An 'easy-on, easy-off' protecting group for the enzymatic resolution of (±)-1-phenylethylamine in an aqueous medium

Article abstract of DOI:10.1016/j.tetasy.2004.06.051

A new approach has been developed for the biocatalytic resolution of (±)-1-phenylethylamine in 100% aqueous medium based on two integrated enzymatic steps: protection and deprotection of the reactive amine enantiomer catalyzed by the same enzyme-penicillin acylase from Alcaligenes faecalis. An 'easy-on, easy-off' protecting group has been introduced using (R)-phenylglycine amide as the acyl donor. (R)-Phenylglycyl-substituted (R)-1-phenylethylamine was poorly soluble and precipitated at enzymatic acylation in an alkaline medium (pH 10-11), driving the synthesis towards high yields. Conversely at pH <7.5, its solubility was continuously increasing, which rendered the subsequent deacylation by the same enzyme highly efficient. In contrast to the resolutions, which employ one biocatalytic step, the new approach made it possible to exploit two sequential enantioselective enzymatic reactions implementing a double enantioselectivity control. Effective enzymatic resolution of (±)-1-phenylethylamine in an aqueous medium was performed with (R)-phenylglycine amide as an acyl donor using the suggested approach.

Full text of DOI:10.1016/j.tetasy.2004.06.051

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 2901–2906
An ꢀeasy-on, easy-off ꢁ protecting group for the enzymatic
resolution of ( )-1-phenylethylamine in an aqueous medium
Dorel T. Guranda,a Andrey I. Khimiuk,a Luuk M. van Langen,b Fred van Rantwijk,b
b
Roger A. Sheldon and Vytas K. Svedas
a,
*
ˇ
aFaculty of Bioengineering and Bioinformatics and Belozersky Institute of Physicochemical Biology, Lomonosov Moscow
State University, Moscow 119992, Russia
bLaboratory of Biocatalysis and Organic Chemistry, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands
Received 28 May 2004; accepted 17 June 2004
Abstract—A new approach has been developed for the biocatalytic resolution of ( )-1-phenylethylamine in 100% aqueous medium
based on two integrated enzymatic steps: protection and deprotection of the reactive amine enantiomer catalyzed by the same
enzyme––penicillin acylase from Alcaligenes faecalis. An ꢀeasy-on, easy-offꢁ protecting group has been introduced using (R)-phenyl-
glycine amide as the acyl donor. (R)-Phenylglycyl-substituted (R)-1-phenylethylamine was poorly soluble and precipitated at enzy-
matic acylation in an alkaline medium (pH10–11), driving the synthesis towards high yields. Conversely at pH <7.5, its solubility
was continuously increasing, which rendered the subsequent deacylation by the same enzyme highly efficient. In contrast to the res-
olutions, which employ one biocatalytic step, the new approach made it possible to exploit two sequential enantioselective enzymatic
reactions implementing a double enantioselectivity control. Effective enzymatic resolution of ( )-1-phenylethylamine in an aqueous
medium was performed with (R)-phenylglycine amide as an acyl donor using the suggested approach.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
lysis of the acylated (ꢀreactiveꢁ) amine enantiomer
requires harsh reaction conditions to liberate the free
amine,6 because enzymatic splitting using lipase or sub-
tilisin is ineffective.9,10
Recently a new strategy based on the unique catalytic
properties, stability and enantioselectivity of the rela-
tively unknown penicillin acylase from Alcaligenes
faecalis (Alcaligenes-PA) has been suggested for the
effective enantioselective acylation of amines in aqueous
media.1 Further development has shown the possibility
to regulate the enantioselectivity of the enzymatic amine
acylation by adding organic cosolvents.2 Very high reac-
tion rates, chemoselectivity and simple product isolation
advantageously distinguish Alcaligenes-PA-catalyzed
amine acylation in aqueous medium from the lipase
or subtilisin-catalyzed amine acylations in organic
solvents.3–8 When comparing applications of different
enzymes in the resolution of amines, it should be noted
that biocatalytic amine acylation in organic solvents
proceeds with a relatively low reaction rate,4 requires
strictly anhydrous reaction conditions and is compli-
cated by nonenzymatic acylation. Moreover, the hydro-
In this respect, the development of the amine resolution
using enzyme(s) for both protection and deprotection
steps could have serious advantages. Such a strategy
of preparing individual amine enantiomers would
encompass three steps: (1) stereoselective enzymatic acyl-
ation of the more reactive enantiomer (e.g., the (R)-
enantiomer as in Alcaligenes-PA-catalyzed acylations)
in the reaction mixture containing racemic amine; (2)
separation of nonacylated (S)-amine from N-acylated
(R)-enantiomer followed by (3) the enantioselective
enzymatic hydrolysis of the latter in order to liberate
the enantiomerically pure (R)-amine.
Herein we present a new biocatalytic approach for
enzymatic resolution of ( )-1-phenylethylamine in an
aqueous medium, which integrates two sequential enzy-
matic steps: protection and deprotection catalyzed by
the same enzyme––Alcaligenes-PA using (R)-phenylgly-
cine amide as acyl donor.
*
Corresponding author. Tel.: +7 0959394484; fax: +7 0959392355;
e-mail: vytas@belozersky.msu.ru
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.06.051

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D. T. Guranda et al. / Tetrahedron: Asymmetry 15 (2004) 2901–2906
2. Results and discussion
dissolved substrate is very strongly suppressed by the
accumulating product.21
Recently we have shown the possibility of highly effi-
cient Alcaligenes-PA-catalyzed stereoselective acylation
of amines in aqueous medium using phenylacetamide
as the acyl donor.1 As a result, the ꢀreactiveꢁ amine
enantiomer is acylated, whereas the other one remains
intact. It should be noted that phenylacetyl derivatives
of hydrophobic amines are poorly soluble in water and
due to this circumstance amine acylation in aqueous
medium can proceed with a high yield leading also to
a simple and effective isolation of the product by filtra-
tion. At the same time, successive hydrolysis of this
poorly soluble phenylacetyl derivative of amines repre-
sents a quite difficult problem. In addition to the low
substrate solubility in water, such a hydrolysis is compli-
cated due to the effective inhibition by the hydrolysis
product––phenylacetic acid,11 which is a well soluble
compound and, therefore, its accumulation slows down
the process sharply [see, e.g., hydrolysis of N-phenyl-
acetyl-(R)-1-phenylethylamine in Fig. 1, dashed line].
Keeping in mind the overall two-step biocatalytic proce-
dure (enzymatic acylation and subsequent enzymatic
deacylation), it would be optimal to have such a protect-
ing group that the protected amine would be poorly sol-
uble at the acylation conditions (making acylation
effective and product separation easy), but would be well
soluble at the hydrolysis conditions, and, as an ideal
property, the hydrolysis product would be a weak,
poorly soluble inhibitor which does not complicate a
deprotection of an ꢀactiveꢁ amine enantiomer. The
(R)-phenylglycyl group, with a pK value close to 7,
turned out an excellent candidate as (R)-phenylglycyl-
1-phenylethylamine appeared to be poorly soluble at
Alcaligenes-PA-catalyzed acylation conditions (pH10)
and precipitated in the course of the enzymatic reaction,
driving synthesis towards high yields in aqueous med-
ium, whereas at pH below 7.5 (R)-phenylglycyl-1-phe-
nylethylamine appeared to be well soluble substrate,
rendering Alcaligenes-PA-catalyzed hydrolysis effective
at these conditions. The solubility of (R)-phenylglycyl-
1-phenylethylamine at pH7.5 surpasses that of N-phe-
nylacetyl-1-phenylethylamine by approximately 2 orders
of magnitude. Moreover, (R)-phenylglycine [the product
of (R)-phenylglycyl-1-phenylethylamine hydrolysis] is a
weak inhibitor of penicillin acylase (Table 1) with a lim-
ited solubility (approximately 10mM at these condi-
tions, Fig. 2).
0.125
0.100
0.075
0.050
0.025
0.000
Table 1. Alcaligenes-PA-catalyzed hydrolysis of N-(R)-phenylglycyl-1-
phenylethylamine [N-(R)-PG-PEA] and N-phenylacetyl-1-phenyleth-
ylamine [N-Phac-PEA]: kinetic parameters, enantioselectivity and
substrate solubility (pH7.5, 25ꢁC)
Substrate
kcat/KM · 103
E
Solubility
(mM)
0
1
2
3
4
5
(Ms)À1
t ( hours )
N-(R)-PG-(R)-PEA
N-Phac-(R)-PEA
5.1
1700
310
22
Figure 1. Alcaligenes-PA-catalyzed deprotection of the ꢀreactiveꢁ amine
enantiomer; hydrolysis of: N-(R)-phenylglycyl-(R)-1-phenylethylamine
in 100% aqueous medium (d, solid line), N-phenylacetyl-(R)-1-
phenylethylamine in 100% aqueous medium (s, dashed line) and in
20% (v/v) methanol (h, dash-dotted line). Reaction conditions: 1.5lM
Alcaligenes-PA, pH7.5, 25ꢁC.
1100
0.65
Slow rates of Escherichia-PA-catalyzed hydrolysis of
phenylacetyl derivatives of amines and alcohols because
of their low solubility were noted in some earlier publi-
cations.12–14 In order to improve the solubility of acyl
derivatives of amines, alchohols and carbinols the
hydrolysis was carried out in water-miscible organic sol-
vents.15–17 The solubility of N-phenylacetyl-1-phenyleth-
ylamine, for example, can be increased in such a system,
but the productivity of hydrolysis cannot be seriously
improved (Fig. 1, dash-dotted line). As another option,
racemic substrates were used, which contain hydrophilic
group in the acyl moiety, such as pyridylacetyl-,18 p-ami-
no-19 and p-hydroxy-phenylacetic acid derivatives.20
However, in all these cases the hydrolysis product (pyr-
idylacetic acid, p-amino- or p-hydroxyphenylacetic acid)
is far more soluble than a corresponding initial substrate
and binds in the enzymeꢁs active site nearly as effectively
as phenylacetic acid; hence, hydrolysis of the partially
Figure 2. pH-dependencies for solubility of (R)-phenylglycyl-1-phen-
ylethylamine [(R)-PG-PEA, solid line], (R)-phenylglycine22 (PG, dash-
dotted line) and N-phenylacetyl-1-phenylethylamine (N-Phac-PEA,
dashed line) at 25ꢁC.
In addition to the inhibition/solubility benefits, the (R)-
phenylglycyl-protecting group brings some more advan-

D. T. Guranda et al. / Tetrahedron: Asymmetry 15 (2004) 2901–2906
Table 2. Comparison of Alcaligenes-PA-catalyzed acylation of ( )-1-
2903
phenylethylamine by (R)-phenylglycine amide [(R)-PGA] and phenyl-
acetamide (PAA) as acyl donors: reaction rates, synthesis/hydrolysis
ratios and enantioselectivities (pH10, 25ꢁC)
vS/E0 (sÀ1
)
(S/H)0
Ec
a
b
Acyl donor
Active amine
enantiomer
(R)-PGA
PAA
R
R
100
210
11.4
7.2
>1000
350
a E0––Alcaligenes-PA concentration in the reaction mixture determined
after titration of the active sites;11 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––enantioselectivity of an enzymatic acylation calculated from the
dependence eep versus degree of racemic amine acylation.
tages: surprisingly, the use of (R)-phenylglycine amide as
the acyl donor instead of phenylacetamide leads to a
remarkably improved enantioselectivity (E >1000) and
a higher synthesis/hydrolysis ratio (S/H >11) of the
Alcaligenes-PA-catalyzed amine acylation (Table 2). In
much the same manner the structure of the protecting
group influences the kinetics and enantioselectivity of
the Alcaligenes-PA-catalyzed hydrolysis of acylated
amines (Table 1). These results undoubtedly demon-
strate the tight relationship between the Alcaligenes-
PA acyl group binding pocket and the nucleophile bind-
ing subsite. A further investigation is needed to study a
mechanism and a scale of available effects. Our results
show that (R)-phenylglycine amide is an excellent acyl
donor for resolution of ( )-1-phenylethylamine and
other hydrophobic amines. A preparative resolution
has been performed according to Scheme 1; the kinetics
of the acylation is presented in Figure 3A. Synthesized
N-(R)-phenylglycyl-(R)-1-phenylethylamine (Fig. 3B)
was quantitatively separated from the intact (S)-1-phen-
ylethylamine by extraction and subjected to the Alcali-
genes-PA-catalyzed hydrolysis leading to a free (R)-1-
phenylethylamine. Both enzymatic steps were fast and
highly enantioselective. Although N-(R)-phenylglycyl-
(R)-1-phenylethylamine seems to be a less reactive sub-
Figure 3. Alcaligenes-PA-catalyzed acylation of ( )-1-phenylethylam-
ine (Æ) with (R)-phenylglycine amide (s) as the acyl donor; N-(R)-
phenylglycyl-(R)-1-phenylethylamine (m), N-(R)-phenylglycyl-(S)-1-
phenylethylamine (.), (R)-phenylglycine (d). Reaction conditions:
3.0lM Alcaligenes-PA, pH10, 25ꢁC. (A)––Time course of the
reaction; (B)––enantiomeric excess of the precipitated N-(R)-phenyl-
glycyl-(R)-1-phenylethylamine (m) versus degree of a racemic amine
acylation; the solid line correspond to the enantiomeric excess of a
product calculated for a biocatalytic acylation with enantioselectivity
equal to 1200.
strate than N-phenylacetyl-(R)-1-phenylethylamine if one
compares kcat/KM values of their hydrolysis (Table 1),
1. Enantioselective enzymatic acylation
NH
NH
2
NH
2
2
NH
2
H
N
Alcaligenes PA
NH
2
H
C
H C
3
3
+
+
aqueous medium, pH 10
O
O
CH
3
e.e. 99.1% (conv. 49.6%)
e.e. 97.8%
2. Separation
Extraction at pH 6.3
3. Enantioselective enzymatic hydrolysis
NH
2
NH
NH
2
2
H
N
Alcaligenes PA
OH
+
H
C
3
aqueous medium, pH 7.5
O
O
CH
3
e.e.>99.1% (conv.>99%)
Scheme 1. Alcaligenes-PA-catalyzed resolution of ( )-1-phenylethylamine by an ꢀeasy-on, easy-off ꢁ approach, which integrates an enantioselective
enzymatic acylation of a racemic amine using (R)-phenylglycine amide as the acyl donor and a successive deprotection of the acylated amine
enantiomer.

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D. T. Guranda et al. / Tetrahedron: Asymmetry 15 (2004) 2901–2906
its preparative conversion (Fig. 1, solid line), how-
ever, proceeds more effectively due to a much higher
substrate solubility and a lower sensitivity to the inhibi-
tion by the product (Table 2). As a result, the resolution
is very effective and both enantiomers of 1-phenylethyl-
amine are produced with a high yield and enantiomeric
excess (see Scheme 1).
nitrile (60%, v/v) and 0.68g/L of sodium dodecylsulfate,
as an eluent. The flow rate was 0.5mL/min. Retention
times (in min): phenylacetamide (5.5), phenylacetic
acid (7.8), N-phenylacetyl-1-phenylethylamine (13.6),
(R)-phenylglycine (5.0), (R)-phenylglycine amide (7.4),
1-phenylethylamine (10.4), N-(R)-phenylglycyl-1-(R)-
phenylethylamine (22.4), N-(R)-phenylglycyl-(S)-1-phen-
ylethylamine (25.0).
3. Conclusion
The enantiomeric excess of the N-phenylacetyl-1-phen-
ylethylamine was determined by HPLC using a Waters
590 pump and a Waters 486 UV detector at 215nm
and the flow rate 0.6mL/min on a Chiralcel OD column
(Daicel Chemical Industries) with hexane–isopropanol
(95:5, v/v) as an eluent. Retention times (in min): N-phen-
ylacetyl-(R)-1-phenylethylamine (42), N-phenylacetyl-
(S)-1-phenylethylamine (55).
Introduction of the ꢀeasy-on, easy-off ꢁ (R)-phenylglycyl-
protecting group allowed us to integrate the enan-
tioselective Alcaligenes-PA-catalyzed acylation of
( )-1-phenylethylamine and the enantioselective Alcali-
genes-PA-catalyzed hydrolysis of its acylated (R)-enanti-
omer into a two-step aqueous biocatalytic amine
resolution process. The suggested approach provided a
key to improve enantioselectivity and effectivity of bio-
catalytic stages and solve a problem, which had arisen
using phenylacetamide as an acyl donor for a resolution
of hydrophobic amines.
The enantiomeric excess of 1-phenylethylamine was
determined by HPLC using a Waters M6000 pump
and a Waters M481 LC detector at 210nm and the flow
rate 0.5mL/min on a Crownpak CR(À) column (Daicel
Chemical Industries) with H2O–HClO4 (pH2.0) as an
eluent. Retention times (in min): (R)-1-phenylethyl-
amine (23.0), (S)-1-phenylethylamine (29.1).
A new strategy has been developed for the biocatalytic
resolution of ( )-1-phenylethylamine in 100% aqueous
medium based on two sequential enzymatic steps: pro-
tection and deprotection of the reactive amine enantio-
mer catalyzed by the same enzyme––Alcaligenes-PA.
The biocatalytic resolution utilizing the ꢀeasy-on, easy-
offꢁ (R)-phenylglycyl-protecting group is highly pro-
ductive, ecologically friendly and involves quite simple
technological solutions. In fact it has a triple chiral con-
trol (two enantioselective biocatalytic steps and a dia-
stereomeric crystallization) resulting in high yields and
enantiomeric purity of the products. In contrast to the
relatively slow biocatalytic processes in an organic med-
ium, Alcaligenes-PA-catalyzed ( )-1-phenylethylamine
acylation and deacylation in an aqueous medium is fast
and efficient, so that answers the principal requirements
for a large-scale resolution of enantiomers. The scope of
this strategy is under investigation.
4.1.1. Preparation of samples for HPLC analysis. Sam-
ples of the heterogeneous reaction mixture were pre-
pared diluting an aliquot (50lL) of the heterogeneous
reaction mixture in 1.95mL of an eluent in order to dis-
solve all reactants and to stop the enzymatic reaction.
An aliquot of the resulting solution was further diluted
by an eluent and subjected to HPLC analysis; when
we needed to perform chiral analysis of 1-phenylethyl-
amine 2M KOH was added to the remaining solution
to adjust the pH to 12 and amine was extracted by
dichloromethane. The organic layer was washed by a
solution of HClO4 (pH2.0), dichloromethane was evap-
orated under vacuum and an aqueous solution of an
amine was subjected to a chiral analysis. The progress
curves for all reaction components (acyl donor, 1-phen-
ylethylamine, N-acylated amine and product of acyl
donor hydrolysis) were documented what provided
additional control due to the balance of the enzymatic
reactions.
4. Experimental
Solution of penicillin acylase from A. faecalis was
obtained from DSM Anti-Infectives, Delft, The Nether-
lands. Concentration of Alcaligenes-PA active sites was
determined as described earlier.11 (R)-Phenylglycine,
racemic ( )-1-phenylethylamine and its individual
enantiomers were obtained from Sigma; (R)-phenylgly-
cine amide from DSM, phenylacetic acid from Aldrich;
phenylacetylchloride from Fluka, phenylacetamide from
Reakhim.
In order to determine enantiomeric excess of a formed
N-phenylacetyl-(R)-1-phenylethylamine another aliquot
of the heterogeneous reaction mixture was taken in the
course of enzymatic acylation, filtrated and washed to
separate the precipitating reaction product from the
solution. After drying the isolated N-phenylacetyl-(R)-
1-phenylethylamine was dissolved in the eluent and sub-
jected to the chiral analysis.
4.1. HPLC analysis
4.2. Synthesis of N-(R)-phenylglycyl-(S)-1-phenylethyl-
amine
Concentrations of the reactants and enantiomeric excess
of N-(R)-phenylglycyl-1-phenylethylamine were deter-
mined by HPLC using a Waters M6000 pump, a
reversed phase Phenomenex Luna 5u C18(2) column
(250 · 4mm, 5lm) and a Waters M481 LC detector at
210nm with 7mM phosphate pH3.0, containing aceto-
N-(R)-Phenylglycyl-(S)-1-phenylethylamine was synthe-
sized using t-butyl oxycarbonyl protection in the follow-
ing way: to an aqueous solution (150mL) of (R)-PG
(25mmol) and triethylamine (35mL, 250mmol) di-t-bu-
tyl dicarbonate (6.55g, 30mmol) in 1,4-dioxane (50mL)

D. T. Guranda et al. / Tetrahedron: Asymmetry 15 (2004) 2901–2906
2905
was added and the mixture was stirred for 2h at 60ꢁC.
The mixture was concentrated under vacuum, cooled
with an ice-salt bath, and the pH was adjusted to 7.0
with 40% HCl. The precipitated salt N-Boc-(R)-
PGÆEt3N was filtrated, washed with a minimum volume
of water and dried under P2O5. The prepared triethyl-
amine salt of t-butylcarbamate (20mmol) was dissolved
in a dry chloroform (100mL), and ethyl chloroformate
(2.4mL, 25mmol) was slowly added to the mixture
cooled with an ice-salt bath. Then a solution of (S)-
PEA (20mmol) in chloroform (30mL) containing trieth-
ylamine (3mL, 21mmol) was added and the mixture was
stirred at room temperature overnight. The solution was
washed consecutively with diluted HCl, sodium bicarbo-
nate solution and water. Evaporation under vacuum
yielded a yellowish oil. Diethyl ether saturated with a
hydrochloric gas was added to the oil under stirring
and the hydrochloric salt of N-(R)-PG-(S)-PEA was
precipitated.
239 (62, M), 120 (49, PhCH2CH(NH)CH3), 105 (100,
PhCCH3), 91 (75, PhCH2), 77 (68, Ph), 65 (61).
4.4. Enzymatic hydrolysis of N-(R)-phenylglycyl-(R)-1-
phenylethylamine
The hydrolysis of obtained N-(R)-phenylglycyl-(R)-1-
phenylethylamine (0.84mmol) was carried out in a
closed thermostated cell of a pH-stat (Titrino 719, Metr-
ohm, Switzerland) at pH7.5, 25ꢁC in 0.02M KH2PO4
aqueous solution (total volume 7mL) in the presence
of 10.5nmol Alcaligenes-PA under permanent stirring
and pH control by adding 2M KOH solution for 2h.
4.5. Enzymatic hydrolysis of N-phenylacetyl-(R)-1-
phenylethylamine
The hydrolysis of N-phenylacetyl-(R)-1-phenylethyl-
amine (0.60mmol) was carried out in a closed ther-
mostated cell of a pH-stat (Titrino 719, Metrohm,
Switzerland) at pH7.5, 25ꢁC in 0.02M KH2PO4 (aque-
ous or containing 20% (v/v) methanol, total volume
5mL) in the presence of 7.5nmol Alcaligenes-PA under
permanent stirring and pH control by adding 2M
KOH solution for 5h.
4.2.1. N-(R)-Phenylglycyl-(S)-1-phenylethylamine hydro-
1
chloride. Yield 50% (2.9g); mp 123–125ꢁC; H NMR
(300MHz, DMSO): d 1.25 (d, 3H, CH3), 4.89 (m, 1H,
CH), 5.09 (br s, CaH), 7.20–7.66 (m, 10H, Ph), 8.82
(br s, 3H, NH3Cl), 9.37 (d, 1H, NH).
4.3. Enzymatic acylation of a racemic ( )-1-phenylethyl-
amine by (R)-phenylglycine amide
4.6. Solubility measurements
The saturated aqueous solutions of N-(R)-phenylglycyl-
(R)-1-phenylethylamine were prepared by stirring a
corresponding suspension during 40–60min in a thermo-
stated cell of a pH-stat (Titrino 719, Metrohm, Switzer-
land) at 25ꢁC and an appropriate pH kept constant by
an automated titration with HCl. Then the suspensions
were centrifuged and the isolated supernatants were sub-
jected to HPLC analysis after dilution with an eluent to
determine solubility value at each particular pH.
The acylation of ( )-1-phenylethylamine was carried out
in a thermostated cell of a pH-stat (Titrino 719, Metr-
ohm, Switzerland) at pH10, 25ꢁC in an aqueous med-
ium (total volume 10mL) with 2.0mmol racemic
amine and 2.5mmol (R)-phenylglycine amide as an acyl
donor in a presence of 30nmol Alcaligenes-PA under
permanent stirring and pH control by adding 2M
KOH solution. Reaction product was precipitating in
a course of acylation and its maximum yield was
achieved in 17min; then 2M KOH was added to the
reaction mixture to adjust the pH to 12 in order to
inactivate enzyme. Then the reaction mixture was acidi-
fied by H3PO4 and (R)-1-phenylglycyl-1-phenylethyl-
amine was extracted by dichloromethane (4 · 3mL) at
pH6.3. The organic layer was evaporated under
vacuum and N-acylated amine was recrystallized from
water.
The solubility of N-phenylacetyl-1-phenylethylamine in
water and in 20% (v/v) methanol was determined in
the same mode by stirring a corresponding suspension
for 1h in a closed thermostated cell of a pH-stat at
pH7.0, 25ꢁC.
Acknowledgements
4.3.1. N-(R)-Phenylglycyl-(R)-1-phenylethylamine. Yield
42% (0.214g); ee 0.991; mp 112–113ꢁC; 1H NMR
(300MHz, DMSO): d 1.37 (d, 3H, CH3), 2.22 (br s, 2H,
NH2), 4.40 (s, 1H, CaH), 4.92 (m, 1H, CH), 7.12–7.43
(m, 10H, Ph), 8.43 (d, 1H, NH). MS m/z: 120 (7.1,
PhCH2CH(NH)CH3), 106 (100, PhCHNH2, PhCH2-
CH3), 79 (50), 77 (39, Ph), 51 (18).
Financial support by the Russian Foundation for Basic
Research (Grant 03-04-48472), DSM Life Sciences
Products and the Netherlands Ministry of Economic
Affairs is gratefully acknowledged.
References
4.3.2. N-Phenylacetyl-(R)-1-phenylethylamine. N-phen-
ylacetyl-(R)-1-phenylethylamine was synthesized in
25min from equimolar (1.6mmol) amounts of racemic
( )-1-phenylethylamine and phenylacetamide with
9.6nmol Alcaligenes-PA. Yield 45% (0.172g); ee 0.985;
mp 117–118ꢁC; H NMR (250MHz, CDCl3): d 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:
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