Highly efficient and enantioselective enzymatic acylation of amines in aqueous medium

Article abstract of DOI:10.1016/S0957-4166(01)00263-4

A new strategy based on the unique catalytic properties, stability and enantioselectivity of the relatively unknown penicillin acylase from Alcaligenes faecalis has been developed for the effective and enantioselective acylation of amines in aqueous medium. In contrast to lipase-catalyzed acylations in organic solvents, the penicillin acylase-catalyzed acylation of amines in aqueous solution is a rapid and chemoselective process leading to a product which can subsequently be deacylated by the same enzyme, imposing secondary enantiocontrol and leading to effective resolution.

Full text of DOI:10.1016/S0957-4166(01)00263-4

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 1645–1650
Highly efficient and enantioselective enzymatic acylation of
amines in aqueous medium
Dorel T. Guranda,a Luuk M. van Langen,b Fred van Rantwijk,b Roger A. Sheldonb and
a,
&
Vytas K. Svedas *
aBelozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119899, Russia
bLaboratory of Biocatalysis and Organic Chemistry, Delft University of Technology, Julianalaan 136,
2628 BL Delft, The Netherlands
Received 30 May 2001; accepted 7 June 2001
Abstract—A new strategy based on the unique catalytic properties, stability and enantioselectivity of the relatively unknown
penicillin acylase from Alcaligenes faecalis has been developed for the effective and enantioselective acylation of amines in aqueous
medium. In contrast to lipase-catalyzed acylations in organic solvents, the penicillin acylase-catalyzed acylation of amines in
aqueous solution is a rapid and chemoselective process leading to a product which can subsequently be deacylated by the same
enzyme, imposing secondary enantiocontrol and leading to effective resolution. © 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
cannot be easily hydrolyzed under basic conditions and
requires harsh treatment to liberate the free amine.9
Although lipases have been developed that tolerate
anhydrous reaction conditions surprisingly well,10 spon-
taneous acylation can be suppressed under special con-
ditions,9 and acyl donors (such as esters of
methoxyacetic or acetic acid), that do not react sponta-
neously with amines, can be used.11–13 However, mas-
sive amounts of enzyme are required to perform the
acylation and the reaction times tend to be long. More-
over, deprotection of the acylated amines remains a
serious problem as enzymatic hydrolysis is ineffective.14
The use of enzymes for the preparation of chiral syn-
thons is one of the most impressive concepts in the
cooperation of organic chemistry and enzymology.
Serious development in this field was stimulated by
employing enzymes in organic media. This approach
has significantly broadened the scope of biocatalytic
reactions used for chiral discrimination by adding, inter
alia esterification and transesterification reactions cata-
lyzed by lipases and esterases to the pool of the syn-
thetic chemoenzymatic methods.1–7 As a result, the
stereoselective acylation of alcohols by lipases in
organic solvents has become a useful strategy in the
development of chirotechnology.8
It would seem that the productivity could be seriously
increased by performing the acylation reaction in
aqueous solution. To be feasible, such an approach
would require an enzyme that is stable and active in
alkaline media as well as capable of transferring an acyl
group from the acyl-enzyme intermediate to the amine
rather than to a water molecule as a competing nucleo-
phile (Scheme 1). In this respect our attention was
drawn towards penicillin acylases (PA), which are
widely used as industrial catalysts in the biocatalytic
production of 6-aminopenicillanic acid.15 Due to its
wide substrate specificity, PA from E. coli (PA-E. coli )
was used also for enantioselective enzymatic hydrolysis
of N-phenylacetylated a-, b- and g-amino acids,16–18
aminophosphonic acids,19 and effective enzymatic
deprotection techniques.20 Attempts to use PA-E. coli
for the hydrolytic resolution of several amines have
Effective enzymatic resolution of the more reactive
amines in organic solvents is hampered by several prob-
lems: (1) their spontaneous acylation with activated
acyl donors commonly used in lipase-catalyzed
acylations; (2) the need for of stringently anhydrous
reaction conditions, as in the presence of even small
amounts of water hydrolysis of the acyl donor would
prevail; (3) the low catalytic activity of most enzymes in
anhydrous organic media; (4) the stability of the
product (amide) formed, which, in contrast to esters,
* Corresponding author. E-mail: vytas@belozersky.msu.ru
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00263-4

1646
D. T. Guranda et al. / Tetrahedron: Asymmetry 12 (2001) 1645–1650
lyzed by relatively unknown penicillin acylase from
Alcaligenes faecalis (PA-A. faecalis) in aqueous
medium.†
2. Results and discussion
Studying the substrate and stereospecificity of different
PAs we have observed that the relatively unknown
PA-A. faecalis has a much higher enantioselectivity in
the hydrolysis of N-phenylacetylated amines compared
with the well known PA-E. coli (Table 1). This enzyme
was also much more active towards most substrates,
including phenylacetamide 1, which could serve as a
potential acyl donor for the enzymatic acylation of
amines. As PA-A. faecalis had a broad pH optimum of
8–10 and maintained nearly 80% of its maximum cata-
lytic activity at pH 11,24 we examined the ability of this
enzyme to acylate different amines (Scheme 2). Surpris-
ingly, we found that PA-A. faecalis, in contrast to
PA-E. coli, is able to catalyze effective and enantioselec-
tive acylation of amines in aqueous solution (Figs. 1
and 2). Acylation of 3-phenylpropylamine 2 was nearly
quantitative (Fig. 1) with 97.5% conversion of amine
and 96.5% conversion of 1 to the precipitated product,
and the acylation rate (Table 2) was more than an
order of magnitude higher compared to the highest
reaction rates observed earlier in PA-A. faecalis-cata-
lyzed hydrolysis of the most specific substrates.24
Acylation of benzylamine 3, as well as other amines
(Table 2), was effective but slower compared to the
extremely fast acylation of 2, and had a lower synthesis/
hydrolysis ratio (S/H). In the acylation of (RS)-1-
phenylethylamine 4 the reaction rapidly came to a
standstill at 50% conversion when all (R)-4 had been
Scheme 1. ‘Minimal’ kinetic scheme for enzymatic acyl trans-
fer reaction from acyl donor S to the enantiomers of nucle-
ophile (amine) NS and NR.
E
–
enzyme, ES
–
enzyme–substrate complex, EA – acylenzyme intermediate,
EAN – acylenzyme–nucleophile(amine) complex, P1 – first
reaction product released at formation of acylenzyme, P2
–
product of acyl donor hydrolysis, PS and PR – acyl transfer
products to (S)- and (R)-enantiomers of nucleophile.
demonstrated surprisingly low stereospecificity21 (see
also Table 1), in contrast to the extremely high
stereospecificity observed in the hydrolysis of deriva-
tives of amino acids and their organoelement
analogues.22
Herein, we report results of our studies on highly
efficient and enantioselective acylation of amines cata-
Table 1. Kinetic parameters and stereospecificity of PA-catalyzed hydrolysis of N-phenylacetylated amines. Reaction condi-
tions: pH 7.5, 25°C
† The development of this work and possibility to regulate enantioselectivity of PA-A. faecalis by adding organic solvents was reported earlier.23

D. T. Guranda et al. / Tetrahedron: Asymmetry 12 (2001) 1645–1650
1647
Scheme 2.
faecalis could also be used for subsequent effective
deacylation of the obtained amide in contrast to harsh
chemical hydrolysis needed at lipase-catalyzed amine
resolution. Moreover, a mild enzymatic method to lib-
erate a free amine imposes secondary chiral control for
effective resolution (see Table 1).
A
kinetic study of the PA-A. faecalis-catalyzed
acylation of the individual enantiomers of 4, 1-(2-
naphthyl)ethylamine 7, and phenylglycinol has
8
demonstrated that high enantioselectivity of enzymatic
acylation is in good agreement with e.e.p values
observed in the acylation of racemic amines (Table 2).
It is worth noting that PA-A. faecalis-catalyzed
acylation of the latter compound was chemoselective
and ester formation was not observed with 8, nor with
(RS)-1-phenylethan-1-ol.
Figure 1. PA-A. faecalis-catalyzed acylation of 2 (ꢀ) with 1
(ꢁ) as an acyl donor; N-phenylacetyl-2 (ꢂ), phenylacetic
acid (ꢃ).
3. Conclusion
PA-A. faecalis-catalyzed acylation of amines is surpris-
ingly efficient and shows a high enantioselectivity for
the chiral amines tested. PA-A. faecalis-catalyzed reso-
lution of chiral amines could become an alternative to
the existing methods due to the very high reaction
rates, simplicity of the product isolation and the possi-
bility to hydrolyze the product formed by the same
enzyme with high enantioselectivity. The further scope
and limitations of this approach are under investiga-
tion.
consumed (Fig. 2). Acylation of 1-(4-chlorophenyl)-
ethylamine 5 and 2-amino-4-phenylbutane 6 was even
more effective taking into account the high S/H ratio.
Neither spontaneous hydrolysis of 1, nor spontaneous
acylation of amines at the conditions of enzymatic
reaction in the absence of PA-A. faecalis was
observed, demonstrating obvious advantage of this
acyl donor. The products of enzymatic amine
acylation could easily be isolated by filtration due to
their low solubility. It should be noted, that PA-A.

1648
D. T. Guranda et al. / Tetrahedron: Asymmetry 12 (2001) 1645–1650
eluent. The flow rate was 1.0 mL/min. Retention times
(in min) for the eluent with 26% CH3CN: phenylac-
etamide (4.3), phenylacetic acid (7.8), N-phenylacetyl-
phenylglycinol (13.8), phenylglycinol (18); retention
times (in min) for the eluent with 40% CH3CN: phenyl-
acetamide (2.9), phenylacetic acid (4.4), benzylamine
(6.1), N-phenylacetyl-benzylamine (10.8), 3-phenyl-
propylamine (10), N-phenylacetyl-3-phenylpropylamine
(20), 1-phenylethylamine (10.5), N-phenylacetyl-1-
phenylethylamine (19), 1-(4-chlorophenyl)ethylamine
(11.7),
N-phenylacetyl-1-(4-chlorophenyl)ethylamine
(26), 2-amino-4-phenylbutane (12.1), N-phenylacetyl-2-
amino-4-phenylbutane (31), 1-benzyl-2,2,2-trifluoro-
ethylamine (15), N-phenylacetyl-1-benzyl-2,2,2-tri-
fluoroethylamine (38), 1-(2-naphthyl)ethylamine (16.6),
N-phenylacetyl-1-(2-naphthyl)ethylamine (33.4).
The enantiomeric excess of the products, e.e.p, was
determined by HPLC using a Waters 590 pump and a
Waters 486 UV detector at 215 nm and the flow rate
0.6 mL/min on a Chiralcel OD column (Daicel Chemi-
cal Industries) with hexane-iso-propanol (95:5, v/v) as
the eluent; retention times, in min: N-phenylacetyl-1-
phenylethylamine: (R)- 42, (S)- 55; N-phenylacetyl-1-
(4-chlorophenyl)ethylamine: (R)- 44, (S)- 58;
N-phenylacetyl-phenylglycinol: (R)- 58, (S)- 49.5; N-
phenylacetyl-2-amino-4-phenylbutane: (R)- 64, (S)- 90;
or a reversed phase Chiralcel OD-RH column (Daicel
Chemical Industries) with CH3CN–water (6:4, v/v) as
the eluent; retention times, in min: N-phenylacetyl-2-
amino-4-phenylbutane: (R)- 8, (S)- 12; N-phenylacetyl-
1-(2-naphthyl)ethylamine: (R)- 8.5, (S)- 11.5.
Figure 2. PA-A. faecalis-catalyzed acylation of (RS)-4 (ꢄ)
with 1 (ꢁ) as an acyl donor; N-phenylacetyl-4 ("), phenyl-
acetic acid (ꢃ).
4. Experimental
Solutions of penicillin acylase from A. faecalis and from
E. coli were obtained from DSM Anti-Infectives, Delft,
The Netherlands. Concentration of penicillin acylase
active sites for both enzyme preparations was deter-
mined as described earlier.24 2-Amino-4-phenylbutane,
1-(4-chlorophenyl)ethylamine and (R)-phenylglycinol
were obtained from Acros; (S)-phenylglycinol, (R)- and
(S)-1-(2-naphthyl)ethylamine, benzylamine was ob-
tained from Fluka; ( )-1-phenylethylamine and its indi-
vidual enantiomers were obtained from Sigma; 3-
phenylpropylamine was obtained from Aldrich;
phenylacetic acid, phenylacetamide and phenyl-
acetylchloride were obtained from Reakhim.
4.2. Synthesis of N-phenylacetylated amines
Acylation of amines was carried out by phenyl-
acetylchloride in the following way: phenylacetylchlo-
ride (2.75 mmol) in acetone (2.75 mL) was added
dropwise to the solution of enantiopure amine (2.5
mmol) and potassium bicarbonate (3 mmol) in aqueous
acetone (1/1 v/v, 10 mL) with stirring at 0–5°C in an ice
bath; the pH value was kept close to 10 by adding 2N
aqueous NaOH. Finally the resulting solution was
stirred for two more hours at room temperature, pH
10, acetone was evaporated under vacuum and the
4.1. HPLC analysis
Concentrations of the reactants were determined by
HPLC using a Waters M6000 pump, a Chrompack
Nucleosil 100 C-18 column (150×4.6 mm, 5 mm) and a
Waters M481 LC detector at 210 nm with 7 mM
phosphate pH 3.0, containing acetonitrile (26 or 40%,
v/v) and a 0.7 g/L of sodium dodecylsulphate, as the
Table 2. PA-A. faecalis-catalyzed acylationa of amines by 1 as an acyl donor: reaction rates, synthesis/hydrolysis ratios and
enantioselectivities
a
b
d
Amine
Enantiomer
6s/E0 (s−1
)
(S/H)0
Ec
e.e.p (%)
2
3
4
5
6
7
8
–
–
730
140
210
160
350
240
130
90
12
7.2
11
13
–
–
350
–
–
R
R
R
R
S
98.5 (47.3)
99.3 (49)
96.0 (45.1)
98.1 (46.8)
98.0 (43)
ꢀ1000
120
250
\100
6.5
3.6
a E0 – PA-A. faecalis concentration in the reaction mixture determined after titration of the active sites;24 vs – initial rate of enzymatic amine
acylation.
b (S/H)0 – synthesis/hydrolysis ratio determined as a ratio of initial rates for accumulation of both reaction products.
c E – estimated from the dependence e.e.p versus degree of racemic amine acylation.
d e.e.p – product e.e. at indicated (in brackets) degree of racemic amine acylation.

D. T. Guranda et al. / Tetrahedron: Asymmetry 12 (2001) 1645–1650
1649
reaction mixture was acidified to pH 7 with 2N aqueous
HCl and washed with ethyl acetate (3×10 mL). The
organic layer was dried with Na2SO4 over 24 h, ethyl
acetate after filtration was evaporated under vacuum
and N-acylated amine was recrystallized from aqueous
ethanol.
analysis. Finally, the reaction product (N-acylated
amine) was filtrated, suspended in aqueous phosphate
buffer (0.01 M pH 7.5, 10 mL) and then extracted by
ethyl acetate (3×10 mL). The organic layer was dried
with Na2SO4 overnight, ethyl acetate was evaporated
under vacuum and N-acylated amine was recrystallized
from aqueous ethanol.
4.2.1.N-Phenylacetyl-(S)-1-(2-naphthyl)ethylamine.Yield:
0.520 g, (72%); [h]D20=−245 (c 1 MeOH); mp 129–
4.3.1. N-Phenylacetyl-3-phenylpropylamine. N-Phenyl-
acetyl-3-phenylpropylamine was synthesised using the
general method with a reaction time of 40 min from
equimolar (1.6 mmol) amounts of 3-phenylpropylamine
and phenylacetamide by using 7.2 nmol PA-A. faecalis.
1
130°C; H NMR (250 MHz, CDCl3): l 1.38 (d, 3H,
CH3), 3.51 (s, 2H, CH2), 5.18 (m, 1H, CH), 5.58 (d, 1H,
NH), 7.13–7.71 (m, 12H, Ar). MS m/z: 289 (71, M),
274 (20, M−CH3), 170 (57, M−PhCH2C(O)), 155 (100,
C10H7CHCH3), 127 (55, C10H7), 91 (75, PhCH2), 65
(18).
1
Yield 0.372 g (92%); mp 73–75°C; H NMR (250 MHz,
CDCl3): l 1.65 (m, 2H, CH2), 2.45 (t, 2H, CH2Ph), 3.12
(q, 2H, CH2NH), 3.45 (s, 2H, PhCH2CO), 5.22 (br. s,
1H, NH), 6.96–7.30 (m, 10H, Ph); MS m/z: 253 (42,
M), 162 (45, M−PhCH2), 149 (83, PhCH2CONHCH2+
H), 118 (83, PhCH2CO−H), 105 (48, PhCH2CH2), 91
(100, PhCH2), 77 (42, Ph), 65 (33).
4.2.2. N-Phenylacetyl-(R)-phenylglycinol. Yield: 0.446 g,
(70%); [h]2D0=−177 (c 1 MeOH); mp 114–115°C; 1H
NMR (250 MHz, CDCl3): l 2.33 (s, 1H, OH), 3.51 (s,
2H, CH2Ph), 3.70 (d, 2H, CH2OH), 4.94 (q, 1H, CH),
6.00 (d, 1H, NH), 7.00–7.30 (m, 10H, Ph). MS m/z: 237
(23, M−H2O), 224 (70, M−CH2OH), 206 (56, M−
CH2OH−H2O), 136 (24, M−PhCH2CO), 132 (58), 120
(66), 119 (50, PhCH(NH)CH2, PhCH2CO), 106 (99,
PhCHNH+H), 91 (100, PhCH2), 77 (73, Ph), 65 (73), 51
(65), 39 (53).
4.3.2. N-Phenylacetylbenzylamine. N-Phenylacetylben-
zylamine was synthesised using the general method with
a reaction time of 60 min from equimolar (1.6 mmol)
amounts of benzylamine and phenylacetamide by using
8.6 nmol PA-A. faecalis. Yield 0.263 g (73%); mp
121–122°C; 1H NMR (250 MHz, CDCl3): l 3.52 (s, 2H,
CH2Ph), 4.31 (d, 2H, NHCH2Ph), 5.57 (br. s, 1H, NH),
7.03–7.28 (m, 10H, Ph). MS m/z: 225 (74, M), 132 (37,
4.3. General procedure for enzymatic acylation
PhCH2CONH−2H,
M−PhCH2−2H),
106
(23,
A typical acylation was carried out in a thermostatted
cell of a pH-stat (Radiometer RTS-622, Copenhagen,
Denmark) at pH 10, 25°C in an aqueous medium (total
volume 8 mL) with equimolar amounts of amine and
acyl donor 1 in the presence of 0.9–2 mM PA-A. faecalis
under permanent stirring and pH control by adding 2
M aqueous KOH solution. Reaction products were
precipitating in the course of acylation and the maxi-
mum yield of amide was achieved in 10–30 min depend-
ing on the amine reactivity. Samples of the
heterogeneous reaction mixture were prepared by
adding an aliquot (50 mL) of the heterogeneous reaction
mixture to a portion of the eluent (1.95 mL) in order to
dissolve all reactants and to stop the enzymatic reac-
tion, then the resulting solution was subjected to HPLC
analysis. Progress curves of the enzymatic acylation
were followed up to high degrees of conversion (from
50 to 100% of amine acylation depending on the enan-
tioselectivity of penicillin acylase to racemic amine
used). As a rule progress curves for all reaction compo-
nents (acyl donor, amine, N-acylated amine and
product of acyl donor hydrolysis) were documented
what provided additional control due to the balance of
the enzymatic reaction and made possible to follow
synthesis/hydrolysis (S/H) ratio in the course of enzy-
matic acylation of amines; (S/H)0 ratio was determined
as a ratio of initial rates for accumulation of both
reaction products.
PhCH2NH), 91 (100, PhCH2), 77 (23, Ph), 65 (39).
4.3.3. N-Phenylacetyl-(R)-1-phenylethylamine.
N-
Phenylacetyl-(R)-1-phenylethylamine was synthesised
using the general method with a reaction time of 25 min
from equimolar (1.6 mmol) amounts of racemic (RS)-1-
phenylethylamine and phenylacetamide by using 9.6
nmol PA-A. faecalis. Yield 0.172 g, (45%); e.e. 98.5%;
mp 117–118°C; 1H NMR (250 MHz, CDCl3): l 1.29 (d,
3H, CH3), 3.47 (s, 2H, CH2), 5.01 (m, 1H, CH), 5.49 (d,
1H, NH), 7.04–7.29 (m, 10H, Ph). MS m/z: 239 (62,
M), 120 (49, PhCH2CH(NH)CH3), 105 (100, PhCCH3),
91 (75, PhCH2), 77 (68, Ph), 65 (61).
4.3.4. N-Phenylacetyl-(R)-1-(4-chlorophenyl)ethylamine.
N-Phenylacetyl-(R)-1-(4-chlorophenyl)ethylamine was
synthesised using the general method with a reaction
time of 37 min from equimolar (0.64 mmol) amounts of
(RS)-1-(4-chlorophenyl)ethylamine
and
phenylac-
etamide by using 13.6 nmol PA-A. faecalis. Yield 0.079
g (45%); e.e. 99.3%; [h]2D0=+110 (c 1 MeOH); mp
1
177–178°C; H NMR (250 MHz, CDCl3): l 1.25 (d,
3H, CH3), 3.46 (s, 2H, CH2), 4.96 (m, 1H, CH), 5.45 (d,
1H, NH), 6.97–7.29 (m, 9H, Ar). MS m/z: 273 (67, M),
154 (25, 4-Cl-PhCH2CH(NH)CH3), 139 (100, 4-Cl-
PhCH2CH(NH)), 103 (80, PhCH2C), 91 (83, PhCH2),
77 (55, Ph), 65 (39).
4.3.5. N-Phenylacetyl-(R)-1-(2-naphthyl)ethylamine. N-
Phenylacetyl-(R)-1-(2-naphthyl)ethylamine was synthe-
sised using the general method with a reaction time of
20 min from equimolar (1.6 mmol) amounts of (RS)-1-
(2-naphthyl)ethylamine and phenylacetamide by using
8.8 nmol PA-A. faecalis. Yield: 0.218 g (47%); e.e.
Another aliquot of the heterogeneous reaction mixture
was taken in the course of enzymatic acylation and
filtrated in order to separate precipitating reaction
product from the solution; isolated N-acylated amine
was dissolved in the eluent and subjected to the chiral

1650
D. T. Guranda et al. / Tetrahedron: Asymmetry 12 (2001) 1645–1650
1
98.1%; mp 127–128°C; H NMR (250 MHz, CDCl3): l
1.38 (d, 3H, CH3), 3.50 (s, 2H, CH2), 5.19 (m, 1H, CH),
5.58 (d, 1H, NH), 7.13–7.71 (m, 12H, Ar). MS m/z: 289
(24, M), 170 (15, M−PhCH2C(O)), 155 (100,
C10H7CHCH3), 127 (13, C10H7), 91 (26, PhCH2).
4. Davies, H. G.; Green, R. H.; Kelly, D. R.; Roberts, S. M.
Biotransformations in Preparative Organic Chemistry;
Academic Press: New York, 1989.
5. Biocatalysis in Non-conventional Media; Tramper, J.; Ver-
mue, M. H.; Beeftink, H. H.; von Stockar, U., Eds;
Elsevier: Amsterdam, 1992.
4.3.6. N-Phenylacetyl-(R)-2-amino-4-phenylbutane. N-
Phenylacetyl-(R)-2-amino-4-phenylbutane was synthe-
sised using the general method with a reaction time of
5 min from equimolar (1.6 mmol) amounts of racemic
(RS)-2-amino-4-phenylbutane and phenylacetamide by
using 16 nmol PA-A. faecalis. Yield 0.17 g (40%); e.e.
6. Faber, K. Biotransformations in Organic Chemistry;
Springer-Verlag: Berlin, 1992.
7. Wong, C. H.; Whitesides, G. M. Enzymes in Synthetic
Organic Chemistry; Pergamon, Elsevier: Amsterdam,
1994.
8. Sheldon, R. A. Chirotechnology: Industrial Synthesis of
Optically Active Compounds; Marcel Dekker: New York,
1993.
9. Takayama, S.; Lee, S. T.; Hung, S. C.; Wong, C. H.
Chem. Commun. 1999, 127–128.
10. Van Rantwijk, F.; Hacking, M. A. P. J.; Sheldon, R. A.
Monatsh. Chem. 2000, 131, 549–569.
11. Jaeger, K.-E.; Liebeton, K.; Zonta, A.; Schimossek, K.;
Reetz, M. T. Appl. Microb. Technol. 1996, 46, 99–105.
12. Balkenhohl, F.; Ditrich, K.; Hauer, B.; Ladner, W. J.
Prakt. Chem. 1997, 339, 381–384.
13. Iglesias, L. E.; Rebolledo, F.; Gotor, V. Tetrahedron:
Asymmetry 2000, 11, 1047–1050.
14. Smidt, H.; Fisher, A.; Fisher, P.; Schmid, R. D. Biotech-
nol. Tech. 1996, 10, 335–338.
1
96%; mp 109–112°C; H NMR (250 MHz, CDCl3): l
0.981 (d, 3H, CH3), 1.54 (m, 2H, CH2Bz), 2.43 (t, 2H,
CH2Ph), 3.43 (s, 2H, CH2Ph), 3.92 (m, 1H, CH), 5.00
(d, 1H, NH), 6.97–7.30 (m, 10H, Ph). MS m/z: 267 (5,
M), 176 (5, M−PhCH2), 163 (26, M−PhCH2CH2+H),
117 (13, PhCH2CH2CH-H), 91 (100, PhCH2), 77 (5,
Ph), 65 (7), 44 (22).
4.3.7. N-Phenylacetyl-(R)-phenylglycinol. N-Phenyl-
acetyl-(R)-phenylglycinol was synthesised using the
general method with a reaction time of 0.5 h from
equimolar (1.6 mmol) amounts of racemic (RS)-phenyl-
glycinol and phenylacetamide by using 11.5 nmol PA-
A. faecalis. Yield 26% 0.106 g (26%); e.e. 98%; mp
1
116–117°C; H NMR (250 MHz, CDCl3): l 2.35 (br. s,
15. Bruggink, A.; Roos, E. C.; de Vroom, E. Org. Process
Res. Dev. 1998, 2, 128–133.
16. Rossi, D.; Lucente, G.; Romeo, A. Experientia 1977, 33,
1557–1559.
17. Soloshonok, V. A.; Fokina, N. A.; Rybakova, A. V.;
Shishkina, I. P.; Galushko, S. V.; Sorochinsky, A. E.;
1H, OH), 3.52 (s, 2H, CH2Ph), 3.69 (br. s, 2H,
CH2OH), 4.94 (q, 1H, CH), 6.01 (d, 1H, NH), 6.99–
7.30 (m, 10H, Ph). MS m/z: 237 (29, M−H2O), 224 (70,
M−CH2OH), 206 (64, M−CH2OH−H2O), 132 (64), 120
(66), 119 (51, PhCH(NH)CH2, PhCH2CO), 106 (92,
PhCHNH+H), 91 (100, PhCH2), 77 (72, Ph), 65 (72), 51
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Financial support by the Russian Foundation for Basic
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