Penicillin acylase-catalyzed peptide synthesis in aqueous medium: A chemo-enzymatic route to stereoisomerically pure diketopiperazines

Article abstract of DOI:10.1016/j.tetasy.2003.08.011

A range of non-natural dipeptides of the general formula D-(-)-phenylglycyl-L-X, where X is a natural α-amino acid, have been prepared by penicillin acylase-catalyzed synthesis in aqueous medium from D-(-)-phenylglycine amide and the corresponding amino acids. The conversion of the dipeptides to the corresponding dipeptide esters, followed by their subsequent spontaneous cyclization afforded the corresponding stereoisomerically pure diketopiperazines.

Full text of DOI:10.1016/j.tetasy.2003.08.011

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 3123–3128
Penicillin acylase-catalyzed peptide synthesis in aqueous medium:
a chemo-enzymatic route to stereoisomerically pure
diketopiperazines
Andrei Y. Khimiuk,a Alexei V. Korennykh,a Luuk M. van Langen,b Fred van Rantwijk,b
b
a,
&
Roger A. Sheldon and Vytas K. Svedas *
aBelozersky Institute of Physicochemical Biology and Department of Bioengineering and Bioinformatics,
Lomonosov Moscow State University, 119992 Moscow, Russia
bLaboratory of Biocatalysis and Organic Chemistry, Delft University of Technology, Julianalaan 136,
2628 BL Delft, The Netherlands
Received 28 May 2003; accepted 7 August 2003
Abstract—A range of non-natural dipeptides of the general formula
D
-(−)-phenylglycyl-
L
-X, where X is a natural a-amino acid,
-(−)-phenylglycine amide and the
have been prepared by penicillin acylase-catalyzed synthesis in aqueous medium from
D
corresponding amino acids. The conversion of the dipeptides to the corresponding dipeptide esters, followed by their subsequent
spontaneous cyclization afforded the corresponding stereoisomerically pure diketopiperazines.
© 2003 Elsevier Ltd. All rights reserved.
1. Introduction
tection and deprotection catalyst.6,7 Additionally, peni-
cillin acylase provides the unique possibility to
The chemical synthesis of dipeptides generally requires
protection and activation steps, which result in lengthy
and wasteful procedures. Enzymatic peptide synthesis,
which is, for example, practiced on an industrial scale
in the synthesis of the sweetener aspartame, is much
more efficient.1 Proteases have been widely applied in
the synthesis of peptides, but they are restricted to
synthesize a wide range of
D
-(−)-phenylglycine dipep-
tides with different amino acids. These can easily be
converted into the dipeptide esters, which, in turn, can
be cyclized to give the corresponding diketopiperazines.
The latter are interesting compounds in their own right;
their application as food additives,8 chitinase
inhibitors,9 anti-allergic agents10 and building block for
anti-viral compounds11 has been claimed. Diketopiper-
azines also catalyze the enantioselective addition of
hydrogen cyanide to aldehydes.12–14
L
-amino acid derivatives and do not convert non-natu-
ral amino acids, such as phenylglycine.
We recently have shown that penicillin acylase (peni-
cillin amidohydrolase, EC 3.5.1.11) from Escherichia
coli can be used for the enzymatic synthesis of non-nat-
ural phenylglycine dipeptides including those contain-
We have recently reported a chemoenzymatic route to
stereoisomerically pure 3,6-diphenyldiketopiperazines.2
Herein we demonstrate the extension of our procedure
ing the
subsite in penicillin acylase, which is highly selective for
phenylacetic acid and derivatives,3 can also accept
-(−)-
D
-(−)-phenylglycyl residue.2 The acyl-binding
to different
D-phenylglycine-derived dipeptides and a
range of the corresponding diketopiperazines.
D
phenylglycine derivatives as the acyl donor. In contrast,
the nucleophile binding subsite is extremely selective for
the
2. Results and discussion
L
-enantiomers of a wide range of amino acids.3–5
Hence, penicillin acylase has been used, in combination
with proteases, in peptide synthesis as a selective pro-
The chemoenzymatic procedure is outlined in Scheme
1. The initial step involves the penicillin acylase-
catalyzed acyl transfer from
(acyl donor) to the appropriate amino acid 2a–h as a
D
-phenylglycine amide 1
* Corresponding author. E-mail: vytas@belozersky.msu.ru
0957-4166/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2003.08.011

3124
A. Y. Khimiuk et al. / Tetrahedron: Asymmetry 14 (2003) 3123–3128
Scheme 1. Synthesis of diketopiperazines.
nucleophile. The reaction is kinetically controlled and
proceeds via an acyl-enzyme intermediate, in which the
-phenylglycyl moiety is covalently bound to the cata-
binding site.3 The reactions were performed at high pH
(9.7) to deprotonate the amino group in 2. The nucleo-
D
phile subsite is highly specific for L
-amino acids;3–5
lytic B1 serine residue. A single product is obtained due
to the unique substrate and stereospecificity of peni-
cillin acylase, combined with a judicious choice of the
reaction conditions. Any tendency of the nucleophile 2
to act as the acyl donor was suppressed by supplying
the former as a free amino acid, which is unable to
acylate penicillin acylase for thermodynamic reasons
and also due to the strict specificity of its acyl group
hence, 1 does not act as an acyl acceptor and no
D
-phenylglycine dimers are formed.2
The penicillin acylase-catalyzed conversion of the reac-
tants into the dipeptides 3a–h was high (>80% on
average) according to HPLC monitoring (see Table 1).
Portionwise addition of 1 avoided the inhibition of the
biocatalyst by high concentrations of 1 as well as the
Table 1. Synthesis of
Nucleophile 2
D-phenylglycine dipeptides according to Scheme 1
R
HPLC yield (%)
Isolated yield (%)
3
3
4
5
Glycine
a
b
c
d
e
f
H
CH3
i-C3H7
i-C4H9
85
88
72
76
85
87
94
85
61
34
41
70
67
53
34
n.d.
n.d.
66
82
81
67
67
n.d.
n.d.
31
42
n.d.
19
n.d.
58
L
L
L
L
L
L
L
-Alanine
-Valine
-Leucine
-Isoleucine
-Phenylalanine
-Tryptophan
-Histidine
s-C4H9
CH2C6H5
3-Indolylmethyl
3-Imidazolylmethyl
g
h
25
26

A. Y. Khimiuk et al. / Tetrahedron: Asymmetry 14 (2003) 3123–3128
3125
irreversible inactivation of the former that takes place
at high pH and is exacerbated at high substrate concen-
trations.15 The isolated yields (Table 1) generally were
much lower but the procedure was performed at a
relatively small scale and was not optimized.
3-(2-methylpropyl)-6-phenylpiperazine-2,5-dione (7.67),
(3R,6S)-3-phenyl-6-(phenylmethyl)piperazine-2,5-dione
(7.61), (3S,6R)-3-(1H-indol-3-ylmethyl)-6-phenylpiper-
azine-2,5-dione (7.80), (3S,6R)-3-(1H-imidazol-4-yl-
methyl)-6-phenylpiperazine-2,5-dione (8.80). Optical
rotations were determined using a Jasco (Japan) DIP-
360 Digital polarimeter.
The dipeptide acids were converted into the corre-
sponding methyl esters 4a–h via a standard procedure
and then incubated in alkaline aqueous solution in
order to give the diketopiperazines 5a–h. All tested
dipeptide esters formed diketopiperazines sluggishly
4.1.3. General method A: enzymatic synthesis of dipep-
tides. 1 (4.4 g, 29.3 mmol) and amino acid 2a–e (80
mmol) were suspended in 200 mL water at 20°C. The
pH was adjusted to 9.7 (25% NH4OH) and immobilized
E. coli penicillin acylase (3.8–5 g) was added. The pH
was maintained at 9.7 while stirring. Two further por-
tions of 1 (4.4 g, 29.3 mmol) were added when the
concentration of 1 was 5–15% from initial. When con-
compared to
ester and -phenylglycyl-
-Phenylglycyl- -isoleucine methyl ester proved to be
D
-phenylglycyl-
L-phenylglycine methyl
L
L
-phenylglycine methyl ester.
D
L
an extreme case and hardly any formation of dike-
topiperazine was observed after nearly 3 months. How-
ever, useful yields of diketopiperazines were obtained
from most of the other dipeptide esters.
version of the by-product (D-phenylglycine) was about
10%, the enzyme was removed by filtration. The pH
was adjusted to 5.5 (3 M H2SO4); the precipitate was
collected and purified several times by dissolution at pH
ꢀ10 followed by precipitation at pH ꢀ5.5
3. Conclusion
Penicillin acylase efficiently catalyzes the synthesis non-
4.1.4. General method B: synthesis of diketopiperazines.
The hydrochloride of the dipeptide methyl ester 4a–e,
3g was dissolved in 75 mL methanol–water (1:1, v/v),
the pH was raised to 8.0 using KOH and the reaction
mixture was stirred at room temperature for several
days. The precipitate was collected and recrystallized
from 1,2-dimethoxyethane.
natural dipeptides from
D-phenylglycine amide and L-
amino acids. The ring closure of the corresponding
dipeptide esters affords the stereoisomerically pure
diketopiperazine derivatives.
4. Experimental
4.2. Synthesis of (R)-3-phenyl-2,5-piperazinedione 5a
4.1. Materials and analysis
4.2.1.
D-Phenylglycyl-glycine 3a. 3a was synthesized
4.1.1. Materials. Immobilised penicillin acylase from E.
coli, Assemblase® (315 U/g), was kindly donated by
using the general method A with a reaction time of 310
min using 4.2 g of immobilized E. coli penicillin acylase.
Yield 10.2 g (49 mmol, 61%): dec. 245°C; [h]2D0=−123.2
DSM Anti-Infectives (Delft, The Netherlands).
Phenylglycine amide was received from DSM (Geleen,
D
-(−)-
1
(c 1, 2.5 M HCl). H NMR (300 MHz) (D2O+DCl):
The Netherlands). The
mon suppliers.
L-amino acids were from com-
+
3.96 (s, 2H, CH2NHCO), 5.20 (s, 1H, CHNH3 ), 7.49
(s, 5H, aromatic protons); 13C NMR (75 MHz) (D2O+
DCl): 31.2, 42.7, 58.1, 129.8, 131.2, 132.0, 133.2, 170.7,
174.3. Anal. calcd for C10H12N2O3: C, 57.69; H, 5.81;
N, 13.45. Found: C, 57.63; H, 5.93; N, 13.31.
4.1.2. Analytical methods. The synthesis of all com-
pounds was monitored by HPLC using a Waters 6000A
pump, a Phenomenex Luna C18(2) column (250×4.6
mm, 5 mm) and a Waters M481 LC detector at 208 nm
with 7 mM phosphate pH 3.0, containing acetonitrile
(from 30 to 45%, v/v) and a 0.7 g/L of sodium dodecyl-
sulphate, as the eluent. The flow rate was 0.5 mL/min.
Retention times (in min) for the eluent with 40%
4.2.2. (R)-3-Phenylpiperazine-2,5-dione 5a. 3a (6 g, 28.8
mmol) was refluxed in 600 mL methanol/HCl for 3 h
and subsequently concentrated in vacuo. The residue
was dissolved in 150 mL methanol–water (1:1, v/v); the
pH was raised to 8.0 (KOH) and the reaction mixture
was stirred at 20°C for 10 days. The precipitate was
collected and recrystallized from 1,2-dimethoxyethane,
yielding 5a (1.7 g, 8.9 mmol, 31%): mp 235°C, [h]2D0=
CH3CN:
(5.34), -phenylalanine (6.7),
tidine (6.33), -phenylglycyl-glycine (8.12),
glycyl- -alanine (9.46), -phenylglycyl- -valine (15.8),
-phenylglycyl- -leucine (21.9), -phenylglycyl-
isoleucine (21.3), -phenylglycyl- -phenylalanine (22.3),
-phenylglycyl- -tryptophan (22.9), -phenylglycyl-
glycine methyl ester (11.7), -phenylglycyl- -alanine
methyl ester (15.2), -phenylglycyl- -valine methyl ester
(28.2), -phenylglycyl- -leucine methyl ester (42.0),
phenylglycyl- -isoleucine methyl ester (42.5), -phenyl-
glycyl- -phenylalanine methyl ester (43.9), (R)-
D
-phenylglycine amide (8.8),
-tryptophan (8.74),
-phenyl-
D
-phenylglycine
L
L
L
-his-
D
D
L
D
L
1
D
L
D
L
-
−74.2 (c 1, DMSO). H NMR (300 MHz) (DMSO-d6):
D
L
3.73 (dd, 1H, CH2, J=3.2, J=17.8), 3.94 (d, 1H, CH2,
J=17.8), 4.87 (d, 1H, CHC6H5, J=2.9), 7.29–7.43 (m,
5H, aromatic protons), 8.14 (s, 1H, CHNHCO), 8.61
(d, 1H, CHNHCO, J=2.2); 13C NMR (75 MHz)
(DMSO-d6): 44.1, 58.4, 126.7, 127.8, 128.4, 138.7,
165.7, 166.1. Anal. calcd for C10H10N2O2: C, 63.15; H,
5.30; N, 14.73. Found: C, 60.81; H, 5.14; N, 13.91. MS
m/z: 190 (26), 169 (5), 162 (8), 147 (100), 132 (9), 118
(46), 104 (72), 91 (24), 77 (42), 69 (17), 63 (8), 56 (6), 51
(29), 44 (7).
D
L
D
D
L
D
L
D
L
D-
L
D
L
3-phenyl-2,5-piperazinedione (4.31), (3S,6R)-3-methyl-
6-phenylpiperazine-2,5-dione (4.96), (3S,6R)-3-isopro-
pyl-6-phenylpiperazine-2,5-dione
(5.6),
(3S,6R)-

3126
A. Y. Khimiuk et al. / Tetrahedron: Asymmetry 14 (2003) 3123–3128
4.3. Synthesis (3S,6R)-3-methyl-6-phenylpiperazine-2,5-
4.4.2. D-Phenylglycyl-L-valine methyl ester 4c. 3c (7 g,
dione 5b
28 mmol) was refluxed in 500 mL methanol/HCl for 3
h and subsequently concentrated in vacuo. The residue
was crystallized from a mixture of hexane, dioxane and
2-propanol, yielding 4c·HCl (6.9 g, 22.9 mmol, 82%):
dec. 123–124°C, [h]2D0=−77.0 (c 1, EtOH). 1H NMR
(300 MHz) (DMSO-d6): 0.63 (dd, 6H, CH(CH3)2, J=
6.9, J=14.1), 1.96 (m, 1H, CH(CH3)2), 3.57 (s, 3H,
OCH3), 4.21 (dd, 1H, CHNHCO, J=6.3, J=8.7), 5.14
4.3.1.
D-Phenylglycyl-L-alanine 3b. 3b was synthesized
using the general method A with a reaction time of 240
min using 3.8 g of immobilized E. coli penicillin acylase.
Yield 6.0 g (27 mmol, 34%): mp 251–252°C, [h]2D0=
1
−119.8 (c 1, 2.5 M HCl). H NMR (300 MHz) (D2O+
DCl): 1.28 (d, 3H, CHCH3, J=7.3), 4.35 (q, 1H,
+
(s, 1H, CHNH3+), 7.33–7.65 (m, 5H, aromatic protons),
+
CHCH3, J=7.3, J=14.6), 5.14 (s, 1H, CHNH3 ), 7.42–
8.8 (s, 3H, NH3 ), 9.03 (d, 1H, CHNHCO, J=8.6); 13C
NMR (75 MHz) (DMSO-d6): 17.5, 18.5, 30.2, 51.8,
54.9, 57.2, 127.5, 128.5, 128.9, 134.4, 167.8, 171.3. Anal.
calcd for C14H21ClN2O3: C, 55.90; H, 7.04; N, 9.31.
Found: C, 55.94; H, 7.15; N, 8.90.
7.55 (m, 5H, aromatic protons); 13C NMR (75 MHz)
(D2O+DCl): 17.4, 31.2, 50.5, 58.0, 129.6, 131.3, 132.0,
133.4, 169.9, 177.6. Anal. calcd for C11H14N2O3: C,
59.45; H, 6.35; N, 12.61. Found: C, 58.38; H, 6.37; N,
13.07.
4.4.3. (3S,6R)-3-Isopropyl-6-phenylpiperazine-2,5-dione
5c. 5c was synthesized using the general method B with
a reaction time of 73 days. Yield 0.23 g (1 mmol, 10%):
4.3.2. D-Phenylglycyl-L-alanine methyl ester 4b. 3b (5 g,
22.5 mmol) was refluxed for 3 h in 600 mL methanol/
HCl and subsequently concentrated in vacuo. The
residue was crystallized from dioxane, yielding 4b·HCl
(3.5 g, 12.8 mmol, 57%): dec. 194°C, [h]2D0=−118.4 (c 1,
1
mp 264°C, [h]2D0=−61.4 (c 1, DMSO). H NMR (400
MHz) (DMSO-d6): 0.7–1.02 (m, 6H, CH(CH3)2), 1.95–
2.32 (m, 1H, CH(CH3)2), 3.8 (m, 1H, CHiPr), 4.95 (s,
1H, CHC6H5), 7.18–7.44 (m, 5H, aromatic protons),
8.14 (s, 1H, CHNHCO), 8.41 (s, 1H, CHNHCO); 13C
NMR (100 MHz) (DMSO-d6): 16.6, 18.1, 31.4, 58.2,
59.1, 126.6, 127.3, 127.7, 128.2, 139.0, 166.8, 167.0.
1
EtOH). H NMR (300 MHz) (DMSO-d6): 1.21 (d, 3H,
CHCH3, J=6.9), 3.64 (s, 3H, OCH3+), 4.26 (m, 1H,
CHCH3, J=7.2), 5.05 (s, 1H, CHNH3 ), 7.38–7.60 (m,
+
5H, aromatic protons), 8.86 (s, 3H, NH3 ), 9.28 (d, 1H,
CHNHCO, J=7.2); 13C NMR (75 MHz) (DMSO-d6):
16.7, 47.9, 51.9, 55.0, 127.7, 128.6, 128.9, 134.0, 167.1,
172.2. Anal. calcd for C12H17ClN2O3: C, 52.85; H, 6.28;
N, 10.27. Found: C, 51.59; H, 6.24; N, 10.50.
4.5. Synthesis of (3S,6R)-3-(2-methylpropyl)-6-phenyl-
piperazine-2,5-dione 5d
4.3.3. (3S,6R)-3-Methyl-6-phenylpiperazine-2,5-dione 5b.
5b was synthesized using the general method B with a
reaction time of 5 days. Yield 1.1 g (5.3 mmol, 48%):
4.5.1.
D-Phenylglycyl-L-leucine 3d. 3d was synthesized
using the general method A with a reaction time of 470
min using 5 g of immobilized E. coli penicillin acylase.
Yield 14.8 g (56 mmol, 70%): mp 262°C, [h]2D0=−95.2 (c
1
mp 262–263°C, [h]D20=−57.2 (c 1, DMSO). H NMR
(300 MHz) (DMSO-d6): 1.29 (d, 3H, CHCH3, J=6.6),
4.00 (m, 1H, CHCH3, J=6.9), 4.90 (d, 1H, CHC6H5,
J=3.3), 7.30–7.42 (m, 5H, aromatic protons), 8.26 (s,
CHNHCO), 8.56 (d, 1H, CHNHCO, J=2.7); 13C
NMR (75 MHz) (DMSO-d6): 18.1, 49.0, 58.8, 126.6,
127.8, 128.4, 138.4, 166.4, 168.7. Anal calcd. for
C11H12N2O2: C, 64.69; H, 5.92; N, 13.72. Found: C,
65.00; H, 5.89; N, 14.00. MS m/z: 204 (21), 176 (14),
161 (100), 133 (32), 118 (26), 106 (85), 91 (28), 77 (34),
63 (6), 56 (12), 51 (21), 44 (80).
1
1, 2.5 M HCl). H NMR (300 MHz) (D2O+DCl): 0.64
(dd, 6H, CH(CH3)2, J=6.6, J=11.5), 1.11 (m, 1H,
CH2CH(CH3)2), 1.54 (m, 2H, CHCH2CH(CH3)2, J=
+
7.4), 4.38 (m, 1H, CHNHCO), 5.17 (s, 1H, CHNH3 ),
7.47 (s, 5H, aromatic protons); 13C NMR (75 MHz)
(D2O+DCl): 21.6, 23.6, 25.7, 31.2, 40.5, 52.9, 58.1, 71.6,
129.4, 131.2, 132.0, 133.3, 170.1, 177.5. Anal. calcd for
C14H20N2O3: C, 63.62; H, 7.63; N, 10.6. Found: C,
62.83; H, 7.97; N, 9.48.
4.5.2. D-Phenylglycyl-L-leucine methyl ester 4d. 3d (7 g,
4.4. Synthesis of (3S,6R)-3-isopropyl-6-phenylpiper-
26.5 mmol) was refluxed in 500 mL methanol/HCl over
3 h and subsequently concentrated in vacuo. The
residue was recrystallized from a mixture of hexane and
dioxane, yielding 4d·HCl (6.8 g, 21.6 mmol, 81%): mp
azine-2,5-dione 5c
4.4.1.
D-Phenylglycyl-L-valine 3c. 3c was synthesized
1
using the general method A with a reaction time of 240
min using 5 g of immobilized E. coli penicillin acylase.
Yield 8.1 g (32.4 mmol, 41%): mp 264–265°C, [h]2D0=
107–110°C, [h]2D0=−94.0 (c 1, EtOH). H NMR (300
MHz) (DMSO-d6): 0.6 (d, 3H, (CH3)CH(CH3), J=6.6),
0.7 (d, 3H, (CH3)CH(CH3), J=6.6), 1.19 (m, 1H,
CH2CH(CH3)2), 1.46 (m, 2H, CHCH2CH(CH3)2), 3.36
(s, 3H, OCH3) 4.26 (m, 1H, CHCH2CH(CH3)2), 5.02 (s,
1
−75.6 (c 1, 2.5 M HCl). H NMR (300 MHz) (D2O+
DCl): 0.66 (dd, 6H, CH(CH3)2, J=6.7, J=8.2), 2.07
(m, 1H, CH(CH3)2), 4.32 (d, 1H, CHNHCO, J=5.7),
+
1H, CHNH3 ), 7.34–7.62 (m, 5H, aromatic protons),
+
+
5.22 (s, 1H, CHNH3 ), 7.49 (m, 5H, aromatic protons);
8.77 (s, 3H, NH3 ), 9.17 (d, 1H, CHNHCO, J=8.1);
13C NMR (75 MHz) (D2O+DCl): 18.3, 19.9, 31.2, 31.6,
58.0, 59.8, 129.4, 129.6, 131.2, 131.9, 133.6, 170.3,
176.3. Anal. calcd for C13H18N2O3: C, 62.38; H, 7.25;
N, 11.19. Found: C, 61.16; H, 7.23; N, 11.22.
13C NMR (75 MHz) (DMSO-d6): 20.7, 22.5, 23.9, 39.5,
50.3, 51.9, 55.1, 127.6, 128.4, 128.9, 134.2, 167.5, 172.2.
Anal. calcd for C15H23ClN2O3: C, 57.23; H, 7.36; N,
8.90. Found: C, 57.61; H, 7.60; N, 9.33.

A. Y. Khimiuk et al. / Tetrahedron: Asymmetry 14 (2003) 3123–3128
3127
4.5.3.
(3S,6R)-3-(2-Methylpropyl)-6-phenylpiperazine-
adjusted to 9.7 (25% NH3) and immobilized E. coli
penicillin acylase (10 g) was added; the pH was main-
tained at 9.7 while stirring. After 280 min the enzyme
was removed by filtration. The pH was adjusted to 5.5
(3 M H2SO4); the precipitate was collected and twice
purified by dissolution at pH ꢀ10 followed by precipi-
tation at pH ꢀ5.5, yielding 3f (6.36 g, 21.3 mmol,
53%): mp 238°C, [h]2D0=−25.4 (c 1, 2.5 M HCl). 1H
NMR (400 MHz) (D2O+DCl): 2.8 (dd, 1H, CH2C6H5,
J=9.9, J=14.1), 3.16 (dd, 1H, CH2C6H5, J=4.5, J=
2,5-dione 5d. 5d was synthesized using the general
method B with a reaction time of 21 days. Yield 0.432
g (1.8 mmol, 19%): dec. 262°C, [h]2D0=−47.0 (c 1,
1
DMSO). H NMR (400 MHz) (DMSO-d6): 0.82–0.93
(m, 6H, CH(CH3)2), 1.65 (m, 2H, CHCH2CH(CH3)2),
1.87 (m, 1H, CH2CH(CH3)2), 3.89 (m, 1H,
CHCH2CH(CH3)2), 4.97 (s, 1H, CHC6H5), 7.3–7.43
(m, 5H, aromatic protons), 8.25 (s, 1H, CHNHCO),
8.52 (s, 1H, CHNHCO); 13C NMR (100 MHz)
(DMSO-d6): 22.0, 22.7, 23.4, 41.4, 52.4, 58.4, 127.2,
127.7, 128.3, 138.6, 166.6, 168.3. Anal. calcd for
C14H18N2O2: C, 68.27; H, 7.37; N, 11.37. Found: C,
70.26; H, 7.57; N, 10.94.
+
14.1), 4.8 (m, 1H, CHNHCO), 5.02 (s, 1H, CHNH3 )
6.83–7.5 (m, 10H, aromatic protons); 13C NMR (100
MHz) (D2O+DCl): 31.2, 37.9, 55.2, 58.1, 71.3, 128.5,
129.3, 130.2, 130.4, 131.3, 131.8, 133.1, 137.6, 169.7,
175.9. Anal. calcd for C17H18N2O3: C, 68.44; H, 6.08;
N, 9.39. Found: C, 67.64; H, 6.02; N, 9.27. MS m/z:
280 (10), 189 (8), 161 (13), 118 (7), 106 (100), 91 (25), 79
(21), 51 (5).
4.6. Synthesis of (3S,6R)-3-((S)-1-methylpropyl)-6-phenyl-
piperazine-2,5-dione 5e
4.6.1. D-Phenylglycyl-L-isoleucine 3e. 3e was synthesized
using the general method A with a reaction time of 315
min using 4.8 g of immobilized E. coli penicillin acylase.
Yield 14.3 g (54.1 mmol, 67%): mp 253°C, [h]2D0=−68.8
4.7.2. D-Phenylglycyl-L-phenylalanine methyl ester 4f. 3f
(3 g, 10 mmol) was refluxed in 450 mL methanol/HCl
for 3 h and concentrated in vacuo. The residue was
crystallized from isopropylalcohol, yielding 4f·HCl
(1.93 g, 5.5 mmol, 55%): dec. 228–229°C, [h]2D0=−40.2
1
(c 0.5, 2.5 M HCl). H NMR (300 MHz) (D2O+DCl):
0.67 (dd, 6H, CH(CH3)CH2CH3, J=7.1, J=13.2),
0.81–1.20 (m, 2H, CH(CH3)CH2CH3), 1.83 (m, 1H,
CH(CH3)CH2CH3), 4.34 (d, 1H, CHNHCO, J=6.0),
1
(c 1, EtOH). H NMR (400 MHz) (DMSO-d6): 2.76–
3.20 (m, 2H, CH2C6H5), 3.62 (s, 3H, OCH3), 4.52 (m,
+
+
5.18 (s, 1H, CHNH3 ), 7.48 (s, 5H, aromatic protons);
1H, CHNHCO), 5.02 (s, 1H, CHNH3 ), 6.83–7.5 (m,
13C NMR (75 MHz) (D2O+DCl): 11.8, 16.5, 25.9, 31.2,
37.9, 58.0, 59.1, 129.1, 131.2, 132.0, 133.6, 170.3, 176.6.
Anal calcd. for C14H20N2O3: C, 63.62; H, 7.63; N, 10.6.
Found: C, 63.13; H, 7.62; N, 10.85.
10H, aromatic protons), 8.75 (s, 3H, NH3 ), 9.2 (d, 1H,
+
CHNHCO); 13C NMR (100 MHz) (DMSO-d6): 36.1,
52.0, 53.5, 55.0, 126.3, 127.7, 128.0, 128.4, 128.7, 128.9,
133.9, 136.4, 167.3, 171.1. Anal. calcd for
C18H21ClN2O3: C, 61.98; H, 6.07; N, 8.03. Found: C,
61.63; H, 6.02; N, 7.86. MS m/z: 161 (6), 117 (17), 106
(100), 91 (54), 77 (31), 65 (14), 51 (23), 45 (70).
4.6.2.
D-Phenylglycyl-L-isoleucine methyl ester 4e. 3e (7
g, 26.5 mmol) was refluxed esterified in 600 mL
methanol/HCl for 3 h and concentrated in vacuo. The
residue was crystallized from a mixture of hexane and
2-propanol, yielding 4e·HCl (4.9 g, 15.6 mmol, 59%);
mp 175–177°C, [h]D20=−76.8 (c 1, EtOH). 1H NMR
4.7.3. (3R,6S)-3-Phenyl-6-(phenylmethyl)piperazine-2,5-
dione 5f. 4f·HCl (1.5 g, 4.3 mmol) was dissolved in 40
mL methanol–water (1:1 v/v), the pH was raised to 8.0
(KOH) and the reaction mixture was stirred at room
temperature. After 14 days the precipitate was collected
and crystallized from isopropyl alcohol, yielding 5f (0.7
g, 2.5 mmol, 58%); mp 243–244°C, [h]2D0=−17.4 (c 1,
(300
MHz)
(DMSO-d6):
0.63
(m,
6H,
CH(CH3)CH2CH3, J=7.5), 0.86–1.19 (m, 2H,
CH(CH3)CH2CH3), 1.72 (m, 1H, CH(CH3)CH2CH3),
3.67 (s, 3H, OCH3), 4.23 (dd, 1H, CHNHCO, J=6.7,
+
1
J=8.6), 5.15 (s, 1H, CHNH3 ), 7.37–7.64 (m, 5H,
DMSO). H NMR (400 MHz) (DMSO-d6): 2.9–3.3 (m,
+
aromatic protons), 8.90 (s, 3H, NH3 ), 9.10 (d, 1H,
2H, CH2C6H5), 4.23 (s, 1H, CHCH2), 4.35 (s, 1H,
CHC6H5), 7.15–7.38 (m, 10H, aromatic protons), 8.24
(s, 1H, CHNHCO), 8.43 (s, 1H, CHNHCO); 13C NMR
(100 MHz) (DMSO-d6): 37.8, 54.9, 58.2, 126.6, 127.7,
127.7, 128.0, 128.2, 130.1, 136.0, 138.9, 166.0, 166.7.
Anal. calcd for C17H16N2O2: C, 72.84; H, 5.75; N, 9.99.
Found: C, 73.00; H, 5.61; N, 10.02. MS m/z: 280 (62),
263 (6), 237 (9), 189 (51), 161 (87), 149 (10), 132 (11),
117 (46), 106 (60), 91 (100), 77 (34), 62 (22), 51 (19), 45
(46).
CHNHCO, J=8.7); 13C NMR (75 MHz) (DMSO-d6):
10.5, 15.1, 24.1, 36.4, 51.8, 54.8, 56.2, 127.5, 128.4,
128.9, 134.2, 167.6, 171.4. Anal. calcd for
C15H23ClN2O3: C, 57.23; H, 7.36; N, 8.90. Found: C,
57.03; H, 7.42; N, 9.22.
4.6.3.
(3S,6R)-3-((S)-1-Methylpropyl)-6-phenylpiper-
azine-2,5-dione 5e. An attempt to synthesize 5e using
the general method B did not succeed during 3 months
of incubation. At higher temperatures and/or pH 8.5
there was increased hydrolysis of the dipeptide ester but
cyclization was not observed.
4.8. Synthesis of (3S,6R)-3-(1H-indol-3-ylmethyl)-6-
phenylpiperazine-2,5-dione 5g
4.7. Synthesis (3R,6S)-3-phenyl-6-(phenylmethyl)-
piperazine-2,5-dione 5f
4.8.1.
D
-Phenylglycyl-
L-tryptophan 3g. 1 (1.76 g, 11.7
mmol) and
L
-(+)-tryptophan (6.54 g, 32 mmol) were
suspended in 80 mL water at 20°C. The pH was
adjusted to 9.7 (25% NH3) and immobilized E. coli
penicillin acylase (8.5 g) was added. The pH was main-
tained at 9.7 while stirring and after 10 and 30 min two
4.7.1.
mmol) and
were suspended in 90 mL water at 25°C. The pH was
D
-Phenylglycyl-
L-phenylalanine 2f. 1 (6.75 g, 45
L
-(+)-phenylalanine (6.69 g, 40.5 mmol)

3128
A. Y. Khimiuk et al. / Tetrahedron: Asymmetry 14 (2003) 3123–3128
subsequent portions of 1 (1.76 g, 11.7 mmol) were
added. After 140 min the product started to precipitate.
The enzyme and precipitated product were separated by
filtration, resuspended in 150 mL of water to dissolve
the product and the enzyme was removed by filtration.
The filtrates were combined; approx. 100 mL of water
was evaporated and ammonia was added, yielding
3g·NH3 (4.07 g, 11.5 mmol, 36%); mp 261–263°C,
J=5.1), 4.70 (d, 1H, CHC6H5, J=2.4), 6.83–7.60 (m,
7H, aromatic protons), 8.19 (s, 1H, CHNHCO), 8.57
(d, 1H, CHNHCO, J=2.2); 13C NMR (75 MHz)
(DMSO-d6): 29.5, 53.9, 58.5, 116.9, 126.9, 127.7, 128.3,
133.1, 134.8, 138.6, 166.2, 167.3. Anal. calcd for
C14H14N4O2: C, 62.21; H, 5.22; N, 20.73. Found: C,
59.32; H, 5.19; N, 20.95. MS m/z: 270 (57), 190 (26),
179 (6), 169 (14), 149 (11), 136 (20), 118 (20), 106 (72),
94 (24), 82 (100), 72 (29), 59 (52), 44 (29). HRMS calcd.
for C14H14N4O2: 270.1117. Found: 270.1123.
1
[h]2D0=−72.8 (c 1, 2.5 M HCl). H NMR (400 MHz)
(D2O+DCl): 2.96 (dd, 1H, CH2C, J=10.0, J=14.8),
3.27 (dd, 1H, CH2C, J=4.4, J=14.8), 4.87 (dd, 1H,
+
CHNHCO, J=4.4, J=10.0), 4.97 (s, 1H, CHNH3 ),
6.5 (s, 1H, NH-aromatic ring Trp), 6.98–7.47 (m, 10H,
aromatic protons); 13C NMR (100 MHz) (D2O+DCl):
28.3, 31.2, 54.2, 58.0, 109.9, 113.4, 119.6, 120.7, 123.2,
125.9, 127.8, 129.0, 131.0, 131.7, 133.1, 137.6, 169.7,
176.4. Anal. calcd for C19H22N4O3: C, 64.39; H, 6.26;
N, 15.81. Found: C, 64.40; H, 6.12, N, 15.78.
Acknowledgements
Financial support by the Russian Foundation for Basic
Research (grant 03-04-48472), INTAS (grant 2001-
0673), DSM Life Science products and the Netherlands
Ministry of Economic Affairs is gratefully acknowl-
edged. Thanks are due to Messrs. A. van Estrik and A.
Sinnema for the NMR analyses and to Mrs. A. Knol
for the MS measurements.
4.8.2.
(3S,6R)-3-(1H-indol-3-ylmethyl)-6-phenylpiper-
azine-2,5-dione 5g. 3g (3 g, 8.9 mmol) was refluxed in
450 mL methanol/HCl for 5 h and subsequently con-
centrated in vacuo. The residue was dissolved in 100
mL methanol–water (80:20, v/v); the pH was raised to
8.0 (KOH) and the reaction mixture was stirred at 20°C
for 28 days. The precipitate was collected and recrystal-
lized from 2-propanol, yielding 5g (0.71 g, 2.23 mmol,
References
1. Cheetham, P. S. J. In Applied Biocatalysis, 2nd ed.;
Straathof, A. J. J.; Adlercreutz, P., Eds.; Harwood:
Amsterdam, 2000; pp. 93–152.
1
25%): mp 271–272°C, [h]2D0=−34.8 (c 1, DMSO). H
NMR (300 MHz) (DMSO-d6): 3.04–3.4 (m, 2H, CH2),
4.3 (s, 2H, CHNHCO), 6.92–7.68 (m, 10H, aromatic
protons), 8.14 (s, 1H, CHNHCO), 8.34 (s, 1H, CHN-
HCO), 10.92 (s, 1H, NH-aromatic ring Trp); 13C NMR
(75 MHz) (DMSO-d6): 28.3, 54.8, 58.4, 108.3, 111.1,
118.3, 118.8, 120.7, 124.5, 127.1, 127.6, 127.7, 128.2,
135.8, 139.0, 166.0, 167.4. Anal. calcd for C19H17N3O2:
C, 71.46; H, 5.37; N, 13.16. Found: C, 68.86; H, 5.18;
N 12.82. MS m/z: 319 (7), 130 (100), 104 (7), 77 (11),
57 (10), 45 (24).
&
2. Van Langen, L. M.; Van Rantwijk, F.; Svedas, V. K.;
Sheldon, R. A. Tetrahedron: Asymmetry 2000, 11, 1077–
1083.
&
3. Svedas, V. K.; Savchenko, M. V.; Beltser, A. I.; Guranda,
D. F. Ann. NY Acad. Sci. 1996, 799, 659–669.
4. Kasche, V.; Michaelis, G.; Wiesemann, T. Biomed.
Biochim. Acta 1991, 50, 237–242.
&
5. Didzˇiapetris, R. J.; Svedas, V. K. Biomed. Biochim. Acta
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6. Didzˇiapetris, R. J.; Drabnig, B.; Schellenberger, V.;
&
Jakubke, H.-D.; Svedas, V. K. FEBS Lett. 1991, 287,
31–33.
4.9. Synthesis of (3S,6R)-3-(1H-imidazol-4-ylmethyl)-6-
phenylpiperazine-2,5-dione 5h
7. Reidel, A.; Waldmann, H. J. Pract. Chem. 1993, 335,
109–127.
1 (4.4 g, 29.3 mmol) and
L
-(+)-histidine (12.41 g, 80
8. Shibuya, K. (Ajinomoto), JP 58162260A, 1983.
9. Izumida, H.; Nishijima, M.; Sano, H.; Yamagishi, M.;
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M. (Kaiyo Bio Technol. Kenkyusho), JP 08277203A,
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10. Shimizaki, M.; Henmi, K.; Hashimoto, S. (Fujisawa), JP
63290868A, 1988.
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US 3560483, 1971
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181–185.
13. Danda, H. Synlett 1991, 263–264.
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1994, 5, 1541–1548.
mmol) were suspended in 200 mL water at 20°C. The
pH was adjusted to 9.7 (25% NH3) and 5 g immobilized
E. coli penicillin acylase was added. The pH was main-
tained at 9.7 while stirring. After 65 and 125 min two
subsequent portions of 1 (4.4 g, 29.3 mmol) were
added. After 200 min the enzyme was removed by
filtration. The filtrate was evaporated to dryness,
refluxed in 600 mL of methanol/HCl for 5 h and
concentrated in vacuo. The crude ester was dissolved in
200 mL methanol–water (1:1, v/v), the pH was raised to
8.0 (KOH) and the resulting solution was stirred at
20°C for 18 days. The precipitate was collected and
purified from ethanol/water by changing pH of the
solution, yielding 5h (5.6 g, 20.7 mmol, 26%): mp
1
222°C, [h]2D0=−40.6 (c 1, DMSO). H NMR (300 MHz)
15. Shcherbakova T. A.; Korennykh A. V.; Van Langen, L.
&
M.; Van Rantwijk, F.; Sheldon, R. A.; Svedas, V. K.,
paper in preparation.
(DMSO-d6): 2.95 (dd, 1H, CH2, J=6.6, J=14.8), 3.07
(dd, 1H, CH2, J=4.1, J=14.8), 4.17 (t, 1H, CHCH2,