Penicillin acylase-catalyzed peptide synthesis: A chemo-enzymatic route to stereoisomers of 3,6-diphenylpiperazine-2,5-dione

Article abstract of DOI:10.1016/S0957-4166(00)00027-6

Chiral dipeptides of phenylglycine were synthesized using immobilized Escherichia coli penicillin acylase. The high selectivity of penicillin acylase for L-amino acids as the nucleophile resulted in the efficient acylation of L-phenylglycine by D-phenylglycine amide at pH 9.7 to give D-phenylglycyl-L-phenylglycine in 69% yield. No isomers or tripeptides were formed. The low enantiospecificity of the enzyme for the acyl donor provided the possibility of preparing the corresponding L,L-dipeptides, starting from L-phenylglycine methyl ester as both donor and acceptor at pH 7.5, resulting in a 63% yield of L-phenylglycyl-L-phenylglycine methyl ester. The product precipitated under the reaction conditions; this effectively prevented the formation of oligomers as well as chemical transformation of the product.The dipeptide esters of phenylglycine easily cyclized to diketopiperazines in aqueous methanol. L-Phenylglycyl-L-phenylglycine methyl ester formed L,L-3,6-diphenylpiperazine-2,5-dione (cis); the achiral trans isomer was obtained from D-phenylglycyl-L-phenylglycine methyl ester. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0957-4166(00)00027-6

Tetrahedron: Asymmetry 11 (2000) 1077±1083
Penicillin acylase-catalyzed peptide synthesis:
a chemo-enzymatic route to stereoisomers of
3,6-diphenylpiperazine-2,5-dione
Luuk M. van Langen,a Fred van Rantwijk,a Vytas K. Svedasb and Roger A. Sheldona,*
aLaboratory of Organic Chemistry and Catalysis, Delft University of Technology, Julianalaan 136,
2628 BL Delft, The Netherlands
bBelozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119899 Moscow, Russia
Received 27 September 1999; revised 13 January 2000; accepted 14 January 2000
Abstract
Chiral dipeptides of phenylglycine were synthesized using immobilized Escherichia coli penicillin acylase.
The high selectivity of penicillin acylase for l-amino acids as the nucleophile resulted in the ecient acyl-
ation of l-phenylglycine by d-phenylglycine amide at pH 9.7 to give d-phenylglycyl-l-phenylglycine in
69% yield. No isomers or tripeptides were formed. The low enantiospeci®city of the enzyme for the acyl
donor provided the possibility of preparing the corresponding l,l-dipeptides, starting from l-phenyl-
glycine methyl ester as both donor and acceptor at pH 7.5, resulting in a 63% yield of l-phenylglycyl-l-
phenylglycine methyl ester. The product precipitated under the reaction conditions; this e€ectively pre-
vented the formation of oligomers as well as chemical transformation of the product.
The dipeptide esters of phenylglycine easily cyclized to diketopiperazines in aqueous methanol. l-Phenyl-
glycyl-l-phenylglycine methyl ester formed l,l-3,6-diphenylpiperazine-2,5-dione (cis); the achiral trans
isomer was obtained from d-phenylglycyl-l-phenylglycine methyl ester. # 2000 Elsevier Science Ltd. All
rights reserved.
1. Introduction
Peptides and their derivatives constitute an interesting class of biologically active compounds,
which ®nd application as food additives and antibiotics;1 one of them, the sweetener aspartame,
has become an industrial commodity. The classical synthesis of peptides requires protection and
activation of the donor and acceptor, whereas enzymatic routes allow the use of simple protecting
and activating groups or even no protecting group at all. Activation of the acyl donor, however,
is, for thermodynamic and kinetic reasons, inevitable.2±4 Enzymatic peptide synthesis using
* Corresponding author. Tel: 31 15 2782675; fax: 31 15 2784700; e-mail: r.a.sheldon@stm.tudelft.nl
0957-4166/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(00)00027-6

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proteases has been extensively studied in order to understand its mechanism as well as to improve
the selectivity and reduce the hydrolytic side reactions in kinetically controlled processes.5±7
Proteases are restricted to l-amino acids, however, and do not convert unnatural amino acids,
such as phenylglycine. We have already shown that penicillin acylase (EC 3.5.1.11) from E. coli,
which is used industrially for the hydrolytic cleavage of penicillin into 6b-aminopenicillanic acid
(6-APA) and phenylacetic acid,8 is a promising catalyst for the kinetically controlled synthesis of
phenylglycine-derived peptides.9 d-Phenylglycine dipeptides have interesting properties and can
be applied, for example, as tumor and tissue-dissolving compounds of low toxicity and as resolv-
ing agents for 1-arylethylamines.10,11
The unique substrate speci®city of penicillin acylase requires careful consideration in this
respect. The acyl donor binding subsite of penicillin acylase has a very high anity for phenyl-
acetic acid12 and can accept derivatives of phenylglycine (a-aminophenylacetic acid), but shows
moderate stereoselectivity.13 The acyl acceptor subsite, however, shows an extremely high pref-
erence for the l-enantiomers of a very broad range of amino acids.12,14,15
In this paper we present a method for the synthesis of stereochemically pure dipeptides of
phenylglycine without any need for protecting groups (Fig. 1). Dipeptide esters are known to readily
undergo ring closure to the corresponding diketopiperazines.16 The latter have interesting phys-
iological properties and can be used as food additives,17 as chitinase inhibitors18 and as synthons
for fungicidal, antiviral19 and anti-allergic compounds.20 We here report the ®rst unambiguous
characterization of two of the three possible stereoisomers of 3,6-diphenylpiperazine-2,5-dione.
Figure 1. Synthesis of phenylglycine containing dipeptides using E. coli penicillin acylase
2. Results and discussion
As noted above, the nucleophile binding subsite of penicillin acylase is l-speci®c. This
characteristic feature simpli®es the synthesis of d-phenylglycyl-l-phenylglycine 1a because any
formation of products with d-phenylglycine at the C-terminus or tripeptides is not to be expected.

L. M. van Langen et al. / Tetrahedron: Asymmetry 11 (2000) 1077±1083
1079
The possible formation of l-phenylglycyl-l-phenylglycine derivatives could be obviated by the
use of free l-phenylglycine, which acts as acceptor at alkaline conditions, but is an extremely
poor donor.4 Accordingly, d-( )-phenylglycine amide and l-(+)-phenylglycine smoothly reacted
at pH 9.7 in the presence of immobilized penicillin acylase. The condensation product 1a was
1
obtained in 69% yield (Table 1) by acidi®cation to pH 5.5. The H and 13C NMR spectra of the
product were compared with its d,d-diastereomer, which had been prepared independently by
chemical means (Fig. 2).11
Table 1
Penicillin acylase-catalyzed peptide synthesis
Figure 2. 1H NMR spectra of obtained products, compared with samples mixed with their diastereomers: (A)
CH(NH3Cl) signal of enzymatically obtained d-phenylglycyl-l-phenylglycine 1a and of a sample to which
d-phenylglycyl-d-phenylglycine has been added; (B) CH(NH3Cl) signal of enzymatically obtained l-phenylglycyl-l-
phenylglycine methyl ester 2b and of a sample to which d-phenylglycyl-l-phenylglycine methyl ester 2a has been added;
(C) CH signal of l,l-3,6-diphenylpiperazine-2,5-dione 3b, obtained by ring closure of 2b and d,l-3,6-diphenylpip-
erazine-2,5-dione 3a with added 3b; (D) CH signal of 3,6-diphenylpiperazine-2,5-dione obtained by heating
d-phenylglycine methyl ester for 18 h at 190ꢀC
Diastereomerically pure l-phenylglycyl-l-phenylglycine methyl ester 2b was enzymatically
synthesized in 63% yield from l-phenylglycine methyl ester, which acted both as activated acyl
donor and nucleophile. The synthesis product precipitated during reaction at pH 7.5, which pre-
vented possible tripeptide formation with 2b acting as a nucleophile. Compound 2b was isolated
by extraction with ethyl acetate. Furthermore, precipitation prevented spontaneous degradation
of the dipeptide ester during the course of its enzymatic synthesis.

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Both 2b and 2a, obtained by esteri®cation of 1a, easily underwent ring closure to the corre-
sponding diketopiperazines as solutions in aqueous alkaline methanol (Fig. 3). 3,6-Diphenyl-
piperazine-2,5-dione has been hitherto obtained by heating phenylglycine esters or their solutions under
basic conditions,21±23 by benzyloxylation and successive hydrogenolysis of 3,6-diphenyl-2,5-
dichloropyrazines23 or by decarboxylative coupling of Leuchs anhydrides catalyzed by aziridine.24
The diketopiperazine of phenylglycine exists in three stereoisomeric forms, two of which are
optically active. The diastereomers could be discriminated by thin layer chromatography, but their
isolation and subsequent stereochemical characterization has never been reported.23 Furthermore,
it was observed that the diastereomers of 3,6-diphenylpiperazine-2,5-dione could not be discriminated
by NMR (100 MHz),23 in contrast to several other 3,6-dialkylpiperazine-2,5-diones.23,25,26
Due to the mild reaction conditions we were able to isolate and characterize stereochemically
pure diketopiperazines obtained by ring closure of the corresponding dipeptide esters. The cycli-
zation of d-phenylglycyl-l-phenylglycine methyl ester 2a to the achiral diketopiperazine 3a was
4.5 times as fast as the cyclization of the l,l-dipeptide ester 2b to 3b (Fig. 4). Furthermore, the
formation of 3a was not accompanied by any side product formation, whereas cyclization of 2b
Figure 3. Diketopiperazine formation by ring closure of phenylglycine dipeptide esters
Figure 4. Rate of diketopiperazine formation by cyclization of d-phenylglycyl-l-phenylglycine methyl ester (~) and
l-phenylglycyl-l-phenylglycine methyl ester (&). Reaction conditions: a solution of 75 mM dipeptide ester in meth-
anol:water (1:1) containing 0.75 M NH3 was stirred at room temperature

L. M. van Langen et al. / Tetrahedron: Asymmetry 11 (2000) 1077±1083
1081
gave, besides 3b, a small amount of 3a, which was removed by recrystallization. Presumably, 3a is
more stable and more easily formed due to the di-equatorial trans con®guration of the phenyl
groups. It has been claimed that d,d-3,6-diphenylpiperazine-2,5-dione was obtained by heating of
d-phenylglycine methyl ester at 180±200ꢀC.19 In our hands, however, this procedure resulted in
the predominant (>90%) formation of the d,l-diketopiperazine 3a (Fig. 2).
3. Conclusion
Two of the possible stereoisomeric phenylglycine dipeptides have been synthesized in high yield
in one step from readily available precursors. Ring closure of the phenylglycine dipeptide esters
a€orded the corresponding diketopiperazines as pure stereoisomers.
4. Experimental
Immobilized E. coli penicillin acylase, Assemblase1 (315 U/g), was kindly donated by Gist-
brocades (Delft, The Netherlands); one unit is de®ned as the amount of enzyme that hydrolyzes 1
mmol of penicillin-G per minute at pH 8.0 and 34ꢀC. l-(+)-Phenylglycine and d-( )-phenyl-
glycine amide were received from DSM (Geleen, The Netherlands) as a gift. l-(+)-Phenylglycine
methyl ester hydrochloride salt was obtained from Aldrich.
4.1. Synthesis of d-phenylglycyl-l-phenylglycine 1a
d-( )-Phenylglycine amide (8 g, 53.3 mmol) and l-(+)-phenylglycine (6 g, 39.7 mmol) were
suspended in 120 ml water at 25ꢀC. The pH was brought to 9.7 using 25% ammonia and 10 g
immobilized E. coli penicillin acylase was added. The reaction was stirred with a turbine stirrer,
while the pH was maintained at 9.7. After 30 min the enzyme was separated from the mixture by
®ltration. The dipeptide was obtained by lowering the pH of the solution to 5.5 using 3 M H2SO4
to yield 7.84 g (27.6 mmol, 69%) of 1a. Dec. 270ꢀC; as HCl salt: dec. 204ꢀC; [ꢀ]D +51.6 (c 1,
methanol). 1H NMR (400 MHz): ꢁ 5.20 (s, 1H, CH(NH3Cl)), 5.40 (d, 1H, CHNHCO), 7.20±7.55
(m, 10H, aromatic protons), 9.42 (d, 1H, NHCO); 13C NMR (400 MHz): 171.05, 170.00, 136.37,
133.87, 128.91, 128.49, 128.33, 127.90, 127.69, 127.01, 56.18, 54.79. Anal. calcd for
C16H17N2O3Cl: C, 59.91; H, 5.34; N, 8.73; Cl, 11.1. Found: C, 59.45; H, 5.21; N, 5.58; Cl, 10.8.
1
IR (KBr) ꢂmax: 1685, 1671, 1516, 1491, 1369 cm .
4.2. Synthesis of l-phenylglycyl-l-phenylglycine methyl ester 2b
l-(+)-Phenylglycine methyl ester hydrochloride (5 g, 24.8 mmol) was dissolved in 60 ml water.
The solution was cooled to 0ꢀC and the pH was brought to 7.5 by addition of 25% ammonia; 5 g
of immobilized E. coli penicillin acylase was added and the mixture was stirred with a turbine
stirrer, while the pH was maintained at 7.5. The product precipitated in the course of reaction.
After 2.5 h 40 ml water was added to the reaction mixture to maintain stirrability. After 4 h 100
ml ethyl acetate was added and the pH was raised to 8.5 using 25% ammonia and the immobi-
lized enzyme was separated by ®ltration. The aqueous layer was extracted twice with 100 ml ethyl
acetate and the combined organic layers washed with 100 ml water and dried over Na2SO4. After

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evaporation of the ethyl acetate under reduced pressure the residue was recrystallized from
hexane, yielding 2.32 g (7.8 mmol, 63%) of 2b. M.p. 89±91ꢀC; [ꢀ]D +144.5 (c 1, methanol). 1H NMR
(300 MHz): ꢁ 3.58 (s, 3H, OCH3), 4.46 (s, 1H, CH(NH2)), 5.22 (s, 1H, CHNHCO), 7.20±7.42 (m,
10H, aromatic protons), 8.92 (s, 1H, NHCO); as HCl salt: dec. 209±210ꢀC; [ꢀ]D +120.0 (c 1,
1
methanol). H NMR (400 MHz): ꢁ 3.53 (s, 3H, OCH3), 5.18 (s, 1H, CH(NH2)), 5.24 (d, 1H,
CHNHCO), 7.38±7.64 (m, 10H, aromatic protons), 9.68 (d, 1H, NHCO); 13C NMR (300 MHz):
170.06, 167.12, 135.37, 133.73, 128.90, 128.66, 128.48, 127.91, 127.77, 56.37, 54.74, 52.21. Anal.
calcd for C17H18N2O3: C, 68.44; H, 6.08; N, 9.35. Found: C, 68.69; H, 6.40; N, 9.32. IR (KBr)
1
ꢂmax: 1736, 1638, 1519, 1324 cm .
4.3. Synthesis of d-phenylglycyl-l-phenylglycine methyl ester hydrochloride 2a
Compound 1a (2 g, 7 mmol) was esteri®ed by 4 h re¯uxing in 25 ml methanol/HCl. The
methanol was evaporated and the product was crystallized from dioxane, yielding 1.0 g (43%)
of 2a hydrochloride. Dec. 145ꢀC; [ꢀ]D +40.4 (c 1, methanol). H NMR: ꢁ 3.66 (s, 3H, OCH3),
1
5.22 (s, 1H, CH(NH2)), 5.47 (d, 1H, CHNHCO), 7.27±7.53 (m, 10H, aromatic protons), 9.64 (d,
1H, NHCO); 13C NMR (300 MHz): 170.31, 167.30, 135.39, 133.83, 128.94, 128.50, 128.24,
127.76, 127.31, 56.13, 54.79, 52.38. Anal. calcd for C17H19N2O3Cl: C, 60.99; H, 5.72; N, 8.37;
Cl, 10.59. Found: C, 60.72; H, 6.02; N, 8.26; Cl, 10.33. IR (KBr) ꢂmax: 1736, 1685, 1553,
1
1453 cm .
4.4. Synthesis of d,l-3,6-diphenylpiperazine-2,5-dione 3a
Compound 1a (2 g, 7 mmol) was esteri®ed by 4 h re¯uxing in 25 ml methanol/HCl. To the
reaction mixture 25 ml water was added and the pH was raised to 8.0 using KOH. After 3 h
stirring at room temperature the product 3a (1.68 g, 90%) was ®ltered o€. Dec. 294±296ꢀC; [ꢀ]D
1
+0.7 (c 1, DMSO). H NMR (400 MHz): ꢁ 5.11 (d, 2H, CH, J=2.0 Hz), 7.34±741 (m, 10H,
aromatic protons), 8.62 (d, 2H, NH); 13C NMR (400 MHz): 166.13, 138.95, 128.30, 127.83,
127.46, 58.51. HRMS calcd for C16H14N2O2: 266.1055. Found: 266.1061. IR (KBr) ꢂmax: 1670,
1
1450, 1303 cm .
4.5. Synthesis of l,l-3,6-diphenylpiperazine-2,5-dione 3b
Compound 2b (1 g, 3.4 mmol) was dissolved in 40 ml methanol:water (1:1) and the reaction
mixture was stirred for 18 h at room temperature. The solvents were evaporated and the crude
product was recrystallized from dimethoxyethane, yielding 0.31 g (34%) of 3b. Dec. 280±282ꢀC;
1
[ꢀ]D +68.5 (c 1, DMSO). H NMR (400 MHz): ꢁ 5.07 (d, 2H, CH, J=3.2 Hz), 7.22±7.32 (m,
10H, aromatic protons), 8.67 (d, 2H, NH); 13C NMR (400 MHz): 165.78, 138.32, 128.00, 127.60,
126.68, 58.13. HRMS calcd for C16H14N2O2: 266.1055. Found: 266.1057. IR (KBr) ꢂmax: 1690,
1
1678, 1448, 1327 cm .
4.6. HPLC analysis
Samples were analyzed by HPLC using a Waters M6000 pump, a Nucleosil C-18 column and a
Shimadzu SPD-6A UV detector at 215 nm. The eluent was prepared by adjusting the pH of a
1 g/l solution of sodium dodecylsulphate in methanol:water (70:30, v/v) to 3.5 with phosphoric

L. M. van Langen et al. / Tetrahedron: Asymmetry 11 (2000) 1077±1083
1083
acid. The ¯ow rate was 1.0 ml/min. Retention times (in minutes) were as follows: l,l-3,6-
diphenylpiperazine-2,5-dione (3.2), phenylglycine (4.5), d,l,-3,6-diphenylpiperazine-2,5-dione (4.8),
phenylglycine amide (5.2), d-phenylglycyl-d-phenylglycine (5.6), phenylglycine methyl ester (6.0),
d-phenylglycyl-l-phenylglycine (6.8), l-phenylglycyl-l-phenylglycine methyl ester (7.6), d-phenyl-
glycyl-l-phenylglycine methyl ester (8.2).
Acknowledgements
Financial support by DSM Life Science Products and the Netherlands Ministry of Economic
A€airs is gratefully acknowledged. This work was ®nancially supported by the Russian Foundation
for Fundamental Research (Grant 97-04-48827). The authors wish to thank Professor Katsuyuki
Ogura (Chiba University, Chiba, Japan) for a sample of chemically prepared d-phenylglycyl-d-
phenylglycine. Thanks are due to Messrs. A. van Estrik and A. Sinnema for the NMR analyses.
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