Biotransformation of new racemic (R,S)-5-benzylhydantoin derivatives by D-hydantoinases from adzuki bean

Article abstract of DOI:10.3109/10242422.2014.893578

In the present work the scope of D-hydantoinase enzyme application was increased towards new racemic (R,S)-5-benzylhydantoin derivatives. Five new substrates for the D-hydantoinase (R,S)-5-(3′-carboxybenzyl)hydantoin, (R,S)-5-(4′-carboxybenzyl)hydantoin, (R,S)-5-(2′-carbomethoxybenzyl) hydantoin, (R,S)-5-(3′-carbomethoxybenzyl)hydantoin and (R,S)-5-(4′(4-ethoxycarboxy)propoxybenzyl)hydantoin were synthesised and converted using a two-step hydantoinase process into their corresponding D-phenylalanine derivatives. In this study two D-hydantoinases from Vigna angularis (adzuki bean) obtained from commercial sources were used: pure, isolated directly from Vigna angularis (V.a.D-HYD) 494 U/g and immobilised, recombinant, cloned and expressed in Escherichia coli (rD-HYD) 53.1 U/g. The results obtained showed that the examined enzymes catalysed hydrolysis of all new substrates into their corresponding N-carbamoyl-D-phenylalanine derivatives. High enantiomeric purities of the resulting D-phenylalanine derivatives were also determined. However, very low conversion yields of (R,S)-5-(3′- carboxybenzyl)hydantoin and (R,S)-5-(2′-carbomethoxybenzyl)hydantoin to corresponding N-carbamoyl-D amino acid were observed. Three D-phenylalanine derivatives: 4-carboxy-D-phenylalanine, 3-carbomethoxy-D-phenylalanine and 4-carbopropoxy-D-phenylalanine were obtained and isolated from the reaction mixtures using ion-exchange chromatography.

Full text of DOI:10.3109/10242422.2014.893578

Biocatalysis and Biotransformation, 2014; 32(2): 117–124
ORIGINAL ARTICLE
Biotransformation of new racemic (R,S)-5-benzylhydantoin
derivatives by D-hydantoinases from adzuki bean
´
GNIEWOMIR LATACZ & KATARZYNA KIEC-KONONOWICZ
Department of Technology and Biotechnology of Drugs, Jagiellonian University, Medical College, Kraków, Poland
Abstract
In the present work the scope of D-hydantoinase enzyme application was increased towards new racemic (R,S)-5-benzyl-
hydantoin derivatives. Five new substrates for the D-hydantoinase (R,S)-5-(3′-carboxybenzyl)hydantoin, (R,S)-5-(4′-
carboxybenzyl)hydantoin, (R,S)-5-(2′-carbomethoxybenzyl)hydantoin, (R,S)-5-(3′-carbomethoxybenzyl)hydantoin and
(R,S)-5-(4′(4-ethoxycarboxy)propoxybenzyl)hydantoin were synthesised and converted using a two-step hydantoinase
process into their corresponding D-phenylalanine derivatives. In this study two D-hydantoinases from Vigna angularis
(adzuki bean) obtained from commercial sources were used: pure, isolated directly from Vigna angularis (V. a .D-HYD) 494
U/g and immobilised, recombinant, cloned and expressed in Escherichia coli (rD-HYD) 53.1 U/g. The results obtained
showed that the examined enzymes catalysed hydrolysis of all new substrates into their corresponding N-carbamoyl-D-
phenylalanine derivatives. High enantiomeric purities of the resulting D-phenylalanine derivatives were also determined.
However, very low conversion yields of (R,S)-5-(3′-carboxybenzyl)hydantoin and (R,S)-5-(2′-carbomethoxybenzyl)hydan-
toin to corresponding N-carbamoyl-D amino acid were observed.Three D-phenylalanine derivatives: 4-carboxy-D-pheny-
lalanine, 3-carbomethoxy-D-phenylalanine and 4-carbopropoxy-D-phenylalanine were obtained and isolated from the
reaction mixtures using ion-exchange chromatography.
Keywords: D-amino acids, D-phenylalanine, Hydantoinase, Dihydropyrimidinase, Capillary electrophoresis
Introduction
However, the hydantoinase process is not only used
The D-hydantoinase enzyme, also known as dihydro-
pyrimidinase or 5,6-dihydropyrimidine amidohydro-
lase (E.C 3.5.2.2), plays a very important role
in pyrimidine catabolism (Syldatk et al. 1999).
D-hydantoinase is commonly used for the two-
step production of D-amino acids from racemic
5-monosubstituted hydantoins. In the first step of
this process, the D-hydantoinase catalyses stereoselec-
tive hydrolysis of (R)-enantiomer of hydantoin ring
yielding optically pure N-carbamoyl-D-amino acid.
The racemisation of 5-monosubstituted hydantoin
derivatives under alkaline conditions, or in the pres-
ence of a racemase, allows high-yield bioconversion
of hydantoins by D-hydantoinase. In the second step,
the N-carbamoyl-D-amino acid is converted into the
corresponding D-amino acid using N-carbamoylase
or a chemical procedure using a diazotisation reaction
(Takahashi et al. 1979; Altenbuchner et al. 2001).
for D-amino acid production, but also used for the
production of natural and unnatural L-amino acids
using modified D-hydantoinase with inverting enanti-
oselectivity (May et al. 2000).
The industrial application of D-hydantoinase in
the pharmaceutical field include biosynthesis of
D-phenylglycine and its 4-hydroxy derivative, starting
from (R,S)-phenylhydantoin or (R,S)-4-hydroxyphe-
nylhydantoin (Takahashi 1986). Nowadays, the
annual production of D-4-hydroxyphenylglycine
(about 10 000 t), an important intermediate for the
synthesis of β-lactam antibiotics, is carried out mostly
using hydantoinase (Ogawa et al. 2012). Apart from
an industrial process, hydantoinase has been used on
a laboratory scale to produce unnatural D-amino
acids for use as ligands of some classes of receptors
or important chiral building blocks for producing
biologically active compounds. For instance, a series
Correspondence: Gniewomir Latacz, Department of Technology and Biotechnology of Drugs, Jagiellonian University, Medical College, Medyczna 9, 30-688
Kraków, Poland. Tel: ϩ48-126205579. Fax: ϩ48-126205596. E-mail: glatacz@cm-uj.krakow.pl
(Received 21 May 2013; revised 2 October 2013; accepted 9 February 2014)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2014 Informa UK, Ltd.
DOI: 10.3109/10242422.2014.893578

118 G. Latacz & K. Kiec´-Kononowicz
of D-phenylglycine derivatives, potential glutamate
receptors effectors, were successfully produced from
racemic-ring-substituted 5-phenylhydantoins (PHs)
using pure enzyme or microorganisms with D-
hydantoinase activity (Garcia & Azerad 1997). The
range of racemic PHs, which can be efficiently con-
verted by D-hydantoinase, were also extended to
include fluorine and trifluoromethyl moieties (Arcuri
et al. 2003). Silicon-containing D-configured N-
carbamoylaminoacidswerealsosuccessfullyobtained
using D-hydantoinase from racemic 5-silylmethylated
hydantoins (Smith et al. 2001). Recently, stereoselec-
tive D-hydantoinase catalysed hydrolysis of aryl-
substituted dihydropyrimidines to the corresponding
N-carbamoyl-β-amino acids for conversion to opti-
cally pure β-amino acids was also investigated (Engel
et al. 2012).
of phenylalanine derivatives were reported to be
a potent ligands of metabotropic (Ma et al.
1998; Pin et al 1999) and ionotropic glutamate
receptors (Szyman´ska et al.2009,2011;Venskutonyte
et al. 2012).
Within this study we determined the substrate
specifity and stereoselectivity of two D-hydantoinases
from adzuki bean towards several new (R,S)-5-
benzylhydantoin derivatives with different substitu-
ents. We also increased the scope of D-hydantoinase
application through production of a new series of
D-phenylalanine derivatives. Additionally, a simple
method for monitoring the biotransformation by
capillary electrophoresis (CE) is also proposed.
Materials and methods
The occurrence of D-amino acids in nature has
been described extensively in recent decades. An
important D-amino acid, which was found in many
naturally occurring, biologically active peptides, is
D-phenylalanine. For instance, pharmaceutically
important peptides with strong antibacterial activity
from Bacillus sp. contain D-phenylalanine: Gramici-
din S, Tyrocidine A–D (Katz & Demain 1977),
Bacitracin A (Ikai et al. 1995), Polymyxin B (Banerjee
& Bose 1963). D-phenylalanine has also been found
in Fungisporin, a cyclooctapeptide isolated from
spores of species of Penicillium and Aspergillus (Studer
1969;Wakayama et al. 2003). Several active peptides
containing D-phenylalanine have also been isolated
from animals: Conotoxins (Buczek et al. 2005),
Ocp-1 (Iwakoshi et al. 2000), cHH (Soyez et al.
2000), Achatin I (Kim et al. 1991). Moreover,
D-phenylalanine was reported to be important for
the design new peptidomimetics because of their
hydrophobic activity, which give better transport
properties of peptidomimetics through cellular mem-
branes (Wang et al. 2002).The other benefits include
better resistance to hydrolysis by peptidases and pro-
teases and antibody recognition of peptidomimetics
containing D-amino acids (Pappenheimer et al.
1994; Chong et al. 1996). D-phenylalanine has been
used for the synthesis of following important pepti-
domimetics: Ac-rfwink-NH2 (Dooley et al. 1994),
Nateglinide (Tentolouris et al. 2007), Octreotide
and Hexarelin (Dasgupta 2004; Melis et al. 2000).
An important group of decapeptides containing not
only D-phenylalanine but also their derivatives,
4-amino-D-phenylalanine and 4-chloro-D-phenyla-
lanine, are the analogues of GnRH hormone (Herbst
2003; Martinez-Rodriguez et al. 2010). Additionally,
D-phenylalanine was shown to possess antidepres-
sant activity in Parkinson’s disease treatment and was
also described as an analgesic in warm-blooded
animals (Heller 1980). Moreover, the enantiomers
Chemicals
Unless otherwise stated, all materials were obtained
from commercial sources and used without further
purification. The benzaldehydes 1a (3-carboxybenzal-
dehyde) and 2a (4-carboxybenzaldehyde) were
obtained from Alfa Aesar. The benzaldehydes 3a, 4a
and 5a were synthesised using 2-, 3- or 4- hydroxyben-
zaldehydes and corresponding bromoesters in the
presence of K2CO3/EtOH (Figure 1). The respective
hydroxybenzaldehyde (0.05 mol) and appropriate
bromo ester (0.05 mol) and K2CO3 (0.025 mol) were
stirred for 8 h in EtOH (50 ml).After removing K2CO3
by filtration, EtOH was evaporated in vacuo. Next, to
the remaining material 2% NaOH was added and
extracted with CH2Cl2. Evaporation of the organic
layer (dried with Na2SO4) gave crude benzaldehydes
3a–5a used for further reactions.The substrates for the
enzymatic reactions 1c–5c: (R,S)-5-(3′-carboxybenzyl)
hydantoin (1c), (R,S)-5-(4′-carboxybenzyl)hydantoin
(2c), (R,S)-5-(2′-carbomethoxybenzyl)hydantoin (3c),
(R,S)-5-(3′-carbomethoxybenzyl)hydantoin (4c) and
(R,S)-5-(4′(4-ethoxycarboxy)propoxybenzyl)hydan-
toin (5c) were synthesised from 1a–5a initially using
Knoevenagel’s condensation to afford 5-arylidenehy-
dantoins as we described previously (Latacz et al.
2006). Next, catalytic hydrogenation of the unsatu-
rated bond in 5-position of the hydantoin ring in the
presence of Pd/C catalyst was performed (Figure 1).
For this reaction the 5-arylidenehydantoins (0.5 mM)
(1b–5b) were dissolved in methanol (250 ml) and, after
addition of 100 mg Pd/C, treated with hydrogen (40
ml/min) at 55°C for 8 h. Pure (R,S)-5-benzylhydan-
toins (1c–5c) were recovered by evaporation of the
methanol in vacuo.The reactions were monitored using
CE at 20 kV, pH 8.9 of the background electrolyte. 1H
NMR spectra, CE and LC/MS analysis confirmed
slight (compound 5c) or almost complete (compounds
3c and 4c) hydrolysis of the ester moiety from the

Biotransformation of (R,S)-5-benzylhydantoin derivatives by D-hydantoinases 119
Figure 1. Synthesis of (R,S)-5-benzylhydantoins.
obtained alkoxybenzaldehyde derivatives after the
Knoevenagel reaction. Complete ester hydrolysis
occurred under conditions of diazotisation (Figure 2).
a MEL-TEMP II apparatus and are uncorrected. 1H
NMR spectra were recorded on aVarian MercuryVX
300 MHz PFG instrument (Varian Inc., Palo Alto,
CA, USA) in [d6]-DMSO at ambient temperature
using the solvent signal as an internal standard.
Chemical shifts are shown in δ. J values are in Hertz
(Hz) and splitting patterns are designated as follows:
s (singlet), d (doublet), t (triplet), q (quartet), qu
(quintet), m (multiplet). Mass spectra were recorded
on LC/MS system consisted of a Waters Acquity
UPLC, coupled to aWatersTQD mass spectrometer
(electrospray ionisation mode ESI-tandem quadru-
pole). A Beckman CE system (P/ACE MDQ)
equipped with diode-array detector was used for all
separations. It was controlled using 32 Karat Soft-
ware version 8.0. An uncoated fused-silica capillary
with total length of 60 cm (50.2 cm to detection
window) and internal diameter 75 μm was purchased
from Beckman. The separations were carried out in
sodium borate buffer pH 8.9 or pH 9.0 (Beckman).
The CE conditions were 25–30 kV, and the
Enzymes
Two D-hydantoinases were used in this study: pure,
isolated directly from (adzuki bean) Vigna angularis
(V. a .D-HYD, 494 U/g using hydantoin as a sub-
strate) and immobilised, recombinant, cloned and
expressed in Escherichia coli (rD-HYD, 53.1 U/g
using hydantoin as a substrate), both obtained from
Sigma-Aldrich.
Analysis
Chemical syntheses were monitored using thin-layer
chromatography (TLC) carried out on 0.25 mm
Kieselgel 60F254 plates (MERCK) and visualised
using UV light. Melting points were determined on
Figure 2. Two-step D-amino acid production using D-hydantoinase and diazotisation.

120 G. Latacz & K. Kiec´-Kononowicz
1.2
1
temperature was 25°C. For the chiral analysis of
4-carboxypropoxy-D-phenylalanine a Beckman Chi-
ral Methods Development Kit was used. The Kit
contained the following: uncoated fused-silica capil-
lary with total length of 30 cm (20 cm to detection
window), phosphate buffer 50 mM pH 2.5, 20% HS-
α-CD and PTS (1,3,6,8-pyrene tetrasulfonate) as an
internal standard. The enantioseparation was per-
formed in 5% solution of HS-α-CD in phosphate
buffer at pH 2.5.The CE conditions were 15 kV, and
the temperature was 25°C.The enantiomeric purities
of all D-phenylalanine derivatives were analysed
using Dionex P580 HPLC instrument equipped with
a ChiroSil (RCAϩ) column containing the chiral sta-
tionary phase prepared by a covalent trifunctional
bonding of (ϩ) or (-)-(18-Crown-6)-tetracarboxylic
acid. The mobile phase was prepared according to
the manufacturer’s instructions dedicated for the
enantioseparation of D,L-phenylalanine and was
composed of 70% methanol/30% 10 mM CH3COOH.
The flow rate was 1.5 ml/min, the temperature 25°C
and the detection wavelength 225 nm. Ion-exchange
chromatography was performed using Amberlite 120
IR/Hϩ cation-exchange resin obtained from Alfa
Aesar. Optical rotations were measured on a Jasco
DIP-1000 spectropolarimeter at the sodium D-line
with a 1 dm path-length analytical cell.
rD-HYD
0.8
0.6
0.4
0.2
0
0
24
48
72
96
120
Reaction time in h
Figure 3. The bioconversion of (R,S)-5-benzylhydantoin
derivatives with rD-HYD in sodium borate buffer pH 8.9, at 37°C
for 96 h: (᭜) 1c, (ᮀ) 2c, (᭝) 3c, (᭡) 4c, (◊) 5c.
and the reaction mixture was acidified with 3.5 M
HCl to pH 1.Then, 5 μl of 3.5 M NaNO2 was added
and the reaction mixture was stored on ice for 6 h.
The reaction was stopped by alkalisation to pH 9.0
with 3.5 M NaOH (Figure 2). The D-amino acids
were not isolated. Enantiomeric excess (ee) of the
obtained D-amino acid was measured using HPLC
and CE (Table I).
Preparation of D-phenylalanine derivatives 1e, 2e, 3e,
4e, 5e by rD-HYD
Biotransformations
To prepare the substrate solutions each (R,S)-5-
benzylhydantoin derivative was dissolved in 100 μl
of DMSO and then sodium borate buffer pH 9
(MERCK) up to a concentration of 1 mM was
added.The calibration curves for compounds 1c–5c
were estimated using CE with the following sub-
strate concentrations: 1, 0.7, 0.5, 0.1 and 0.02 mM
using DMSO as the internal standard.The biotrans-
formation was carried out on the orbital shaker
120 rpm, at 37°C for 96 h and monitored using CE.
All experiments were carried out with 10 mg of
D-hydantoinase suspended in a 2 ml solution of
1 mM 5-benzylhydantoin derivative in sodium borate
buffer pH 9. The course of reaction was followed
by CE determination of concentration of the res-
pective (R,S)-5-benzylhydantoin derivative (1c–5c).
Biotransformation data were collected after 24, 48,
72 and 96 h of the reaction (Figures 3 and 4). The
electrophoreses were performed at room tempera-
ture applying a voltage of 20 kV and at pH 8.9 of the
background electrolyte for reaction mixtures with
rD-HYD and applying a voltage of 30 kV and at pH
9 of the background electrolyte for reaction mixtures
with V.a.D-HYD. In the next step, the D-hydantoi-
nase was removed using a 0.2 μm membrane filter
For the larger-scale reaction (R,S)-5-benzylhydan-
toin derivatives (0.2 mmol) were dissolved in 200 ml
sodium borate buffer pH 8.9 and rD-HYD (1 g) was
added and incubated with shaking (120 rpm)
for 144 h in 37°C.The reaction was monitored using
1.2
V.a.D-HYD
1
0.8
0.6
0.4
0.2
0
0
24
48
72
96
120
Reaction time in h
Figure 4. The bioconversion of (R,S)-5-benzylhydantoin
derivatives with V.a.D-HYD in sodium borate buffer pH 8.9, at
37°C for 96 h: (᭜) 1c, (ᮀ) 2c, (᭝) 3c, (᭡) 4c, (◊) 5c.

Biotransformation of (R,S)-5-benzylhydantoin derivatives by D-hydantoinases 121
tm ϭ 4.20 min MS calculated for [Mϩ H]ϩ:
C11H13NO5 m/z: 240.08 found 240.15;
Table I. Biotransformation efficiency and enantioselectivity.
rD-HYD V.a.D-HYD
Conversion
Conversion
3-carbomethoxy-D-phenylalanine (4e). Prepared from
4c as brown powder mp 222–224°C; HPLC:
tLϭ3.40 min [minor, (L)-isomer], tDϭ4.15 min
[major, (D)-isomer], 91.9% ee; CE: tm ϭ4.06 min;
MS calculated for [MϩH]ϩ: C11H13NO5 m/z:
240.08 found 240.15; 1H-NMR [D2O] δ 2.81–2.89
(dd, J1 ϭ5.90, J2 ϭ8.46, 1H, H-C-H); 3.06–3.78
(dd, J1 ϭ9.23 Hz, J2 ϭ5.00 Hz, 1H, H-C-H); 3.72–
3.76 (q, Jϭ4.44 Hz, 1H, CH-NH2); 4.36 (s, 2H,
O-CH2-COOH); 6.72–6.80 (m, 3H, Ph-2-H,
Ph-4-H, Ph-6-H); 7.19 (t, Jϭ7.82 Hz, 1H, Ph-5-H);
[α]D ϭϩ20.1 (cϭ0.001, H2O);
Substrate
eea (%)
(%)
eea (%)
(%)
1c
2c
3c
4c
5c
83.0
98.5
94.5
91.9
98.5b
97.3c
23
86
22
67
76.0
97.5
98.7
99.4
18
97
17
81
81
99.4
97
aee (%) estimated using HPLC for 1e–5e or CE for 5e.
bHPLC.
cCE.
CE every 24 h. The reaction was stopped after
144 h by removing rD-HYD using a 0.2 μm mem-
brane filter. Next, the reaction mixture was acidified
with 3.5 M HCl to pH 1 and 250 μl of 3.5 M NaNO2
was added. After 6 h of cooling on ice 3.5 M NaOH
was added to pH 9.0 and the D-phenylalanine
derivatives were deposited on Amberlite 120 IR/Hϩ
cation-exchange resin.The D-amino acids were next
eluted with 0.9 M NH4OH and recovered by
lyophilisation. The 1H NMR analyses were per-
formed for the compounds 2e, 4e and 5e.The pres-
ence of all obtained D-amino acids was confirmed
using CE, HPLC and LC/MS. Due to the low con-
version yield of compounds 1c and 3c to the corre-
sponding N-carbamoyl-D-phenylalanine derivatives
and low ion-exchange chromatography efficiency,
no 1H NMR data of 3-carboxy-D-phenylalanine
(1e) and 2-carbomethoxy-D-phenylalanine (3e) were
obtained.
4-carbopropoxy-D-phenylalanine (5e). Prepared from
5c as brown powder mp. 248–250°C, HPLC:
tLϭ2.10 min [minor, (L)-isomer], tDϭ2.59 min
[major, (D)-isomer], 99.36% ee; CEchiral: tm ϭ4.15
min [major, (D)-isomer tm ϭ5.2 min [minor, (L)-
isomer]; CE: tm ϭ3.56 min MS calculated for
[MϩH]ϩ: C13H17NO5 m/z: 268.11 found 268.16;
1H-NMR [D2O] δ 1.90 (qu, Jϭ6.86 Hz, 2H, -OOC-
CH2-CH2-CH2-O); 2.26 (t, Jϭ7.31 Hz, 2H, -OOC-
CH2-CH2-CH2-O); 2.87–2.95 (dd, J1 ϭ6.67 Hz,
J2 ϭ7.95 Hz, 1H, H-C-H), 3.06–3.78 (dd, J1 ϭ9.75
Hz,J2 ϭ5.00 Hz,1H,H-C-H);3.78–3.83 (q,Jϭ4.36
Hz, 1H, CH-NH2); 3.95 (t, Jϭ6.41 Hz, 2H, OOC-
CH2-CH2-CH2-O); 7.23 (d, Jϭ8.72 Hz, 2H,
Ph-2-H, Ph-6-H), 7.11 (d, Jϭ8.46 Hz, 2H, Ph-3-H,
Ph-5-H); [α]D ϭϩ16.2 (cϭ0.001, H2O)
Results
3-carboxy-D-phenylalanine (1e). Prepared from 1c.
HPLC:tLϭ3.34 min [minor,(L)-isomer],tDϭ4.68
min [major, (D)-isomer], 83.03% ee; CE: tm ϭ4.42
min; MS calculated for [MϩH]ϩ: C10H11NO4 m/z:
210.07 found 210.19;
FromTable I and Figures 3 and 4 it can be seen that
the conversion yield of 1c to the corresponding
N-carbamoyl-D-amino acid after 96 h of biotransfor-
mation was only 23% with the V.a.D-HYD enzyme
and 18% with rD-HYD enzyme. Chiral HPLC anal-
ysis of 3-carboxy-D-phenylalanine prepared from
substrate 1c by the hydantoinase process showed the
lowest enantiomeric purity among all the D-amino
acids obtained (83% ee for rD-HYD and 76% ee for
V.a.D-HYD). The lowest bioconversion efficiency
was observed for compound 3c (22% conversion
with rD-HYD, 17% conversion with V.a.D-HYD).
However, the resulting 2-carbomethoxy-D-phenyla-
lanine showed a high optical purity (94.5% ee for
rD-HYD and 98.7% ee for V.a.D-HYD, respec-
tively). The highest bioconversion efficiency of the
examined substrates was shown for 2c (86% conver-
sion with rD-HYD, 97% conversion with V.a.D-HYD)
and 5c (81% conversion with rD-HYD, 97% conver-
sion with V.a.D-HYD). Both D-hydantoinases
4-carboxy-D-phenylalanine (2e). Prepared from 2c as
brown powder. mp. 315–317°C; HPLC: tLϭ3.16
min [minor, (L)-isomer], tDϭ3.76 min [major, (D)-
isomer], 98.5% ee; CE: tm ϭ4.02 min; MS calculated
for [MϩH]ϩ: C10H11NO4 m/z:210.07 found 210.12;
1H-NMR [D2O] δ 2.96–3.03 (dd, J1 ϭ6.15 Hz,
J2 ϭ8.46 Hz, 1H, H-C-H), 3.18–3.25 (dd, J1 ϭ9.75
Hz, J2 ϭ4.87 Hz, 1H, H-C-H);3.78–3.83 (q, Jϭ4.45
Hz,1H,CH-NH2);6.86 (d,Jϭ8.21 Hz,2H,Ph-2-H,
Ph-6-H); 7.70 (d, Jϭ8.46 Hz, 2H, Ph-3-H, Ph-5-H);
[α]D ϭϩ17.8 (cϭ0.0005, H2O)
2-carbomethoxy-D-phenylalanine (3e). Prepared from
3c. HPLC: tLϭ2.74 min [minor, (L)-isomer],
tDϭ3.25 min [major, (D)-isomer], 94.5% ee; CE:

122 G. Latacz & K. Kiec´-Kononowicz
showed very high enantioselectivity towards 5c
with enantiomeric excesses of the corresponding
4-carboxypropoxy-D-phenylalanine 99.4% ee
(V.a.D-HYD) and 98.5% ee (rD-HYD). Addition-
ally, the ee of the 4-carboxypropoxy-D-phenylalanine
obtained from rD-HYD was calculated using chiral
CE and compared with the HPLC result (98.5% ee
for HPLC and 97.3% ee for chiral CE respectively).
The conversion yield of 4c to the corresponding
N-carbamoyl-D-phenylalanine derivative was 67%
with rD-HYD as catalyst and 81% with V.a.D-HYD.
V.a.D-HYD also showed very high enantioselectivity
towards 4c.The ee of 3-carbomethoxy-D-phenylala-
nine obtained from 4c by using V.a.D-HYD
was 99.4% ee, whereas using rD-HYD it was only
91.9% ee.
1992; Schnell et al. 2003). In addition, it was shown
that the activity of D-hydantoinase is also affected
by product inhibition (Kikugawa et al. 1994; Soong
et al. 1999).To our knowledge, the D-hydantoinases
examined during this study were able to hydrolyse
all new substrates into corresponding N-carbamoyl
amino acids D-selectively with significant enantio-
meric purity. However, the efficiency and enantiose-
lectivity differed depending on the enzyme and on
substrate. Generally, V.a.D-HYD revealed higher
hydrolysis efficiency than rD-HYD, which is in
accordance with manufacturer specifications. Sur-
prisingly, very high enantioselectivity and efficiency
of 5c hydrolysis by using rD-HYD and V.a.D-HYD
was observed. This substrate has a very long side
chain in the 4 position of the phenyl ring. Similar
results were obtained for 2c, the substrate with a
carboxylic substituent in the 4 position of the
phenyl-ring. On the other hand, very low hydrolysis
efficiency and the lowest enantioselectivity among
all substrates rD-HYD and V. a .D-HYD were
exhibited towards 1c.This substrate possess the same
carboxylic substituent as 2c but at the 3 position of
the phenyl ring. Surprisingly, another substrate with
a larger substituent in the 3 position, 4c, was hydro-
lysed by the D-hydantoinases examined with signifi-
cant conversion yield and high enantioselectivity.
Substrate 3c, which has a similar substituent was
converted with very low efficiency. However, chiral
analysis of the phenylalanine derivative obtained
from 3c showed a high ee of the D-enantiomer.
Discussion
A broad range of hydantoin derivatives which can be
converted non- or stereoselectively to N-carbamoyl-
amino acids has been reported in the literature.
However, the substrate specificity, activity and ste-
reoselectivity of hydantoinases vary with the type of
substrate and the source of the enzyme (Syldatk
et al. 1999; Sharma & Vohra 1999). For instance,
the D-hydantoinase obtained from adzuki bean
(Vigna angularis) prefers PH as the substrate rather,
than 5-(4-hydroxyphenyl)hydantoin (pHPH). It was
shown, that specific activity towards PH was 5.3–7-
fold higher of that towards pHPH (Fan & Lee 2001).
The hydantoinases were also shown to be substrate-
dependent in terms of enantioselectivity.The hydan-
toinase from Arthrobacter aurescens DSM 3745 was
reported to be highly L-selective for indolylmethyl-
hydantoin, non-selective for methylthioethylhy-
dantoin (with preference for the D-enantiomer)
and D-selective for methylhydantoin (May et al.
1998). Recent analyses of substrate docking and
re-engineering of the D-hydantoinase binding pocket
using mutagenesis showed that the activity, efficiency
and substrate specificity of D-hydantoinase depends
on the size and spatial orientation of the substrate.
In that study it was shown that modification of the
enzyme’s hydrophobic binding pocket allows better
access of substrates to the catalytic centre increasing
enzyme activity (Lee et al. 2009; Cai et al. 2009).
However, the efficiency of hydantoin-derivative
hydrolysis catalysed by D-hydantoinase may also be
reduced due to the low racemisation rate of the sub-
strate. The velocity of chemical racemisation of
monosubstituted hydantoins bearing an aromatic
substituent in 5-position may vary according to the
electronic nature of the substituent (Syldatk et al.
Conclusions
rD-HYD and V.a.D-HYD enantioselectivity and
hydrolysis efficiency towards new (R,S) 5-benzylhy-
dantoin derivatives is substrate-dependent. The enz-
ymes have a preference for substrates with substituents
in the 4 position of the phenyl ring regardless of their
size. However, the exact reasons for differences in the
hydrolysis efficiency and enantioselectivity of the sub-
strates examined remain to be elucidated.
Acknowledgement
The authors gratefully acknowledge the financial
support of the Polish Ministry of Health and Higher
Education grant N405 2518 33.
Declaration of interest: The authors report no
declarations of interest. The authors alone are
responsible for the content and writing of the
paper.

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