A green and expedient synthesis of enantiopure diketopiperazines via enzymatic resolution of unnatural amino acids

Article abstract of DOI:10.1016/j.tetlet.2014.06.105

Dipeptides comprising a d-phenylglycyl moiety coupled to the l-enantiomer of 2-amino butyric acid, norvaline, norleucine, and homocysteine were successfully synthesized from d-phenylglycine amide and the racemate of the corresponding unnatural amino acid.

Full text of DOI:10.1016/j.tetlet.2014.06.105

Accepted Manuscript
A green and expedient synthesis of enantiopure diketopiperazines via enzymatic
resolution of unnatural amino acids
Pedro C. Pereira, Isabel W.C.E. Arends, Roger A. Sheldon
PII:
S0040-4039(14)01118-6
DOI:
http://dx.doi.org/10.1016/j.tetlet.2014.06.105
TETL 44830
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 February 2014
4 June 2014
26 June 2014
Please cite this article as: Pereira, P.C., Arends, I.W.C., Sheldon, R.A., A green and expedient synthesis of
enantiopure diketopiperazines via enzymatic resolution of unnatural amino acids, Tetrahedron Letters (2014), doi:
http://dx.doi.org/10.1016/j.tetlet.2014.06.105
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A green and expedient synthesis of enantiopure diketopiperazines via
enzymatic resolution of unnatural amino acids
Pedro C. Pereira, Isabel W.C.E. Arends, Roger A. Sheldon
Section of Biocatalysis and Organic Chemistry, Department of Biotechnology, Delft University of Technology,
Julianalaan 136, 2628 BL, Delft, The Netherlands
Corresponding author: r.a.sheldon@tudelft.nl
ABSTRACT
Dipeptides comprising a D-phenylglycyl moiety coupled to the L-enantiomer of 2-amino
butyric acid, norvaline, norleucine and homocysteine were successfully synthesized from D-
phenylglycine amide and the racemate of the corresponding unnatural amino acid. The
reaction is catalyzed by an immobilized form of penicillin G acylase in an aqueous medium.
The dipeptides were subsequently converted into the corresponding enantiopure
diketopiperazines in overall isolated yields of 22% to 33%.

GRAPHICAL ABSTRACT
H
N
Penicillin
acylase
OH
OH
NH2
O
R
O
O
O
+
+
R
R
2 steps
NH2
O
H2N
N
H
H2N
R Abu (-CH2CH3); Nva (-CH2CH2CH3); Nle (-CH2CH2CH2CH3); Hcy (-CH2CH2SH)
2

Keywords: Diketopiperazine synthesis; Unnatural amino acids; Kinetic resolution; Penicillin
acylase
Piperazine-2,5-diones represent a rich source of biologically interesting compounds.1 The 2,5-
diketopiperazine motif has been the subject of significant attention due to its biological
properties.2,3 These include alteration of blood clotting functions,2 antitumor,4 antiviral
including HIV-I antagonists,5,6,7 antifungal,8 antibacterial,9 antihyperglycemic,10 and
analgesic,11 amongst others.12 Diketopiperazines are now seen as ideal compounds for the
rational design of new therapeutic agents.13,14
The applicability of these cyclic dipeptides extends beyond this, as they are also known for
possessing catalytic activity in the hydrocyanation of aldehydes15 and in the Strecker
synthesis of amino acids.16,17 In the former case it was shown that several diketopiperazines
have enantioselective catalytic activity,18 with enantiomeric excesses reaching 90%. In
addition to this there is great potential displayed by simple peptides in asymmetric catalysis,19
which has even been considered of prebiotic relevance.20 The versatile scope of these
compounds, together with the recent growth in research developed around them, suggests
their full potential is yet to be explored.21
Most biologically active diketopiperazines have been obtained from natural sources.12 One
representative example is the case of the (R,R)-diketopiperazines obtained from marine
bacteria. These show inhibitory activity against Vibrio anguillarum, a pathogenic bacterium
responsible for vibriosis, suffered by wild and farmed bivalves, crustaceans and fishes, which
in turn gives rise to severe economic losses, as it is a limitation in the development of
aquaculture.9
Diketopiperazines can also be synthesized. Although they have frequently been deemed as
unwanted side products, research regarding cyclic dipeptides has actually contributed to
peptide synthesis in general.22 This goal could be achieved by solid-phase strategies or
solution-phase syntheses. Representative examples of solution-phase syntheses are thermal
condensation of a dipeptide ester,23 coupling of protected amino acids followed by
deprotection and condensation,24,25 a multicomponent approach (Ugi reaction),26 and an
innovative microwave-based procedure has also been reported.27 Solid-phase strategies are
more widely used and have been frequently employed in building libraries of these
compounds.12
3

Biocatalysis is today a widely accepted method for the production of chemicals in general and
enantiopure compounds in particular.28-31 Enzyme-catalyzed transformations proceed under
mild conditions of temperature and pressure at physiological pH in water as solvent, and are
often highly chemo-, regio-, and enantioselective in addition to being atom and step
economic.32 An illustrative example is provided by the industrial synthesis of the artificial
sweetener aspartame, a dipeptide, catalyzed by the metalloprotease, thermolysin.33
We have previously described a chemo-enzymatic route for the synthesis of some enantiopure
diketopiperazines.34 The key enzymatic step involved coupling of two enantiopure species,
namely D-phenylglycine amide and an L-amino acid. This step was catalyzed by an
immobilized form (Assemblase 7500®) of penicillin G acylase (penicillin amidohydrolase EC
3.5.1.11) from Escherichia coli, which is used in the manufacture of β-lactam antibiotics.35,36
The resulting dipeptide was subsequently esterified followed by reaction with sodium
hydroxide in methanol, to afford the corresponding diketopiperazine (Scheme 1). We have
recently broadened the scope of this procedure by testing eleven new enantiopure amino
acids, all with chemically functionalized side-chains.37
This work describes a further expansion of the scope and synthetic utility of this concise and
practical methodology but uses a different approach. This consists of starting with a racemic
mixture rather than a pure enantiomer, thereby integrating a kinetic resolution into the
procedure. To this end, we have proceeded by ascertaining the viability of this route with
substrates that had not been previously tested in this route and could afford interesting
products, for example, new compounds or compounds with specific biological activity. 5
Here we report the results of these studies, in which we tested the racemates of unnatural
amino acids as one of the reactants. In addition to fulfilling the above mentioned pre-
requisites, these racemic mixtures are significantly less expensive than the corresponding pure
enantiomer (Scheme 2).
Dipeptide Syntheses
The enzymatic coupling reaction was performed at pH 9.8, in order to maximize the
concentration of reactive amine nucleophile. The acyl donor was added in portions, in order to
prevent D-phenylglycine amide induced inactivation, which is known to occur at high
concentrations and high pH.38 Only one coupling product was obtained in all the coupling
reactions, consistent with the known selectivity of the enzyme catalyst and demonstrating the
feasibility of the starting hypothesis (Table 1).39
4

The dipeptide derived from D-phenylglycine amide and L-norvaline coupling was obtained in
pure form according to NMR spectroscopy, with no need for any further purification. The
other three linear dipeptides were obtained with minor impurities, though they were easily
purified. NMR analysis showed 10-20% impurity for D-phenylglycyl-L-norleucine, 10-11%
for D-phenylglycyl-L-homocysteine and not quantifiable for the case of D-phenylglycyl-L-
amino-butyric acid. The impurities were identified by NMR as unreacted amino acid in the
first three cases and mass spectra indicated traces of D-phenylglycine in the last example.
Nevertheless, this was not reflected in the melting points. The isolated yields (Table 1) fit
within the window described previously.34
Table 1: Isolated yields of step a and steps b and c in the chemo-enzymatic dipeptide
synthesis (see Scheme 2) a
Dipeptide
Residue
-CH2CH3
Linear Dipeptide
Diketopiperazine
36%
46%
52%
32%
62%
64%
D-PG-L-Abu
D-PG-L-Nva
D-PG-L-Nle
D-PG-L-Hcy
-CH2CH2CH3
-CH2CH2CH2CH3
-CH2CH2SH
63%
not determined
a
Step a isolated yield was calculated as η=100 x (product/substrate) x 2; steps b and c isolated yield was
calculated as η=100 x (product/substrate)
Although the trend in our experiments suggests that nucleophiles with more carbon atoms
seem to be preferred, the isolated yields depend strongly on solubility. It was previously
found that the yield after crystallization is frequently lower than the observed HPLC yield and
that this varies with the amino acid residue.34, 35
These reactions were carried out on a relatively small scale and were not optimized,
suggesting there is ample room for further improvement in yields and product purity. The
thiol function showed no hindering effect, as already observed with other chemical
functionalities. 34, 35
Both steps b and c (Scheme 2) afforded the desired compounds, as was shown previously with
similar compounds.34,35,39 The methyl ester intermediates were not isolated and were used
directly in the condensation step, without any further purification or characterization. The
final product precipitated from solution. All the expected diketopiperazines were obtained and
1
three of the proposed four were obtained in pure form according to H-NMR spectroscopy.
5

The diketopiperazine containing the L-homocysteine residue could not be dissolved for NMR
analysis but was positively identified through mass spectrometry.
In conclusion, we have developed a green, cost-effective and chemo-enzymatic route for the
synthesis of enantiopure 2,5-diketopiperazines containing unnatural amino acids, starting
from the corresponding racemic amino acids. This was demonstrated in the synthesis of four
dipeptides which were subsequently converted into the corresponding 2,5-diketopiperazines,
5
all eight targeted compounds, some of which have known antiviral activity, were isolated
and/or identified. This suggests that the approach may be applicable to other cases where the
route is already feasible with enantiopure amino acids.
Of the four coupling products, one was obtained pure and three were obtained with minor
impurities. Three of the four possible diketopiperazines were obtained in pure state.
The presence of the D-enantiomer of the nucleophilic species did not seem to hinder the
formation of the desired enantiopure linear dipeptide, allowing for a synthetic strategy
involving a kinetic resolution. The dipeptide, D-PG-L-Abu, and its diketopiperazine were not
previously known compounds.
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8

NH2
NH OH
Penicillin
acylase
NH2
NH2
O
O
+
R
pH 9.7, 20o
O
NH2
OH
R
O
MeOH, HCl
NH2
NH
H
N
O
NaOH
O
O
H2O / MeOH
O
O
N
H
R
R
Scheme 1: Penicillin G acylase catalyzed diketopiperazine synthesis from enantiopure
natural amino acids
Step a
NH2
Penicilin
acylase
NH2
O
O
O
R
OH
O
OH
OH
+
+
H2N
H2N
HN
pH 9.8, 20o
O
NH2
R
R
Steps b and c
MeOH, SOCl2
i)
ii)
H2O / MeOH, NaOH
R:
-CH2CH3
-CH2CH2CH3
-CH2CH2CH2CH3 Nle
-CH2CH2SH Hcy
Abu
Nva
H
N
O
O
N
H
R
Scheme 2: Proposed penicillin G acylase catalyzed peptide synthesis with racemic unnatural
amino acids
9