after 120 h. This could result from retention by the
biomass, metabolism by the cells or bioconversion by an
extracellular enzyme.
After 2 days the ketone 2 was reduced with slight
selectivity (26% ee) in favour of formation of the (S)-
amidoalcohol 1. However after 4 and 6 days of bio-
transformation the amidoalcohol 1 became increasingly
enriched in the (R)-enantiomer. The overall yields of
amidoalcohol 1 and ketone 2 were particularly low,
again indicating the presence of a competing degrada-
tion process. These results collectively suggest that one
enzyme carries out enantioselective oxidation of the
(S)-amidoalcohol 1 to the ketone 2, leaving the (R)-
amidoalcohol 1 untouched. A second enzyme reduces
the ketone 2 with partial selectivity to the (S)-amido-
ketone-1. However because the oxidation step is highly
During the first 32 h of the biotransformation there is a
slow decrease in the concentration of (R)-amidoalcohol
1 with a much faster decrease in the amount of (S)-1
concomitant with a rise in the amount of ketone 2. The
ketone level then falls off after 32 h, with a sharp rise in
the level of (R)-1. These results suggest a competing
process that consumes the (S)-amidoalcohol 1 enantio-
selectively and/or the ketone 2. The addition of glucose
OH
OH
O
OH
enantioselective
Ph
Ph
Ph
Ph
+
+
HN
O
HN
O
HN
O
HN
O
(S)-oxidation
Ph
Ph
Ph
Ph
(R)-1
(S)-1
(R)-1
2
partially selective
reduction to (S)-alcohol
Scheme 2.

G. J. Cardus et al. / Tetrahedron: Asymmetry 15 (2004) 239–243
241
Table 3. Resting culture biotransformations of ( )-1 at various pHÕs
60
50
40
30
20
10
0
100
90
80
% (R)-1
% (S)-1
% ketone 2
70
60
% e.e. (R)-1
50
40
30
20
10
0
% alcohol 1
% ketone 2
0
2
4
6
8
24
hrs
32
48 72
96 120
5
6
7
pH
8
9
Figure 1. Time course study for deracemisation of (ꢀ)-1 with
C. echinulata.
Reactions were carried out according to Section 4.5 of the Experi-
mental over 6 days using resuspended cells in the appropriate pH
phosphate buffer (100 mM).
Table 2. Glucose addition study showing the affect of addition of
glucose (2 g/L) throughout the biotransformation of (ꢀ)-1
Glucose addition % Ee (R)-1
points (days)
% Alcohol 1
% Ketone 2
With the possibility of improving product recovery and
to examine the effect of pH, we also used resting cultures
C. echinulata for the biotransformation. The cells were
spun down from the normal growth media (pH 5) and
resuspended in 100 mM phosphate buffer at a range of
pHÕs (5–9) (Table 3). Surprisingly the results show that
C. echinulata 1384 has a broad tolerance to pH. The
yields of amidoalcohol 1 from all the biotransforma-
tions, with the exception of pH 9, were significantly
increased when compared to the growing cell biotrans-
formations. However the result at pH 5 was the most
striking, with yields of 90% alcohol 1 (90% ee) and 3%
ketone 2. This result is significant as it confirms that
there is extracellular activity in the supernatant of the
normal growing cell biotransformation that is respon-
sible for the lower yields observed. The resting culture
screening was carried out on 25 mg of substrate (0.5 mg/
mL). Subsequently the resting culture biotransformation
at pH 5 was scaled-up to 200 mg of substrate to afford an
increased 98% ee and an isolated yield of 82% for the
amidoalcohol 1. The slight decrease in yield when
compared to the small scale biotransformations can be
attributed to isolation and purification procedures.
0, 2, 4
2, 4
4
44
74
76
51
43
10
1
100
1
Reactions were run over 6 days according to Section 4.5 of the exper-
imental with the addition of glucose supplements as shown in Col-
to fermentations can provide additional reducing
equivalents, leading to improved kinetics in bioreduc-
tions. We reasoned that, if the reduction component of
the deracemisation is rate limiting and facilitates a
competing process, additional reducing equivalents
might speed up the reduction thus reducing loss of
material. We therefore investigated the effect of sup-
plementing the bioconversion with glucose (2 g/L) at
various points throughout the reaction (Table 2). All
reactions were run for 6 days and glucose supplements
were added at 0, 2 and 4 days, 2 and 4 days or at 4 days
only.
The results from this group of biotransformations show
a simple trend. The addition of glucose at regular
intervals (0, 2 and 4 days) throughout the biotransfor-
mation results in a dramatic decrease in the enantio-
meric excess observed for (R)-amidoalcohol 1. However
the more glucose addition points there are, the higher
the yield of recovered alcohol. These findings provide
additional evidence that the reductase activity is selec-
tive for formation of the (S)-amidoalcohol 1 and when
provided with more reducing equivalents, it is able to
over-ride the effect of (S)-selective oxidation and the
competing process that is responsible for loss of mate-
rial. The results also point to the ketone being the main
substrate for further metabolism. As would be pre-
dicted, incubation of the ketone 2 with glucose supple-
mentation at 0, 2 and 4 days gave the (R)-amidoalcohol
1 in higher yield (34% vs 27%) but lower ee (12% vs 72%)
(when compared to the same reaction without glucose
supplements).
Obviously there is a limitation in scale for use of resting
cells in this type of biotransformation, since cofactor
regeneration is limited. We therefore investigated the
possibility of inhibiting the competing activity in the
growing cell biotransformations. We strongly suspected
the presence of an amidase/protease in the supernatant,
which may cleave the amide bond of the ketone 2 or the
amidoalcohol 1 leading to the more polar amino alco-
hol, which would not extract into organic solvent. We
therefore ran the biotransformation in the presence
of the known protease inhibitor phenylmethylsulfonyl
fluoride (PMSF). This inhibitor has a half-life of
approximately 5 h so needed to be added to the reaction
daily. In the presence of this inhibitor, the biotransfor-
mation gave excellent yields of the amidoalcohol 1
(86%) and ketone 2 (12%) (overall recovery 98%) and an
86% ee for the amidoalcohol after 5 days. In the absence
of PMSF, the amidase/protease may be selective for the

242
G. J. Cardus et al. / Tetrahedron: Asymmetry 15 (2004) 239–243
(S)-amidoalcohol, thereby making the apparent rate of
deracemisation faster. However, when PMSF is used,
the competing enzyme is suppressed, thereby increasing
the total alcohol yield, but decreasing the enantiomeric
excess.
ing points were taken on a Gallenkamp melting point
apparatus and are uncorrected. HPLC analysis was
performed on a Waters 2690 separations module
equipped with a Chiralcel OJ column. Samples were
recorded using a Waters 996 photodiode array detector.
Integration was performed using Waters Millenium32
software. All reagents were purchased from Aldrich,
Lancaster and Fluka chemical companies and were used
without purification. Flash column chromatography
was performed using Sorbsil C 60 (40–60 lm mesh) silica
gel. Analytical thin layer chromatography (TLC) was
carried out on 0.25 mm pre-coated silica gel plates
(Macherey–Nagel SIL g/UV254) and compounds were
visualised using UV fluorescence or permanganate.
DCM was distilled from CaH2 and diethyl ether and
THF from sodium/benzophenone. Light petrol ether
refers to that portion boiling between 40 and 60 ꢁC.
3. Summary
In summary, we have shown that whole cells of the
fungus C. echinulata NRRL 1384 can deracemise the
model substrate amidoalcohol 1 by sequential (S)-
selective oxidation and (S)-selective reduction. The first
oxidation step appears to be highly selective and rela-
tively fast. The second reduction step is slower and only
partially selective, allowing high levels of the (R)-
amidoalcohol to accumulate over the course of the
biotransformation. Supplementation of the bioconver-
sion with glucose gives higher yields but lower ee for the
(R)-amidoalcohol. Yield and selectivity can be further
improved using static cells over the pH range 5–8, with
the optimum pH being pH 5 giving the (R)-amidoalco-
hol 1 in 82%, 98% ee. An extracellular amidase/protease
enzyme is likely to be responsible for reducing the
recovered yields of amidoalcohol and ketone in the
growing cell reactions and can be suppressed using
the protease inhibitor PMSF, leading to a slower
deracemisation but a very high overall recovery (98%)
and a reasonable 86% ee for the amidoalcohol 1.
4.2. ( )-N-(1-Hydroxy-1-phenylethyl)benzamide 120
To a stirred solution of racemic 2-amino-1-phenyl-
ethanol (2.0 g, 14.60 mmol) in 1 M sodium hydroxide
(50 cm3) was added benzoyl chloride (2.1 cm3,
18.09 mmol). The solution was stirred at rt for 3 h.
whereupon the sticky residue was extracted into ethyl
acetate (2 · 30 cm3) and washed with 1 M hydrochloric
acid (2 · 40 cm3), water (2 · 40 cm3), saturated aqueous
sodium hydrogen carbonate (2 · 40 cm3) and water
(2 · 40 cm3). The organic layer was dried (MgSO4) and
concentrated under reduced pressure. Purification by
recrystallisation from ethyl acetate–petrol gave racemic
benzamide 1 (2.52 g, 71%) as a white solid; Rf (ethyl
acetate, 100%) 0.72; mp 143–145 ꢁC (lit.21 146–147 ꢁC);
Found: C, 74.5; H, 6.2; N, 5.7. C15H15NO2 requires C,
74.7; H, 6.2; N, 5.8%; mmax (Nujol) cmꢁ1 3335 (NH),
3300 (br, OH), 1635 (CONH); dH (400 MHz, CDCl3)
3.22 (1H, s, br, OH), 3.52–3.57 (1H, m, H-2a), 3.90–3.96
(1H, m, H-2b), 4.96–4.99 (1H, m, H-1), 6.57 (1H, s, br,
NH), 7.26–7.76 (10H, m, Ar-H); dC (100 MHz, CDCl3)
48.2 (CH2), 74.2 (CH), 126.2, 127.4, 128.4, 129.0, 129.5
and 132.1 (Ar-H), 134.5 and 142.2 (ipso Ar), 169.0 (CO);
m=z (CI) 242 (100%, [M+H]þ), 224 (87, [M)H2O+H]þ).
HPLC Chiralcel OJ, tR 13.2 min, 15.1 min, eluent 9.2:0.8
hexane–isopropyl alcohol, flow rate 1 cm3/min,
k 210 nm.
We are currently extending the biotransformation for
deracemisation of a range of related amidoalcohols and
exploring the possibility of replacing the (S)-selective
dehydrogenase with an enzyme of opposite selectivity
using recombinant methods in order to accelerate the
deracemisation.
4. Experimental
4.1. General
Elemental microanalyses were performed using a Carlo
Erba module 1106 elemental analyser. NMR spectra
were recorded on a Gemini-300 (1H, 300 MHz; 13C,
75 MHz) and a Gemini-400 (1H, 400 MHz; 13C, 75 MHz)
spectrometer. Chemical shifts are described in parts per
million downfield shift from SiMe4 and are reported
consecutively as position (dH or dC), relative integral,
multiplicity
(s ¼ singlet,
d ¼ doublet,
t ¼ triplet,
4.3. N-(1-Oxo-1-phenylethyl)benzamide 222
q ¼ quartet, dd ¼ doublet of doublets, sep ¼ septet,
m ¼ multiplet and br ¼ broad), coupling constant (J,
Hz) and assignment (numbering according to the IU-
PAC nomenclature for the compound). Infra-red spec-
tra were recorded on a Perkin–Elmer 1710 FT-IR
spectrometer in the range of 4000–600 cmꢁ1. The sam-
ples were prepared as either Nujol mulls, or neat,
between sodium chloride discs. The frequencies (m) as
To a stirred mixture of molecular sieves (1.0 g) and
pyridinium dichromate (0.80 g, 2.10 mmol) in dry DCM
(40 cm3) was added acetic anhydride (0.65 cm3,
6.82 mmol). Benzamide 1 (0.50 g, 2.07 mmol) was added
and the reaction mixture was stirred at rt under nitrogen
for 4 h. Ether (100 cm3) was added and the reaction
mixture was filtered through a small pad of silica. The
filtrate was concentrated under reduced pressure to yield
the crude product as a colourless liquid, which was
redissolved in toluene and concentrated under reduced
pressure to remove the excess acetic anhydride. This was
repeated several times until a solid residue was obtained.
absorption maxima are given in wave numbers (cmꢁ1
)
relative to a polystyrene standard. Mass spectra and
accurate mass measurements were recorded on a VG
70-070E, or a TRIO 1000. Major fragments were given
as percentages of the base peak intensity (100%). Melt-

G. J. Cardus et al. / Tetrahedron: Asymmetry 15 (2004) 239–243
243
Purification by recrystallisation from petrol–ethyl acet-
Acknowledgements
ate gave the title compound as a white solid (0.32 g,
60%); mp 122–124 ꢁC (lit.23 125–126 ꢁC); HRMS: found
[M+H]þ 240.10209. C15H14NO2 requires 240.10245; mmax
(Nujol) cmꢁ1 3356 (NH), 1690 (CO), 1634 (CONH); dH
(400 MHz, CDCl3) 4.95 (2H, d, J 4.1 Hz, H-2), 7.26–
7.66 (6H, m, Ar-H), 7.88 (2H, d, J 7.3 Hz, Ar-H), 8.03
(2H, d, J 7.54 Hz, Ar-H); dC (100 MHz, CDCl3) 47.3
(CH2), 127.5, 127.8, 128.7, 129.1, 130.0 and 130.4 (Ar-
H), 134.6 and 134.8 (ipso Ar), 170.1 and 194.7 (CO); m=z
(CI) 240 (100%, [M+H]þ), 139 (18), 122 (29), 105 (25).
HPLC Chiralcel OJ, tR 25.9 min, eluent 9.2:0.8 hexane–
isopropyl alcohol, flow rate 1.0 cm3/min, k 220 nm.
We gratefully acknowledge Degussa AG for financially
supporting this work through a fully funded studentship
(G.C.).
References and notes
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