Conformational studies of new pseudotripeptide with pyrazine amidoxime motif and simplified analogs using IR, NMR spectroscopy, and molecular dynamic simulations

Article abstract of DOI:10.1007/s11224-016-0870-2

Solution structures of new pyrazine-based pseudotripeptide with amidoxime function and simplified pseudodipeptide analogs were determined by a combination of IR and NMR spectroscopic studies and molecular dynamic simulations using explicit chloroform as a

Full text of DOI:10.1007/s11224-016-0870-2

Struct Chem
DOI 10.1007/s11224-016-0870-2
ORIGINAL RESEARCH
Conformational studies of new pseudotripeptide with pyrazine
amidoxime motif and simplified analogs using IR, NMR
spectroscopy, and molecular dynamic simulations
1
,2
2
1
3
Olga Ovdiichuk & Olga Hordiyenko & Evgenia Fotou & Caroline Gaucher
&
1
1
Axelle Arrault & Marie-Christine Averlant-Petit
Received: 17 June 2016 /Accepted: 27 October 2016
#
Springer Science+Business Media New York 2016
Abstract Solution structures of new pyrazine-based
pseudotripeptide with amidoxime function and simplified
pseudodipeptide analogs were determined by a combination
of IR and NMR spectroscopic studies and molecular dynamic
simulations using explicit chloroform as a solvent. It was
found that proline-phenylalanine dipeptide residue and
amidoxime moiety in o-position are essential for intramolec-
ular hydrogen bonding including a seven-membered γ-turn
formation. In addition, a cis/trans equilibrium study was pres-
Introduction
Amidoximes have been shown to possess a widespread of
biological activities [1]. They are also well known precursors
in the synthesis of 1,2,4-oxadiazoles and oxadiazole-derived
peptidomimetics, compounds displaying a variety of pharma-
ceutical applications [2–5]. Moreover, amidoximes represent
potent pharmacophores due to the ability of their in vivo ox-
idation to amides with a subsequent release of NO
(antiaggregatory activity), or their reduction to amidines
[6–10]. The latter are important pharmacophores since they
can imitate arginine residue in biological structures [11]. The
use of amidoximes as amidine prodrugs is an alternative to
overcome unfavorable physicochemical and pharmacokinetic
properties [12, 13].
ent for prolyl amides in polar solvents (D O and DMSO). A
2
phenylalanine substituent was found to exhibit profound ef-
fect on thermodynamic parameters in prolyl peptides. The
presence of intramolecular hydrogen bonds dramatically in-
creases the amount of trans isomer in non-hydrogen-bonding
CHCl and significantly favorcis isomer in hydrogen-bonding
3
solvents such as DMSO and D O. All molecules are not cy-
totoxic therefore they can be further studied in relation to
potent biological activities.
The proline residue is known to play a key role in protein
and peptide folding [14]. The cyclic nature of the backbone
means that cis and trans isomers of a typical peptide bond are
closer in energy for peptidyl-proline bonds than for other pep-
tide bonds [15, 16]. It is thus of particular interest to investi-
gate intra- and/or intermolecular effects that might be the sig-
nificant factors contributing to a difference in the cis/trans
equilibrium [17]. Moreover, the introduction of conformation-
al restrictions in small peptides leads to stabilizing biological-
ly active conformation thus favoring pharmacological proper-
ties of peptide-based drugs and decreasing toxicity [18–20].
In a preliminary work, we have demonstrated the synthesis
of pyridine and pyrazine-based pseudodipeptides bearing
amidoxime function as a replacement of amidine one [21,
2
.
.
Keywords Amidoxime FT-IR spectroscopy NMR
.
.
spectroscopy Molecular dynamics Cytocompatibility
Electronic supplementary material The online version of this article
(doi:10.1007/s11224-016-0870-2) contains supplementary material,
which is available to authorized users.
*
Marie-Christine Averlant-Petit
marie.averlant@univ-lorraine.fr
1
Laboratoire de Chimie Physique Macromoléculaire, UMR7375
CNRS Université de Lorraine, 1 rue Grandville, BP 20451,
22]. In this study, we decided to extend this protocol in order
5
4001 Nancy, France
to synthesize novel pseudotripeptide with the N-terminal pyr-
azine amidoxime motif in the backbone. Proline-
phenylalanine dipeptide was chosen to discover the steric ef-
fects of the C-terminal substitution in proline on the amide
isomer equilibrium of the model prolyl surrogate. Likewise,
2
3
Chemistry Department, Taras Shevchenko National University of
Kyiv, 64/13 Volodymyrska str, Kyiv 01601, Ukraine
CITHEFOR, EA3452, Faculté de Pharmacie, Université de Lorraine,
5
rue Albert Lebrun, BP 80403, 54001 Nancy, France

Struct Chem
1
3
o-substitution in non-peptidic fragment with dipeptide can
provide additional conformational rigidity. Therefore, study-
ing a relationship between structure geometry and biological
properties is particularly important.
4.09 (s, 3H, OMe) ppm. С NMR (75 MHz, DMSO-d6,
25 °C): δ = 162.8, 147.7, 146.8, 146.6, 131.4, 115.0,
54.5 ppm.
2. A mixture of methyl 3-cyanopyrazine-2-carboxylate
(
(
0.978 g, 6.0 mmol), MeOH (50 mL), and 1 M NaOH
12 mL) were stirred at room temperature for 3 h and then
Experimental
concentrated in vacuo. The residue was diluted with water
and CH Cl , and then acidified with 1 М НСl under
2
2
Materials and measurements
cooling. The resulting precipitate was collected, washed
with H O and hexane, and dried in vacuo. Pale-yellow
2
1
Melting points were measured on a Buchi M–560 Melting
Point apparatus and are uncorrected. IR spectra were recorded
with a Bruker Tensor 27 spectrometer. All FT-IR spectra (64
solid; yield: 0.742 g (82%); mp 157–158 °C. Н NMR
(300 MHz, DMSO-d6, 25 °C): δ = 9.00 (d, J = 2.3 Hz, H,
H ), 8.96 (d, J = 2.3 Hz, H, H ) ppm. С NMR
1
1
13
Ar
Ar
−
1
scans) were obtained at 2 cm resolution using a 50 μm CaF2
solution cell and a dry air-purged Bruker Tensor 27 equipped
with liquid nitrogen cooled MCT-detector. All samples were
dissolved at the concentration of 10 mM in spectrophotomet-
ric grade chloroform (≥99.8%, Sigma-Aldrich). NMR spectra
(75 MHz, DMSO-d6, 25 °C): δ = 164.5, 155.1, 146.6,
144.6, 128.1, 116.7 ppm.
The scheme of synthesis of latter acid is depicted in
Scheme 1a.
1
were measured with a Bruker Avance 300 (300 MHz for H
1
3
and 75 MHz for C) with tetramethylsilane as standard. In the
cases where a spectrum reflects the presence of two species
arising from trans and cis rotamers about the X-Pro amide
bond, the following conventions have been adopted for
Synthesis of Boc-Pro-Phe-OMe (2a)
Compound is commercially available, but was synthesized by
the method reported previously [24]. It was prepared by cou-
pling of Boc-Pro-OH hydrochloride (1.08 g, 5 mmol) and H-
Phe-OMe (1.08 g, 5 mmol) using EDC (0.97 g, 5.05 mmol),
HOBt (0.77 g, 5 mmol), and triethylamine (1.4 mL, 10 mmol)
in CH Cl (30 mL). The mixture was stirred at room temper-
1
reporting: for H spectra, chemical shift (multiplicity, protons,
trans/cis
13
assignment/s
); for C spectra, signals are clearly part of
a pair they are listed, X.X (X.X), where the first signal refers to
the trans species and the second, in parentheses, to the cis. Dry
THF was obtained by distillation over sodium and benzophe-
none. Preparative column chromatography was carried out
with silica gel 60 (40–63 μm). Electron spray ionization mass
spectra (ESI-MS) were recorded on a Brucker MicroTof-Q
HR spectrometer, Faculté des Sciences et Techniques,
Vandoeuvre-lès-Nancy, France. The equilibrium constants
2
2
ature overnight and then washed with a solution of 0.1 M HCl
(3 × 15 mL), brine (20 mL), dried with MgSO , and then the
4
solvent was removed in vacuo. The product was purified by
flash chromatography, eluting with AcOEt/Petroleum ether, 2/
1
1; yield: 92% (1.73 g). H NMR (300 MHz, CDCl , 25 °C):
3
1
δ = 7.32–7.07 (m, 5H, H ), 6.46 (s, H, NH), 4.97–4.77 (m,
J = 1.4 Hz, H, CH ), 4.37–4.10 (s, H, CH Pro), 3.72 (s, 3H,
Phe
OCH ), 3.47–3.21 (m, 2H, CH
Ph
1
1
α
(
K ) for the trans/cis rotamer ratios were determined by inte-
gration of all well-resolved peaks in the 1D H NMR spectra,
and averaging of these results.
t/c
1
δ
2 Pro
), 3.10 (ddd, J = 20.8, 13.9,
3
β,γ
6.3 Hz, 2H, CH2Phe), 2.33–1.67 (m, 4H, CH CH Pro), 1.43
2
2
1
3 3 3
3
(
s, 9H, (CH ) ) ppm. C NMR (75 MHz, CDCl , 25 °C):
Synthesis of 3-cyanopyrazine-2-carboxylic acid (1)
δ = 172.4, 172.7, 136.6, 129.9, 129.2, 127.7, 81.4, 61.8
60.5), 53.3 (53.9), 52.9, 47.6, 38.8, 30.3 (31.2), 28.9, 25.1
(
Compound is commercially available, but was prepared in a
similar manner as described in [22, 23]:
(24.1) ppm.
Synthesis of H-pro-Phe-OMe TFA salt (2b)
1
. To a stirred suspension of pyrazine-2,3-dicarboxylic acid
monoamide (10.s86 g, 65 mmol) in CH Cl (100 mL) at 0
Compound is commercially available, but was synthesized from
Boc-Pro-Phe 2a (0.752 g, 2 mmol) by treatment with 30%
trifluoroacetic acid–dichloromethane (10 mL) during 1 h [25].
The solvent was then evaporated in high vacuum. A beige solid
2
2
°С were added TEA (19.00 mL, 136.5 mmol) and methyl
chloroformate (11.05 mL, 143 mmol), and the mixture
was allowed to reach room temperature and stirred for
1
2
h. Water and NaHCO (sat.) ~1:1 were added, and the
2b (0.772 g, 99%) was obtained. H NMR (300 MHz, MeOD,
3
mixture was extracted with CH Cl . The organic layer
25 °C): δ = 7.32–7.18 (m, 5H, H ), 4.67 (t, J = 6.8 Hz, 1H,
2
2
Ph
1
was washed with H O, dried over MgSO , and concen-
CH), 4.52 (dd, J = 8.5, 3.9 Hz, H, CH), 3.82–3.72 (m, 2H,
2
4
cis/trans
trated to give the methyl 3-cyanopyrazine-2-carboxylate.
CH ), 3.70, 3.66 (2 × s, 3H, OCH
), 3.24–2.91 (m, 2H,
2
3
1
13
Yellow solid; yield: 9.22 g (87%); mp 70–72 °C. Н NMR
CH ), 2.32–1.67 (m, 4H, CH CH ) ppm. C NMR (75 MHz,
2 2 2
(
300 MHz, DMSO-d , 25 °C): δ = 8.89 (s, 2H, 2 × H ),
MeOD, 25 °C): δ = 173.0 (173.3), 138.0 (138.3), 130.5 (130.2),
6
Ar

Struct Chem
−
1 1
1
29.6, 128.0, 119.8 (116.0), 62.7 (61.8), 55.5 (55.2), 52.7
1679, 1670, 1656, 1653 (C = O, C = N) cm . H NMR
1
(52.9), 38.4 (38.1), 30.2 (33.4), 26.0 (22.2) ppm.
(300 MHz, CDCl , 25 °C, K = 13.30): δ = 9.42 (s, H,
3
t/c
1
trans/cis
The scheme of synthesis of the dipeptide is depicted in
Scheme 1b.
OH), 8.55 and 8.49 (d, J = 2.4 Hz, H, H
8.41 (d, J = 2.4 Hz, H, H
), 8.52 and
Ar
1
trans/cis
1
), 7.78 (d, J = 9.2 Hz, H,
Ph), 5.68 and 5.42 (2 × s,
, NH ), 5.10 and 4.69 (td, m, J = 9.4, 6.5 Hz,
2
Ar
trans/cis
NH), 7.27–6.97 (m, 5H, 5 × H
trans
cis
Synthesis of methyl (2S)-2-[({(2S)-1-[(3-cyanopyrazin-2-yl)
carbonyl]pyrrolidin-2-yl}carbonyl)amino]
2H, NH
H, CH
2
trans
1
cis
1
trans
cis
CH Phe), 4.77 and 4.43 (m, H, CH
CH
,
,
trans
3
trans/cis
cis
-
3-phenylpropanoate (3)
Pro), 3.69 and 3.52 (s, 3H, OCH
, OCH3 ), 3.21–2.84
trans/cis
(
m, 4H, CH2
Phe CH -δ
Pro), 2.06–1.87 (m,
Pro), 1.61–1.52 and 1.11–0.96 (2 × m, 2H,
Pro) ppm. C NMR (75 MHz, CDCl , 25 °C):
3
,
2
trans/cis
To a solution of dipeptide 2b (0.390 g, 1 mmol), acid 1
2H, CH -β
2
trans/cis
13
(
(
0.149 g, 1 mmol), and HOBt (0.168 g, 1.1 mmol) in THF
5 mL) at 0 °C, HATU (0.380 g, 1 mmol) and DIEA (0.4 mL,
CH -γ
2
δ = 175.8, 171.9 (171.6), 167.1 (167.7), 150.3 (150.0), 148.4,
144.2, 143.7, 141.6, 136.6 (137.4), 129.8, 129.2, 127.7, 60.7
(63.0), 53.6 (52.9), 48.2 (47.1), 39.1 (37.9), 30.1 (31.1), 24.0
(22.8) ppm. HRMS (ESI): m/z = calcd. 479.1448 (for
2
.1 mmol) were added. After 15 min at 0 °C and 10 h at room
temperature, the solvent was evaporated and the crude product
was purified by flash column chromatography (AcOEt/
CH Cl , 7:3) to give 3 as a light-yellow oil (0.830 g, 68%).
+
C H KN O [M + K] ), found 479.1440.
2
2
21 24
6 5
1
H NMR (300 MHz, CDCl , 25 °C, K = 1.73): δ = 8.77 (s,
3
t/c
1
trans
cis
4
H, H
), 8.62 (d, J = 2.3 Hz, H, H Ar), 8.35 (d,
X-ray diffraction study of compound AOPzPhe (methyl
(2S)-2-({[3-(N′-hydroxycarbamimidoyl)pyrazin-2-yl]
carbonyl}amino)-3-phenylpropanoate)
Ar
1
cis
trans/cis
J = 2.2 Hz, H, H Ar), 7.31–7.05 (m, 18H, H
Ph), 6.58
1
(
d, J = 7.8 Hz, H, NH), 4.87–4.74 and 4.68 (m, ddd, J = 13.8,
trans/cis
trans/cis
7
.4, 4.8 Hz, 6H, CH
Phe, CH-α
Pro), 3.71, 3.69
trans
cis
trans/cis
and 3.85–3.51 (s, s, m, 16H, OCH3 , OCH3 , CH -δ
Pro), 3.23–3.05 and 2.96–2.88 (2 × m, 6H, CH2 , CH2
Phe), 2.32–1.78 and 1.67–1.50 (2 × m, 16H,
The structure was solved by direct method using OLEX2 soft-
ware. Visualization and analysis of crystal structure were done
in Mercury 3.8 [26]. Positions of the hydrogen atoms were
located from electron density difference maps and refined by
Briding^ model with Uiso = nUeq of the carrier atom (n = 1.5
for methyl and OH groups, n = 1.2 for other hydrogen atoms).
Full-matrix least-squares refinement of the structure against
F2 in anisotropic approximation for non-hydrogen atoms
was converged to wR2 = 0.104 (R1 = 0.0390 for 3341 reflec-
tions with I > 2σ(I), S = 1.033). The final atomic coordinates
and crystallographic data have been deposited with the
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge, CB2 1EZ, UK (fax: +44 1223 336033; e-mail:
deposit@ccdc.cam.ac.uk), and are available on request quot-
ing the deposition number CCDC 1475179 for AOPzPhe.
Compound AOPzPhe (C H N O ): monoclinic crystals;
2
trans
cis
trans/cis
13
CH CH -β,γ
Pro) ppm. C NMR (75 MHz, CDCl3,
5 °C): δ = 172.3 (172.2), 170.6 (171.8), 163.4 (163.1), 152.5
151.5), 146.2 (145.9), 145.6 (144.6), 136.6, 130.4 (131.1),
2
2
2
(
1
6
30.0 (129.5), 129.0 (129.3), 127.4 (127.8), 115.2 (115.5),
1.5 (63.1), 54.2 (53.8), 52.9 (53.0), 49.9 (48.6), 38.3
(
37.9), 28.3(32.3), 25.8(22.4)ppm.HRMS(ESI): m/z=calcd.
+
4
46.1232 (for C H KN O [M + K] ), found 446.1225.
21 21 5 4
The scheme of synthesis of the nitriles is depicted in
Scheme 1c.
Synthesis of methyl (2S)-2-({[(2S)-1-({3-[(Z)-(hydroxyamino)
(
imino)methyl]pyrazin-2-yl}carbonyl)pyrrolidin-2-yl]
carbonyl}amino)-3-phenylpropanoate (4)
1
6 17 5 4
at 110 K, a = 8.6226(2) Å, b = 10.4629(2) Å, c = 9.5228(2) Å,
β = 105.842(2)°, V = 826.49(3) Å3, Mr = 343.34, Z = 2, space
group P2 , D = 1.3795 g/sm-3, μ(CuKα) = 0.854 mm-1,
F(000) = 361.274. Intensities of 15,088 reflections (3341 in-
dependent, Rint = 0.0340) were measured on a SuperNova
Agilent diffractometer equipped with a copper microsource
(Cu Kα radiation with mirror optics, Atlas CCD detector,
ω-scanning, 2θmax = 76.43°). The frames were integrated
with the Agilent CrysAlisPro software package.
The target molecule was prepared utilizing the same strategy
elaborated before for the nicotinic and pyrazinic acid-based
pseudodipeptides bearing an amidoxime residue [21, 22]. A
synthetic route to desired pseudotripeptide is shown in
Scheme 1c. Triethylamine was added (0.3 mL, 2 mmol) to a
solution of nitrile 3 (0.407 g, 1 mmol) in MeOH (15 mL)
followed by hydroxylamine hydrochloride (0.139 g, 2 mmol),
and the reaction mixture was stirred at room temperature over-
night. The mixture was then evaporated to dryness. The resi-
due was dissolved in ethyl acetate (100 mL) and washed with
1
c
Molecular dynamics
water (20 mL). The organic layer was dried with MgSO , and
4
the solvent was removed in vacuo. The residue was purified
by flash column chromatography (CH Cl /MeOH, 95:5) to
give 4 as a light-yellow solid (0.669 g, 76%); mp 59–62 °C.
IR (ATR): νmax = 3517, 3402, 3319 (NH, OH), 1743, 1722,
Molecular dynamic calculations on the molecule 4 and on the
simplified pseudodipeptide analogs AOPzPro and AOPzPhe
were run according to the following protocol: 2 μs with 2 fs
steps molecular dynamics simulations were calculated in
2
2

Struct Chem
N
N
CO H
2
a)
N
N
CO H
2
i); ii)
CN
CONH2
1
O
b)
O
iii)
Ph
+
N
HN
N
OH
-
+
Cl H N CO Me
R
CO2Me
Boc
3
2
iv) 2a R = Boc (92%)
b R = H, TFA salt (98%)
2
O
c)
O
N
N
HN
Ph
HN
Ph
N
N
N
N
vi)
O
N
v)
O
CO Me
2
1
+ 2b
CO Me
2
OH
CN
NH2
76% yield
6
8% yield
3
4
trans/cis - 98/2
trans/cis - 63/37
Scheme 1 Synthesis of pseudotripeptide 4. (i) Methyl chloroformate, Et
CF COOH, CH Cl ; (v) HATU, HOBt, DIEA, THF; (vi) NH OH HCl, Et N, MeOH
3
N, CH
2 2 3 2 2
Cl ; (ii) NaOH, MeOH; (iii) EDC, HOBt, Et N, CH Cl ; (iv)
.
3 2 2 2 3
explicit solvent (chloroform or DMSO) with constant pressure
periodic boundary conditions, pressure relaxation time of
250 μL DMSO was added under stirring for 10 min to extract
the formazan crystals. The absorbance was read at 570 nm with
a reference at 630 nm using EL 800 microplate reader (Bio-
TEK Instrument, INC®, France). Metabolic activity in the
presence of pyrazine amidoxime derivatives was compared to
the control condition (culture medium alone).
2
μs, and constant temperature at 300 K, using the package
of molecular dynamic simulation programs AMBER 10.0.
The starting molecules were constructed using
MarvinSketch (ChemAxon) Antechamber [27] and Xleap
[27] programs. Molecular simulation was done with Sander
program [27] with general AMBER force field (GAFF) and
included amino acid parameters (ff99SB) [27]. Ptraj was
employed for the analysis of 25,000 structures in order to
assess the possibility of a hydrogen bond formation.
Statistical analysis
Results are shown as mean ± standard error of the mean (sem)
values. Statistical comparisons were performed using the two-
way ANOVA with Bonferroni posttest. p < 0.05 was consid-
ered significantly different. Statistical analyses were per-
formed using the GraphPad Prism software (GraphPad
Software version 5.0, San Diego, USA).
Cytocompatibility with vascular smooth muscle cell
The cytocompatibility of pyrazine amidoxime derivatives
AOPzPro, AOPzPhe, and AOPzProPhe (6) were evaluated
on rat smooth muscle cell (SMC) line (A-10) ATCC® CRL-
1
476™. SMCs were grown in complete medium made of
Results and discussion
Conformational analysis
Dulbecco’s Modified Eagle’s Medium (DMEM) supplement-
ed with 10% (v/v) FBS, 2% (v/v) glutamine, 100 U/mL
penicillin and 100 μg/mL streptomycin mixture, and 4 mM
sodium pyruvate. Cells were cultivated at 37 °C under 10% (v/
Structural properties of the pyrazine amidoxime motif incor-
porated at N-terminus of the peptide chain were investigated
in order to determine its use in future applications.
Information concerning the conformation adopted in solution
v) of CO in a humidified incubator. SMC were seeded in 96-
2
well plates at 60,000 cells/well 48 h before exposure to pyr-
azine amidoxime derivatives for 24 h (complete medium was
used as control). Pyrazine amidoxime derivatives were firstly
1
by pseudotripeptide was obtained by combining H NMR and
−1
resuspended in 100% methanol at a concentration of 10 ,
FT-IR absorption spectroscopy analyses. Unfortunately, it has
been difficult to perform NOESYor ROESYexperiments with
enough structural informations to calculate 3D structures of
our molecules, neither good quality mono crystals could be
obtained. The mobility of the peptidic part is probably a rea-
son for these factual situations in both cases. Especially,
−2
−3
−4
10 , 10 , and 10 M. Finally, pyrazine amidoxime deriva-
tives were diluted in complete medium for final concentrations
of 10 , 10 , 10 , and 10 M. Cytocompatibility through
metabolic activity was checked with MTT assay. Briefly,
−6
−5
−4
−3
0.5 mg/mL MTT was incubated with the cells for 4 h. Then,

Struct Chem
several conformations have been observed in NMR spectra.
The strategy, repeatedly used successfully by our group and
others, is to highlight the presence of hydrogen bonds [18–22,
proton is involved in the hydrogen bond [28–32]. In the
carbonyl region of the spectrum, six bands are observed
−
1
at 1743, 1722, 1679 1670, 1656, and 1653 cm (Fig. 1,
,
2
8–32]. For that purpose, two spectroscopic methods were
Table 1).
used and combined to molecular dynamic calculations. First,
FT-IR spectroscopy in solution was applied in order to ob-
serve the NH and CO stretching vibrations at around 3500–
The number of bands indicates that at least two of the three
carbonyl groups of the molecule are involved in hydrogen
bonds. According to their wavelengths, the two bands at
−1
−1
−1
3
1
200 cm and 1600–1800 cm region, respectively. Second,
1743 and 1722 cm were assigned to the ester C = O free
1
D H–NMR spectra in CDCl /DMSO-d varying ratio high-
and involved in the hydrogen bond, respectively. The next
four bands were assigned using the absorption spectra of the
shorter peptides with one amino acid and the pyrazine
amidoxime motif previously synthesized by our laboratory
(Table 1, Fig. 2, and Figs. S1, S2 in SI) [22].
3
6
lights the labile protons not influenced by the environment
changes because they are involved in hydrogen bonds.
FT-IR experiment of 4 at 25 °C shows a large peak at
1
3
319 cm− which means that at least one NH and/or OH
Fig. 1 FT-IR spectra of
Experimental spectrum
Deconvoluate spectrum
Deconvoluate band
pseudotripeptide 4 in CHCl
3
3
(
c = 10− M)
0,40
0,35
0,30
0,25
0,20
0,15
0,10
0,05
0,00
1800 1780 1760 1740 1720 1700 1680 1660 1640 1620 1600
-1
Wavenumber cm
NH-free
NH-bonded
0
,25
,20
,15
,10
,05
,00
0
0
0
0
0
3550
3500
3450
3400
3350
3300
3250
Wavenumber, cm-1

Struct Chem
Table 1 FT-IR adsorption in the C = O stretching regions for 10 mM
concentration samples of AOPzPhe, AOPzPro, and AOPzProPhe (4) in
pure chloroform at room temperature
ν, cm−
1
ν, cm−1
ν, cm−1
Product
(C = O ester)
(C = O amide)
(C = N)
AOPzPhe
1743/1691
1743
1680
1655
1679
1659
1642
1653
AOPzPro
AOPzProPhe
1743/1722
Therefore, the amide stretching vibration at 1679 cm−1 cor-
responds to C = O of the peptide bond between proline and
phenylalanine as it is close to the value obtained for AOPzPhe.
3
Fig. 3 Effect of varying ratios of solvent in the mixture of CDCl and
DMSO-d on the chemical shifts of the NH proton of 4 (c = 3 × 10 M)
6
−3
−1
The bands at 1670 and 1656 cm were attributed to the amide
bond of the pyrazine ring and proline-phenylalanine dipeptide
in both free and bonded forms, respectively. The C = N
stretching vibration of the amidoxime group was then
The superposition of the three structures issued from
simulation in CHCl3 shows that the amidoxime-
pyrazine moiety introduces rigidity of the molecules
−1
(
Fig. 4a). The superpositions of the structures two by
assigned to the band at 1653 cm .
two show the good conservation of the structure of the
motif since the rmsd on the position of amidoxime-
pyrazine atoms are below 0.5 Å (Figs. S14–S16 in SI).
φ, ψ and, ω dihedrals were measured over the trajectories
of molecular dynamic simulations for all three molecules
AOPzPhe, AOPzPro, and AOPzProPhe 4 (Fig. S8 in SI).
The results show that all ω dihedral angle values are in favor
of the trans peptide bonds in our simulations (Table 2,
Figs. S9–S12 in SI). The mean φ and ψ angles for the proline
residue for AOPzProPhe pseudopeptide 4 are depicted on the
left-hand side of the Ramachandran plot in the favored region
Results issued from IR spectroscopy experiments were
complemented by H NMR experiments in solvent mixtures,
1
which showed that two sets of signals are present at every
solvent mixture ratio (Fig. 3). This observation means that in
solution, the structure of 4 is in equilibrium between at least
two conformations. These results are consistent with the pres-
ence of the cis/trans isomers about the X-Pro peptide bond.
Moreover, in both sets, the amide proton of phenylalanine
reveals no significant shift, which means that for both confor-
mations, the NH proton is involved in an intramolecular hy-
drogen bond. This result is consistent with the large band
below 3400 cm− in the IR spectrum, which probably is the
envelope of several bands.
1
(Figs. S8, S13 in SI), which is close to the distribution of these
average value of angles for proline residues measured in nat-
ural peptides for the α-region in Ramachandran plot (φ,
ψ = −61°, −35°) [33]. This correlation suggested that
pyrazine-proline motif induced torsion when placed before
proline residue and it could be particularly suited for
peptidomimetic construction. The dihedral angle φ in crystal-
line AOPzPhe is −108.2(2)° according to the results of the X-
Ray diffraction study.
Hence, the NH amide group of phenylalanine is involved in
the hydrogen bond with the carbonyl group of pyrazine close
−1
to it (band at 1656 cm ) and adopt a seven-membered γ-turn
conformation (Fig. 2).
Therefore, to confirm our observations, molecular model-
ing calculations in explicit solvent and without restraints have
been undertaken.
However, we noted a major point of difference in the struc-
tures: the carbonyl group near the pyrazine ring is oriented
differently in AOPzPhe pseudopeptide compared to
AOPzPro and AOPzProPhe 4. According to molecular model-
ing of AOPzPhe, the NH group is projected inward expecting
a hydrogen bond with a nitrogen atom of the amidoxime
group (Fig. S3 in SI). However, in the crystal structure of
AOPzPhe, carboxamide fragment is turned relatively the pyr-
azine ring in such way that the formation of the NH…N in-
tramolecular hydrogen bond is impossible (Fig. 4b). It is con-
firmed by the N…H distance 3.14 Å and the N–H…N angle
value 78°. In AOPzProPhe 4 and AOPzPro, the proline amino
acid imposes an additional constraint associated with the steric
hindrance due to the cyclic residue. This forces the proline
carbonyl in 4 that have been angled outwards and promotes
Molecular modeling
Molecular dynamic calculations on the molecule 4 and on the
simplified pseudodipeptide analogs AOPzPro and AOPzPhe
(
Fig. 2) were run in CHCl and DMSO.
3
O
CO Me
2
O
N
N
C7
O
N
Ph
N
N
N
N
N
N
NH
N
CO Me
2
O
N
H
CO Me
2
N
OH
OH
OH
NH2
AOPzPhe
NH2
AOPzPro
NH2
AOPzProPhe (4)
Fig. 2 Pseudotripeptide 4 and its pseudodipeptide analogs

Struct Chem
COR ROC
N
N
N
N
N
O
N
O
N
OH
N
OH
R = -OMe (AOPzPro)
-NH-Phe-OMe (4)
NH2
NH2
trans
cis
Fig. 5 Trans and cis isomers of the prolyl peptide bond for AOPzPro and
AOPzProPhe (4)
were observed: first, between carbonyl oxygen atom of the C-
terminal ester group and the hydrogen atom of the amidoxime
OH; and second, between the oxygen atom of the amide car-
bonyl next to the pyrazine ring and the hydrogen of the amide
group of phenylalanine with occurrence ratios of 98 and 42%,
respectively (Fig. 4c). Moreover, the strong hydrogen bond
observed in molecular modeling of AOPzProPhe 4 is corrob-
orated by the infrared experiments in solution. In the shorter
molecule AOPzPhe, only one hydrogen bond was observed
with an occurrence ratio of 31%, between ester C = O and OH
of the amidoxime group. The turns observed might be related
to the β N-O turn defined by Chang and co-workers [32], even
if in our case a OH group is involved instead of an amide
group. However, as it follows from crystallography data, this
hydrogen bond is not observed in all pseudopeptides, partic-
ularly, and it has not been observed in the crystal structure
(Fig. 4b and Fig. S3 in SI). No hydrogen bond has been dem-
onstrated in AOPzPro molecule in solution (Fig. S4 in SI), and
no crystal was obtained. Simulations in DMSO, as expected,
show that DMSO does not promote the establishment of hy-
drogen bonds except for AOPzProPhe 4 where the bond be-
tween the N-term OH and the C-term carbonyl group exist
with an occurrence ratio of 96% (Fig. S17 in SI).
With regard to the proline residue, two conformers were
observed in NMR spectra of the proline derivatives and we
decided to investigate further forward the cis/trans isomeriza-
tion (Fig. 5). In particular, we decided to consider the thermo-
dynamics for trans and cis isomers of the prolyl peptide bond.
Fig. 4 Solution conformations of AOPzProPhe 4 and crystal structure of
AOPzPhe. a) Superposition of 4 (blue) and its pseudodipeptide analogs
AOPzPhe (green) and AOPzPro (pink). Graphic representations were
done in the PyMOL Molecular Graphics System, Version 1.8
Schrödinger, LLC. b) Molecular structure of compound AOPzPhe
according to results of the X-Ray diffraction study. c) Hydrogen bonds
3
(orange dots) in pseudotripeptide 4 in solution (CHCl ) as obtained from
molecular dynamics simulation. As an example, φ,ψ, and ω dihedral
angles are presented in b)
the hydrogen bonding between phenylalanine amide proton
and the carbonyl next to the pyrazine ring (Figs. 4a, c). For
molecule 4, results show that two potential hydrogen bonds
Table 2 Mean dihedral angles of AOPzProPhe 4, AOPzPhe, and
AOPzPro calculated from 25,000 structures issued of the molecular
dynamics simulations
φPro
φPhe
ψ
ωPro
ωPhe
AOPzProPhe
4)
−71 ± 9
139 ± 18
|19 ± 14|
|171 ± 6|
|169 ± 7|
(
AOPzPro
AOPzPhe
|169 ± 7|
–
–
–
–
|168 ± 7|
–
–
|94 ± 57|
|156 ± 21|
6 2
Fig. 6 Van’t Hoff plots for AOPzPro in DMSO-d and 4 in D O and
DMSO-d
6

Struct Chem
Table 3 Thermodynamic
parameters for compounds
AOPzPro [22] and AOPzProPhe
0
0
0
‡
K
t/c at
ΔH
ΔS
ΔG 300K
ΔG
−
1
−1 −1
−1
−1
300 K
(kJ mol )
(J mol K )
(kJ mol )
(kJ mol )
(
4). Additionally, the trans/cis
AOPzPro–DMSO-d
4–DMSO-d6
6
2.73
1.12
1.37
−3.43
1.82
−2.97
6.96
−2.53
−0.27
−0.58
–
equilibrium constant at 300 K is
given
–
2
4–D O
3.75
14.43
75.3
Cis/trans isomerization study
Although both molecules demonstrate a temperature de-
pendence, Pro-based dipeptide has a slight positive slope
Hydrogen bonds in CHCl solution of 4 augment extremely
while the tripeptide 4 shows negative slope in both D O and
3
2
the trans rotamer population up to 98%. Therefore, variable
temperature H NMR studies of the cis/trans conformational
DMSO-d . In the case of AOPzPro, the difference in entropy
favors the cis isomer, and the trans isomer therefore is favored
by enthalpy.
6
1
equilibrium were performed on the synthesized tripeptide 4.
The effects of temperature (in the range 25–90 °C) on the
values of Kt/c are illustrated by Van’t Hoff plots in Fig. 6.
The corresponding results were compared to those obtained
for the simple Pro-containing amidoxime AOPzPro [22].
Interestingly, an increased amount of the cis conformation
of 4 in polar solvents was observed. The trans/cis ratio of
about 52:48, according to the relative intensities of their
As we can see, the Kt/c values of 4 are dependent on
temperature such that the trans isomer becomes favored
as the temperature increase, in other words, it is favored
by enthropy. The cis rotamer is favored by enthalpy
showing opposite behavior to the smaller proline deriva-
tive AOPzPro. Hence, C-terminal proline substitution
with the phenylalanine amino acid has significant influ-
ence on the cis/trans equilibrium and thermodynamics
of 4.
1
well-resolved peaks in the H NMR spectra, does show im-
portant difference to the 73:27 ratio for AOPzPro in DMSO
solution. The increase of the cis isomer of 4 and the difference
in Kt/c between AOPzPro and AOPzProPhe (4) may be due in
part to interaction of the NH group with either the imide ni-
trogen lone pair or the imide carbonyl oxygen [15, 34].
The thermodynamic parameters of the cis/trans isomeriza-
tion for prolyl amide bond in two molecules were calculated
from linear least-squares fits of the data in these plots to Eq. 1
‡
The free energy of activation (ΔG ) was determined to be
75.3 kJ/mol at the coalescence temperature 70 °C in D O
2
(Table 3, Fig. S5 in SI). The rate of interconversion at
−1
T = 70 °C was found to be 24.4 s and the interconversion
time 41 × 10 s.
c
−3
Viability assays
(
Table 3).
ꢀ
. ꢁꢀ . ꢁ
.
The cellular toxicity of compounds was tested in order to
verify a possibility of their use as therapeutics (Fig. 7).
Cells bearing a mitochondrial activity between 100 to 80%
are considered as viable (Fig. 7). AOPzPro and AOPzProPhe
.
○
○
lnK
¼ ‐ΔH
R
1 T þΔS
R
ð1Þ
t
c
(
4) were non-toxic whatever the concentration was. However,
−4
−3
AOPzPhe was toxic from 10 to 10 M, which promotes the
use of proline containing pyrazine amidoxime scaffold in
therapeutics.
Conclusions
A new ProPhe pyrazine-based pseudotripeptide was investi-
gated with respect to conformation and rigidity. NMR and FT-
IR absorption techniques were used to describe the conforma-
tional preferences of the molecule. Based on the experimental
data and molecular dynamics calculations, we have elucidated
the major solution structure of pseudotripeptide in comparison
with the solution structure of two simplified pseudodipeptide
analogs. The amidoxime residue in AOPzProPhe reduces the
mobility and promotes the hydrogen bond formation between
the proton of the OH and the carbonyl oxygen of the C-
Fig. 7 In vitro cytocompatibility of the three different pyrazine
amidoxime derivatives (AOPzPhe, AOPzPro, and AOPzProPhe (4)) on
smooth muscle cell line (A-10) compared to control cells (culture
medium). A-10 cells were treated with the indicated concentrations of
pyrazine amidoxime derivatives for 24 h at 37 °C. Viability was estimated
by the MTT assay. Data are expressed as mean ± sem (n = 3). *p 0.05
versus control two-way ANOVA, Bonferroni posttest

Struct Chem
1
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hydrogen bond is present in DMSO as well. Likewise, the
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up to 98% was observed in weakly polar solvent, which is
4
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