Microwave-Assisted Cyclization of Unprotected Dipeptides in Water to 2,5-Piperazinediones and Self-Assembly Study of Products and Reagents

Article abstract of DOI:10.1055/s-0037-1612376

Dipeptides and their cyclized 2,5-piperazinedione (or diketopiperazine, DKP) derivatives are attractive building blocks for supra-molecular hydrogels. The Phe-Phe, (p -nitro)-Phe-Phe, and Phe-Val dipeptides and their corresponding DKPs are studied for sel

Full text of DOI:10.1055/s-0037-1612376

SYNTHESIS
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Georg Thieme Verlag Stuttgart · New York
2019, 51, A–F
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Synthesis
M. Kurbasic et al.
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Microwave-Assisted Cyclization of Unprotected Dipeptides in
Water to 2,5-Piperazinediones and Self-Assembly Study of
Products and Reagents
Marina Kurbasica
Sabrina Semeraroa
Ana M. Garciaa
Slavko Kralja,b
Evelina Parisia
Caterina Deganuttia
Rita De Zorzia
Silvia Marchesan*a
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a
Chemical & Pharmaceutical Sciences Dept., University of Trieste,
Via L. Giorgieri 1, 34127 Trieste, Italy
Materials Synthesis Department, Jožef Stefan Institute, Jamova
b
39, 1000 Ljubljana, Slovenia
smarchesan@units.it
Published as part of the Bürgenstock Special Section 2018 Future
Stars in Organic Chemistry
Received: 03.02.2019
Accepted after revision: 08.03.2019
Published online: 25.03.2019
its initial dissolution at the concentration required to gel an
aqueous buffer. In light of these findings, we reasoned that
9
DOI: 10.1055/s-0037-1612376; Art ID: ss-2019-z0070-op
the compound (p-nitro)-Phe-Phe could be an interesting
candidate to achieve a stable hydrogel without the use of
organic solvents. Indeed, short peptides composed of 2–4
amino acids are highly attractive as hydrogelators thanks to
their inherent biocompatibility, low-cost and ease of prepa-
ration also on a large scale, and benign fate in the environ-
ment. The applications are numerous and diverse, and
range from the delivery of agents for plant growth stimula-
Abstract Dipeptides and their cyclized 2,5-piperazinedione (or diketo-
piperazine, DKP) derivatives are attractive building blocks for supra-
molecular hydrogels. The Phe-Phe, (p-nitro)-Phe-Phe, and Phe-Val di-
peptides and their corresponding DKPs are studied for self-assembly in
water. The DKPs were obtained in high yields by microwave-assisted
cyclization of the dipeptides in water, demonstrating that use of their
methyl ester derivatives as reported in the literature is not necessary for
successful cyclization. Single-crystal XRD structures are reported for
two DKPs as well as stable hydrogels at neutral pH.
tion,10 imaging, and drugs, to supramolecular catalysts,
11
12
13
14
and organocatalysts, to cite a few recent examples. How-
ever, Phe-Phe is notorious for its poor water solubility and
it typically requires organic solvents for its dissolution, thus
limiting the scope of this compound.
Key words dipeptide, diketopiperazine, self-assembly, hydrogels,
amyloid, microwaves, 2,5-piperazinedione
Since the early report on Science in 2003 by Reches and
Dipeptides have been reported to cyclize to 2,5-pipera-
zinediones (or diketopiperazines, DKPs), at a range of pH
values, and upon heating. DKPs are found in numerous
natural products, as well as in processed food and beverag-
es, and occur also as secondary metabolites in microorgan-
isms and fungi. DKPs are very interesting for medicinal
chemistry, thanks to their ability to mimic a peptidic turn
that is a common feature of many bioactive compounds,
1
Gazit, diphenylalanine (Phe-Phe) has become a very popu-
2
3
15
lar self-assembling compound on its own, in mixtures, or
4
as a motif in longer peptides and their derivatives. A num-
ber of approaches have been successfully used to modulate
the supramolecular behavior of related compounds, includ-
5
ing use of D-amino acids in D,L-heterochiral sequences, N-
terminus protection with fluorenylmethyloxycarbonyl
6
7
(
Fmoc) protecting group or other aromatic moieties, as
and indeed they have been reported to have anticancer, an-
tibiotic, antiviral properties, to cite a few.1
5–17
They are also
well as phenylalanine substitution with electron-donating
or electron-withdrawing groups to stabilize dipolar interac-
attractive as chiral catalysts in organic chemistry.15 Recent-
ly, they have raised interest even in the area of the origins of
life, since they have been reported to form spontaneously
from proline-containing dipeptides under alkaline condi-
8
tions between neighboring aromatic rings. In particular, p-
nitro substitution of the Fmoc-Phe benzene ring proved to
be the most effective amongst the substitutions tested, and
8
18
resulted in the most rapid self-assembly. Phe-Phe was also
tions and in simulated prebiotic conditions. In the area of
reported to self-assemble into metastable hydrogels, al-
though it required use of an organic solvent to assist with
supramolecular chemistry, DKPs have been long known for
their ability to pack into high-order structures, both as
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M. Kurbasic et al.
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crystals and in the (organo)gel phase.19 From a synthetic
point of view, the most recent dipeptide cyclization strate-
gies are microwave-assisted and have been reported in
water starting from dipeptides protected at the C-terminus
Table 1 Microwave-Assisted Dipeptide Cyclization in Water to DKP
O
O
R1
MW
R2
H
(
180 °C, 10 min, 250 W)
HN
N
COOH
H2N
20
NH
as ester derivatives, and either N-protected or N-depro-
R1
R2
H2O
O
21
tected. Use of protected dipeptides as reagents is sensible
in light of the fact that their synthesis requires use of pro-
tecting groups. Key advantages relative to the more tradi-
tional thermal heating are 1) short reaction times, 2) ab-
sence of epimerization, and 3) generally high yields, al-
though amino acids with steric demand near the peptide
backbone, such as Phe and Val, have been reported to be
Dipeptide
p-nitro)-Phe-Phe
DKP
R1
R2
Yield (%)
(
1
2
3
4-nitrobenzyl
benzyl
benzyl
quantitative
Phe-Phe
Phe-Val
benzyl
95
90
benzyl
2-propyl
20,21
less reactive and result in lower yields.
In light of the
prominent role of Phe in self-assembling peptide sequences
and their derivatives, similar water-based and facile proto-
cols with quantitative yields also for Phe-bearing dipeptides
would be highly attractive. An additional advantageous fea-
ture would be the possibility to directly use unprotected di-
peptides as reagents, since they are widely available as
products from enzymatic reactions, or in extracts from nat-
ural sources.
Figure 1 Single-crystal XRD structures of DKP1 (cyan) and DKP2
green)
(
This work reports the microwave-assisted synthesis in
water of DKPs from unprotected dipeptides, namely (p-ni-
tro)-Phe-Phe, Phe-Phe, and Phe-Val, all in excellent yields
with a reaction time as short as 10 minutes. Products were
ed network of hydrogen bonds involving DKP rings (Figure
1
13
24
characterized by H NMR, C NMR, ESI-MS, HRMS, melting
point, FT-IR, circular dichroism, polarimetry, and single-
crystal XRD [see sections 1, 5, and 7 of Supporting Informa-
tion (SI)]. In addition, we assessed the supramolecular be-
havior of the DKPs and parent dipeptides, and report on
self-assembling systems in phosphate buffer at neutral pH,
characterized by circular dichroism, Thioflavin T fluores-
cence as amyloid stain, transmission electron microscopy
2).
Having achieved a water-based synthesis of the desired
DKPs, we sought to compare the self-assembly behavior
among the three parent dipeptides and their corresponding
DKPs. To avoid use of organic solvents, each dipeptide was
first dissolved in a phosphate solution at pH 12, followed by
addition of an equal amount of a phosphate buffer at pH 6
to achieve a final pH of ~7.3, following a standard proce-
dure.25 To our delight, both (p-nitro)-Phe-Phe and Phe-Phe
could be dissolved with this approach without the need for
organic solvents at the final concentration of 15 mM that
was sufficient to obtain a self-supporting hydrogel (see Fig-
ure S25 of Section 2 in SI). Oscillatory rheology confirmed
rapid gel formation for both compounds (Figure S35 in SI).
TEM micrographs (see section 3 of SI) revealed elongated
nanostructures compatible with nanotubes as previously
(TEM), and rheometry (see sections 2–6 of SI).
(p-Nitro)-Phe-Phe was synthesized by Fmoc-based sol-
id-phase peptide synthesis, and purified by reversed phase
HPLC, while Phe-Phe and Phe-Val are commercially avail-
able. Each dipeptide was dissolved in water and underwent
microwave-assisted cyclization as detailed in Table 1. Each
compound was successfully converted into the correspond-
ing 2,5-diketopiperazine in nearly quantitative yield within
1
13
1
1
0 minutes, as confirmed by H NMR, C NMR, ESI-MS,
reported for Phe-Phe (Figures S26 and S27 in SI). On the
HRMS, FT-IR, circular dichroism, polarimetry, and single-
crystal XRD. In particular, the latter showed that the pres-
ence of the nitro group did not affect the conformation of
cyclo(Phe-Phe) (Figure 1). In both DKP1 and DKP2, the typi-
contrary, oscillatory rheology (Figure S35 in SI) confirmed
that Phe-Val remained soluble as seen by naked eye at con-
centrations as high as 100 mM (see Figure S25-C in SI). This
is not too surprising, considering that Phe-Val is notably
more hydrophilic than the other two dipeptides, as appar-
ent from reverse-phase HPLC retention times, which are an
experimental indication of hydrophobicity. They corre-
sponded to 8.4 and 8.1 minutes for (p-nitro)-Phe-Phe and
Phe-Phe, respectively, and to just 3.5 minutes for Phe-Val. A
difference between the supramolecular behavior of (p-ni-
tro)-Phe-Phe and Phe-Phe was that the latter displayed sy-
neresis over time, in agreement with previous reports on
metastable gels, which were formed upon dissolution of
22
cal turn-mimetic structure was seen, with DKP in planar
conformation, as expected for benzyl derivatives, which
15
feature the aromatic substituent folded on top. The CH-
23
interaction reported for DKP2 between the two side
chains maintained the same distance in DKP1 and DKP2, re-
gardless of the nitro-substitution, thus revealing absence of
electronic effects on molecular conformation due to the
presence of the nitro group (Figure 1). In terms of molecular
packing, both DKP1 and DKP2 featured the typical extend-
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Synthesis
M. Kurbasic et al.
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sponded to 11.6, 11.2, and 10.1 minutes for DKP1-3, respec-
tively (see Figures S10, S16, and S22 in SI). Dilution of the
DKP solutions with phosphate buffer to a final pH of ~7.3
and a final concentration of 15 mM led to a self-supporting
hydrogel only in the case of DKP1 (see Figure S25-D in SI),
confirming once again a positive role for the 4-nitro substi-
tution on Phe to promote self-assembly and hydrogelation.
Self-assembly of DKP1 and DKP3 was unreported to date,
while DKP2 was previously reported to self-assemble into
fibers and organogels, and noted for its poor solubility in a
number of solvents.26 Oscillatory rheology revealed a gel
nature for all DKPs 1–3, although gelation was rapid for
DKP1–2, while a lag time of several minutes was observed
for DKP3. TEM micrographs revealed the presence of elon-
gated nanostructures compatible with fibrils in all cases, al-
beit DKP1 and DKP2 displayed evident bundling into heter-
ogeneously-sized fibers (see Figures S28 and S29 in SI),
while DKP3 formed homogenously-sized nanofibrils (see
Figure S30 in SI). Thioflavin T fluorescence (see Figure S31
in SI), which is often used as a stain to detect amyloid struc-
tures,27 indeed was significantly higher for DKP3 than for
any other compound tested (i.e., DKP1–2 and the three par-
ent dipeptides). These data agree with previous reports on
increased fluorescence for the case of thin fibrils, as op-
posed to fibril bundles, due to an overall higher amyloid
surface available for dye binding.28
In conclusion, we have reported the rapid and high-
yield cyclization of unprotected dipeptides in water under
microwave irradiation. This work confirmed a positive role
played by p-nitro substitution on the benzene ring of phe-
nylalanine for self-assembly both of the linear dipeptide
Phe-Phe and for the corresponding DKP derivative, as previ-
ously reported for Fmoc-Phe by means of stabilization of di-
pole interactions.8 Single-crystal XRD structures revealed
absence of electronic effects due to the nitro group on DKP
molecular conformation and intramolecular CH- interac-
tion. These data further support the hypothesis that the
stabilizing role played by the nitro group in self-assembly is
due to electronic effects in terms of intermolecular, as op-
posed to intramolecular, interactions.
This work also investigated the self-assembly behavior
in water of the three dipeptides and their corresponding
DKPs, revealing that the 5 more hydrophobic compounds of
the 6 tested formed supramolecular structures of elongated
nanomorphology in aqueous buffer at neutral pH. In partic-
ular, only in the case of (p-nitro)-Phe-Phe and its DKP deriv-
ative, the compounds self-assembled into stable and self-
supporting hydrogels at neutral pH, confirming a positive
role played by the nitro group on hydrogelation. In the case
of Phe-Val, dipeptide cyclization favored self-assembly by
increasing hydrophobicity and allowed formation of ho-
mogenously sized nanofibrils for cyclo(Phe-Val).
Figure 2 Molecular packing of DKP1 (A) and DKP2 (B), and hydrogen
bonding network for DKP1 (C) and DKP2 (D)
Phe-Phe in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and
subsequent dilution with an aqueous buffer at pH 8 with
9
the assistance of ultrasonication. We were pleased to note
that, instead, the unreported (p-nitro)-Phe-Phe hydrogel
see Figure S25-A in SI) was stable for 7 days, thus support-
(
ing the reported role of 4-nitro substitution in stabilizing
the dipolar interactions between aromatic rings, hence the
6
aromatic-interaction driven self-assembly in water. Inter-
estingly, we noted that after 7 days of self-assembly, 20% of
(
p-nitro)-Phe-Phe had spontaneously converted into DKP1,
1
while Phe-Phe (and Phe-Val) had not, as revealed from H
NMR spectra of the freeze-dried hydrogels (Figures S42 and
S43 in SI).
The corresponding DKPs were also thus assessed for
their supramolecular behavior. Unfortunately these com-
pounds are more hydrophobic than their parent dipeptides,
hence they could only be dissolved at high concentration in
1,1,1,3,3,3-hexafluoroisopropanol (20% v/v of the final vol-
ume). Indeed their higher hydrophobicity relative to their
parent dipeptides could be assessed experimentally by
their reversed-phase HPLC retention times, which corre-
Indeed, all DKPs were notably more hydrophobic than
their parent linear dipeptides. On one hand, the increased
hydrophobicity was an issue since all DKPs displayed poor
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M. Kurbasic et al.
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solubility, and it was not possible to avoid use of organic
solvents to allow dissolution of these compounds into aque-
ous systems. On the other hand, cyclization of more hydro-
philic dipeptides could allow to obtain self-assembling mo-
tifs with defined conformation from non-assembling se-
quences. Future application of this approach to other
dipeptides could thus provide the means for the facile
preparation of DKPs in water, and for the introduction of a
variety of hydrophilic functional groups in geometrically-
defined self-assembling motifs for potential biological ap-
plications.
LC-MS Analysis
Flow 0.3 mL/min. The gradient used consisted of MeCN/H O with 0.1%
formic acid with the following program: t = 0–2 min, 25% MeCN;
t = 17 min, 95% MeCN; t = 17–20 min, 95% MeCN.
2
Hydrogel Preparation
The peptides were dissolved in sodium phosphate buffer (0.1 M pH
11.8) at a concentration of 30 mM; subsequent addition of an equal
volume of mildly acidic sodium phosphate buffer (0.1 M pH 5.8) re-
sulted in a final concentration of 15 mM and a pH of 7.3 ± 0.2. DKPs
were dissolved first in HFIP (20% v/v final volume) and then sodium
phosphate buffer (0.1 M, pH 7.3) was added on top (80% v/v).
Circular Dichroism
2
-Chlorotrytil resin, O-benzotriazole-N,N,N,N′-tetramethyluronium
To follow the self-assembly kinetics, the CD signal was monitored
over a range of wavelengths from 185 to 280 nm at 25 °C (Peltier) ev-
ery 5 min for 1 h. Samples were freshly prepared directly in the CD
cell and the spectra immediately recorded. CD spectra were acquired
for peptides at 15 mM (self-assembly conditions) and at 1 mM (condi-
tions under minimum gelling concentration).
hexafluorophosphate (HBTU), HOAt, and Fmoc-protected L-phenylal-
anine were purchased from GL Biochem (Shanghai) Ltd. Fmoc-pro-
tected (p-nitro)-L-phenylalanine (CAS 95753-55-2), Phe-Phe (CAS
2577-40-4), and Phe-Val (CAS 3918-90-9) dipeptides were purchased
from Sigma-Aldrich. All solvents were purchased of analytical grade
from Merck. Piperidine, trifluoroacetic acid (TFA), N,N-diisopropyl-
ethylamine (DIPEA), and triisopropylsilane (TIPS) were from Acros.
NaH PO and Na HPO4 were from BDH AnalaR. High purity Milli-Q
H O (MQ H O) with a resistivity greater than 18 M Ω cm was obtained
from an in-line Millipore RiOs/Origin system. Microwave-assisted re-
actions were carried out in a Discover SP MW reactor from CEM Cor-
Thioflavin T Fluorescence
2
4
2
Gel precursor solutions (0.12 mL) were prepared as described above
and immediately put on wells of Greiner 96 U Bottom Black P. After 4
h, 30 L of Thioflavin T (22.2 M in 20 mM glycine/NaOH pH 7.5, fil-
tered with a 0.2 m filter) solution was added in the wells. After 15
min, the fluorescence emission was analyzed, selecting an excitation
wavelength of 446 nm and an emission wavelength of 490 nm, with a
bandwidth of 20 nm. Each condition was repeated thrice in triplicate
2
2
1
poration (Matthews, NC, USA). H NMR spectra were recorded at 400
13
MHz and C NMR spectra were recorded at 101 MHz on a Varian In-
nova Instrument with chemical shifts reported as ppm (in DMSO with
TMS as internal standard). LC-MS analysis data were acquired on a
Agilent 6120 LC-MS system with a C-18 analytical column (Luna 5
(n = 9). Average and standard deviations were calculated and plotted
with Excel (see SI).

m, 100Å, 150 × 2 mm). Electrospray HRMS data were obtained on a
Bruker microTOF-Q. FT-IR spectra were recorded on a PerkinElmer
Paragon 1000 FT-IR spectrophotometer, with solid compounds com-
pressed into KBr pellets. Melting points were measured on a Gallen
Kamp apparatus. Polarimetric measurements were carried out on
compound solutions on a Jasco P-2000 Polarimeter. Circular dichro-
ism spectra were acquired in 0.1 mm quartz cells in a Jasco J815 Spec-
tropolarimeter, with 1s integrations, 1 accumulation, and a step size
of 1 nm with a bandwidth of 1 nm. The fluorescence emission was
analyzed using a Tecan Infinite M1000 pro. Rheological analyses were
performed on a Malvern Kinexus-Pro oscillatory rheometer with 20
mm-wide parallel plate geometry. Transmission electron microscopy
analyses were performed on JEM 2100 (Jeol, Japan) operated at 100
kV.
Oscillatory Rheometry
Each hydrogel was prepared in situ applying a gap of 1 mm. Time
sweeps were recorded for 1 h at 1 Pa and 1 Hz, frequency sweeps
were recorded at 1 Pa from 0.1 to 10 Hz and stress sweeps were re-
corded at 1 Hz from 1 Pa until the breaking point typical for every hy-
drogel, recognizable by the inversion of G′ and G′′ values. Each analy-
sis was repeated at least 2 times.
Transmission Electron Microscopy (TEM)
The small amount of supramolecular material was precisely deposit-
ed on a TEM grid (copper-grid-supported lacey carbon film), dried for
1
7
5 min at r.t., and contrasted by aq tungsten phosphate solution (pH
.4). TEM micrographs were acquired on at least 25 different spots on
Peptide Synthesis
TEM grid.
(
p-Nitro)-Phe-Phe was not commercially available, and it was pre-
pared from the corresponding amino acids by Fmoc-based solid-
Peptide Crystallization
25
phase peptide synthesis, under standard conditions.
,5-Piperazinediones; General Procedure
Crystals of DKP1 and DKP2 were grown using the sitting-drop vapor
diffusion method. A small vial with a solution of (p-nitro)-Phe-Phe or
DKP2 was introduced into a larger vial containing the reservoir solu-
tion. Thus, 800 L of the dipeptide solution (1 mM in MeOH) or DKP2
2
Each dipeptide [10.7, 9.4, 8.1 mg for (p-nitro)Phe-Phe, Phe-Phe, and
Phe-Val, respectively) was introduced in a 10 mL MW glass vial with
(<1 mg/mL in MeCN) was deposited in a small vial and sealed with a
reservoir containing either MeOH (3 mL, for DKP1) at 30% (v/v) in H O
or MeCN (for DKP2) at 70% (v/v) in H O, to allow vapor diffusion until
equilibration. Single crystals were grown for 3 weeks.
of milliQ H O (2 mL) to reach a final concentration of 15 mM. The
2
2
mixtures were dispersed with the assistance of ultrasonication in a
water-bath for 5 min at r.t. The vials were sealed and placed in the
MW reactor for 10 min at 180 °C and 250 W. The precipitate was vac-
2
uum filtered, washed with H O, and dried in vacuo to afford a white
Single-Crystal X-ray Diffraction
2
powder (DKP1, 10.2 mg, quantitative yield; DKP2, 8.6 mg, 95% yield;
DKP3, 6.6 mg, 90% yield).
Single crystals of DKP1and DKP2 were collected with a loop, cryopro-
tected by dipping the crystals in glycerol, and stored frozen in liquid
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Synthesis
M. Kurbasic et al.
Paper
N . The crystals were mounted on the diffractometer at the synchro-
(3S,6S)-3,6-Dibenzyl-2,5-piperazinedione (DKP2)
2
tron Elettra, Trieste (Italy), beamline XRD1, using the robot present at
White powder; yield: 9 mg (95%); mp 315 °C; []D20 +52 (c 0.15,
DMSO).
the facility. Temperature was kept at 100 K by a stream of N on the
2
crystals. Diffraction data were collected by the rotating crystal meth-
od using synchrotron radiation, wavelength 0.70 Å, rotation interval
IR (KBr): 3318, 3205, 3162, 3088, 2968, 2927, 2876, 1675, 1662, 1497,
–1
1
460, 1339 cm .
1°/image, crystal-to-detector distance of 85 mm. A total of 360 imag-
1
H NMR (400 MHz, DMSO-d ):  = 7.90 (s, 2 H, NH), 7.28–7.16 (m, 6 H,
es were collected for DKP1, and 360 for DKP2. Reflections were in-
6
2
9
ArH), 7.13–7.08 (m, 2 H, ArH), 3.96–3.93 (m, 2 H, CH), 2.57–2.52 (m,
2 H, CH), 2.23–2.18 (m, 2 H, CH).
dexed and integrated using the XDS package, space groups P2 for
1
30
DKP1 and P2 2 2 for DKP2, were determined using POINTLESS and
the resulting data set were scaled using AIMLESS. Phase information
were obtained by direct methods using the software SHELXS. Re-
finements cycles were conducted with SHELXL-14,
through the WinGX GUI, by full-matrix least-squares methods on
F2. Unit cell parameters and scaling statistics are reported in Table S1
1
1
31
13
C NMR (101 MHz, DMSO-d ):  = 166.4 (C=O), 137.0, 130.1, 128.6,
6
32
126.9 (Ar), 55.8 (C), overlapped with DMSO signal (C).
33
operating
+
MS (C18H18N O ): m/z = 395.1 [M + H] ; requires: 395.1.
2
2
34
HRMS: m/z [M + Na]+ calcd for C18H18N O Na: 317.1260 [M + Na] ;
+
2
2
found: 362.1261.
(see SI). For DKP1, a phenyl group was refined in 2 statistical posi-
tions at partial occupancy, occupancy of each position was refined to
give a total occupancy of 1. All the atoms except the H atoms within
(3S,6S)-3-Benzyl-6-(2-propyl)-2,5-piperazinedione (DKP3)
White powder; yield: 7 mg (90%); mp 270 °C; []D20 –68 (c 0.15, DM-
2
the asymmetric unit have been refined with anisotropic thermal pa-
rameters. H atoms were added at geometrically calculated positions
SO).
2
and refined isotropically. Refinement statistics are reported in Table
S1 (see SI).
IR (KBr): 3195, 3056, 2969, 2888, 1955, 1672 (br), 1613, 1456, 1346,
35
–1
1315 cm
.
1
H NMR (400 MHz, DMSO-d ):  = 8.06 (s, 1 H, NH), 7.87 (s, 1 H, NH),
6
(
4-NO )-Phe-Phe
2
7
.24–7.13 (m, 5 H, ArH), 4.18 (dd, J = 6.5, 4.9 Hz, 1 H, CH), 3.50 (dd,
J = 3.7, 1.9 Hz, 1 H, CH), 3.13 (dd, J = 13.5, 5.0 Hz, 1 H, CH), 2.85 (dd,
J = 13.5, 5.0 Hz, 1 H, CH), 1.67 (m, 1 H, CH), 0.62 (d, J = 7.1 Hz, 3 H,
CH), 0.24 (d, J = 6.8 Hz, 3 H, CH).
White powder; yield: 55 mg (85%); mp 290 °C; []D20 –25 (c 0.15, DM-
SO).
IR (KBr): 3306, 3091, 3038, 2935, 1714, 1669, 1599, 1557, 1523, 1437,
1
–1
397, 1352 cm
.
13
C NMR (101 MHz, DMSO-d ):  = 167.0, 166.8 (C=O), 136.8, 130.7,
6
1
H NMR (400 MHz, CD OD):  = 8.21–8.16 (m, 2 H, ArH), 7.60–7.45
128.5, 128.4, 126.9 (Ar), 59.6, 55.5 (C), 38.2, 31.5 (C), 18.7, 16.6 (C).
3
(


1
m, 2 H, ArH), 7.36–7.10 (m, 5 H, ArH), 4.71 (dd, J = 8.6, 5.3 Hz, 1 H,
CH), 4.15 (dd, J = 7.9, 5.8 Hz, 1 H, CH), 3.35 (dd, J = 14.3, 5.8 Hz, 1 H,
CH), 3.27–3.14 (m, 2 H, CH), 3.02 (dd, J = 14.1, 8.6 Hz, 1 H, CH).
+
MS (C14H18N O ): m/z (%) = 247.1 [M + H] ; requires: 247.1.
2
2
HRMS: m/z [M + Na]+ calcd for C14H18N O Na: 269.1260 [M + Na] ;
+
2
2
found: 269.1260.
3
C NMR (101 MHz, CD OD):  = 172.4, 167.7 (C=O), 147.6, 141.6
3
136.7, 130.5, 128.8, 128.1, 126.5, 123.5 (Ar), 53.9, 53.4 (C), 39.2, 38,6
(
C).
Funding Information
+
MS (C18H19N O ): m/z = 358.1 [M + H] ; requires: 358.1.
3
5
+
+
Italian Ministry of University and Research (MIUR) through the Scien-
tific Independence of Young Researchers (SIR) program (‘HOT-SPOT’
project, personal research grant no. RBSI14A7PL to S.M.), Ramón Are-
ces Foundation (A.M.G.’s fellowship), and Beneficentia Stiftung Funda-
HRMS: m/z [M + H] calcd for C18H19N O : 358.1397 [M + H] ; found:
3
5
358.1397.
(
3S,6S)-6-Benzyl-3-(4-nitrobenzyl)-2,5-piperazinedione (DKP1)
White powder; yield: 10 mg (quant); mp 300 °C; []D20 –20 (c 0.15,
tion.
M
i
n
i
stero
d
e
l
lʼ
I
stru
z
i
o
n
e
,
d
elʼ
U
n
i
v
ersità
e
d
e
l
l
a
R
i
c
erc
a
(R
B
S
I
1
4
A
7
P
L)
DMSO).
IR (KBr): 3306, 3095, 3038, 2935, 1713, 1669, 1599, 1510, 1438, 1397,
Acknowledgment
–1
1392 cm .
The authors would like to acknowledge Dr. Paolo Pengo for useful sci-
entific discussions and Dr. Fabio Hollan for HRMS technical assis-
tance.
1
H NMR (400 MHz, DMSO-d ):  = 8.15 (d, J = 1.0 Hz, 1 H, NH), 8.07–
.98 (m, 3 H, 1 H, NH, 2 H, ArH), 7.23–7.16 (m, 5 H, ArH), 7.14–7.04
6
7
(m, 2 H, ArH), 4.12 (dd, J = 5.8, 4.2 Hz, 1 H, CH), 4.01 (dd, J = 5.8, 5.3
Hz, 1 H, CH), 2.81 (m, J = 13.5, 4.3 Hz, 1 H, CH), 2.69 (m, J = 13.4, 4.9
Hz, 1 H, CH), 2.55–2.49 (m, 1 H, CH), 2.20 (dd, J = 13.5, 6.4 Hz, 1 H,
Supporting Information
CH).
13
C NMR (101 MHz, DMSO-d ):  = 166.7, 166.4 (C=O), 146.6, 145.5
Supporting information for this article is available online at
6
136.58, 131.2, 130.6, 128.5, 126.8, 123.53 (Ar), 55.6, 55.2 (C), 39.2,
https://doi.org/10.1055/s-0037-1612376.
S
u
p
p
orit
n
g Inform ati
o
n
S
u
p
p
orit
n
g Inform ati
o
n
38,6 (C).
+
MS (C18H17N O ): m/z = 358.1 [M + H] ; requires: 357.1.
3
4
References
HRMS: m/z [M + Na]+ calcd for C18H17N O Na: 362.1111 [M + Na] ;
+
3
4
found: 362.1111.
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©
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