Concomitant polymorphism in the pseudo-peptide Me2N-pC 6H4C(O)-Phe-OEt

Article abstract of DOI:10.1016/j.molstruc.2012.07.026

Pseudo-peptide Me₂N-pC₆H₄C(O)-Phe-OEt (1) exhibits two polymorphic forms which crystallizes in non-centrosymmetric space groups, monoclinic 1m (P2₁) and orthorhombic 1o (P2₁2 ₁2₁). Both forms occur concomitantly or as a pure phase depending on the solvent of crystallization. Single crystal X-ray diffraction revealed equivalent two-dimensional layers in both 1m and 1o, the different modes of layer stacking are caused by CH?π interactions specific for each polymorph. Both solid forms are transparent in visible spectral region, with different absorbance patterns in lower UV region. Thermodynamic relations between two polymorphs were unambiguously established using room-temperature competitive slurry experiments, mechanochemistry and heating.

Full text of DOI:10.1016/j.molstruc.2012.07.026

Journal of Molecular Structure 1031 (2013) 160–167
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Concomitant polymorphism in the pseudo-peptide Me2N-pC6H4C(O)-Phe-OEt
a
b
b
a
b,
⇑
ˇ
´
´
´
´
Krunoslav Uzarevic , Zoran Kokan , Berislav Peric , Nikola Bregovic , Srecko I. Kirin
a Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia
b
-
ˇ
Ruder Boškovi´c Institute, Bijenicka cesta 54, HR-10000 Zagreb, Croatia
h i g h l i g h t s
"
"
"
"
"
Monoclinic (1m) and orthorhombic (1o) polymorphs of pseudo-peptide 1 were studied.
1m and 1o reveal equivalent 2D structures with a different mode of layer stacking.
Thermodynamic relations between two polymorphs were unambiguously established.
Both polymorphs were prepared in a pure form from solution or by solvent-free methods.
Kinetically stable 1m transfers to thermodynamically stable 1o by grinding or heating.
a r t i c l e i n f o
a b s t r a c t
Article history:
Pseudo-peptide Me2N-pC6H4C(O)-Phe-OEt (1) exhibits two polymorphic forms which crystallizes in non-
centrosymmetric space groups, monoclinic 1m (P21) and orthorhombic 1o (P212121). Both forms occur
concomitantly or as a pure phase depending on the solvent of crystallization. Single crystal X-ray diffrac-
tion revealed equivalent two-dimensional layers in both 1m and 1o, the different modes of layer stacking
Received 23 March 2012
Received in revised form 14 July 2012
Accepted 16 July 2012
Available online 25 July 2012
are caused by CAHꢀ ꢀ ꢀ
p interactions specific for each polymorph. Both solid forms are transparent in vis-
ible spectral region, with different absorbance patterns in lower UV region. Thermodynamic relations
between two polymorphs were unambiguously established using room-temperature competitive slurry
experiments, mechanochemistry and heating.
Keywords:
Pseudo-peptide
Phenylalanine
Ó 2012 Elsevier B.V. All rights reserved.
Polymorphism
Crystal structure
Solid-state transformations
Mechanochemistry
1. Introduction
Compounds that crystallize in non-centrosymmetric groups are
widely studied due to their non-linear optical properties and po-
Polymorphism, well known solid-state phenomenon wherein
one chemical substance forms two or more crystalline phases
[1,2], remains one of the most important topics in modern solid-
state chemistry [3,4]. It is mainly manifested through different
conformations in flexible molecules and/or different packing
arrangements in the crystal structure which molecules assume to
achieve free energy minimum. Interest for this phenomenon arises
from the fact that polymorphs can display different physicochem-
ical properties, such as crystal habitus, thermal stability, solubility,
bioavailability, hygroscopicity, magnetism, color, and electric con-
ductivity. For this reason, polymorphism has developed as a solid
state property of paramount importance in materials chemistry
[5,6] and pharmaceutical industry, where patenting and material
specifications rely on above mentioned properties [7,8].
tential applications in laser devices and signal transduction
[9,10]. The main prerequisites for applications, such as thermal sta-
bility and optical properties, are directly dependable on the orien-
tation of the molecules in the crystal lattice [11]. In some cases,
crystallization experiment yields more than one polymorph in
the same batch, which is known as concomitant polymorphism
[12]. Although such systems result in a batch sample of incoherent
physical properties and make control of crystallization process
even more demanding, concomitant polymorphs provide valuable
data on the thermodynamic and kinetic aspects of the explored
system which are inaccessible if only one phase crystallizes.
Recently, we used chiral pseudo-peptides Ph2P-pC6H4C(O)-Aan-
OR, Aa = amino acid, as ligands in Rh(I) catalyzed asymmetric
hydrogenation [13]. As an extension of that work, we are interested
in the synthesis and structural features of nitrogen analoges Me2N-
pC6H4C(O)-Aa-OR. In this paper we present solid-state and solution
study of two concomitant polymorphs of Me2N-pC6H4C(O)-Phe-OEt
(1), that crystallize in non-centrosymmetric space groups,
⇑
Corresponding author. Tel.: +385 (0) 1 4561189; fax: +385 (0) 1 4680098.
E-mail address: Srecko.Kirin@irb.hr (S.I. Kirin).
0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2012.07.026

ˇ
´
161
K. Uzarevic et al. / Journal of Molecular Structure 1031 (2013) 160–167
monoclinic P21, 1m, and orthorhombic P212121, 1o. The analysis of
common and distinct intermolecular interactions was based on X-
ray single crystal structures. Solution properties were investigated
by NMR and UV–Vis spectrometry, while solid-state UV–Vis mea-
surements were employed to determine the absorbance cut-off of
crystalline phases. Competitive slurry experiments [14,15] were
combined with solvent-free methods (grinding and heating) [16]
to precisely establish relative thermodynamic relations between
two polymorphs.
Retsch MM200 ball mill (25 Hz) for 30 min. After such treatment
PXRD patterns were collected. Differential scanning calorimetry
(DSC) was performed on the Mettler–Toledo DSC823e calorimeter
with STARe SW 9.01 in the range from 25 to maximally 250 °C
(5 °C minꢁ1) under the nitrogen stream. Thermogravimetric analy-
sis (TGA) was performed on a Mettler–Toledo TGA/SDTA851e ther-
mobalance using alumina crucibles under nitrogen stream with the
heating rate of 5 °C minꢁ1. In all experiments the temperature ran-
ged from 25 to 250 °C. The results were processed with the Mettler
STARe 9.01 software.
2.2. Synthesis
2. Experimental section
4-Dimethylaminobenzoic acid (412 mg, 2.5 mmol), TBTU
(800 mg, 2.5 mmol) and HOBt (339 mg, 2.5 mmol) were dissolved
in acetonitrile (100 ml, p.a.) in a 250 mL round bottomed flask at
room temperature. DIPEA (1.7 mL, 10 mmol) was added and the
2.1. General remarks
Synthesis was carried out in ordinary glassware; chemicals
were used without further purification. Nuclear magnetic reso-
nance spectra (NMR) were collected with
clear greenish solution was continued stirring for 1 h. Then L-phen-
a Bruker Avance
ylalanine ethyl ester (574 mg, 2.5 mmol) was added and the stir-
ring was continued overnight. Acetonitrile was evaporated in
vacuo and ethyl-acetate (100 ml) was added to the oily residue,
washed with NaHCO3 (100 mL, sat. aq.) and H2O (2 ꢂ 100 mL),
dried over anhydrous Na2SO4, filtered and evaporated to dryness
to give a pale green solid residue. The residue was purified by col-
umn chromatography (silica, 50 g, Et2O/MeOH (100:1), TLC:
Rf = 0.36) and eluted with diethyl ether (50 mL) and then with
Et2O/MeOH (100:1). Peak fractions were evaporated to dryness to
give 624 mg (77%) of white crystalline product. 1H NMR
(300 MHz, CDCl3) d/ppm: 1.26 (t, 3H, J = 7 Hz, HEt), 3.02 (s, 6H,
300 MHz spectrometer. Chemical shifts, d/ppm, indicate a down-
field shift from tetramethylsilane, TMS, the internal standard. Cou-
pling constants, J, are given in Hz. Individual peaks are marked as:
singlet (s), doublet (d), triplet (t), quartet (q) or multiplet (m). UV–
Vis measurements were carried out on Varian Cary 5 double beam
spectrophotometer equipped with a thermostat device. Infrared
spectra (IR) were recorded on a PerkinElmer Spectrum RXI FT-IR
spectrometer from dried samples dispersed in KBr pellets (4000–
400 cmꢁ1 range, step 2 cmꢁ1). Positive ion electrospray mass spec-
tra (ES + MS) were measured on an Agilent 6410 Triple Quadrupole
Mass Spectrometer. Powder X-ray diffraction (PXRD) experiments
HNMe), 3.18–3.30 (m, 2H, Hb), 4.19 (q, 2H, J = 7 Hz, HEt), 5.06 (dt,
were performed on a Philips PW 3710 diffractometer, Cu Ka radi-
1H, J1 = 7.5 Hz, J2 = 5.5 Hz, H ), 6.44 (d, 1H, J = 7.5 Hz, HNH), 6.63–
a
ation, flat plate sample on a zero background in Bragg–Brentano
geometry, voltage 40 kV, and current 40 mA. The patterns were
collected in the angle region between 4° and 40° (2h) with a step
size of 0.02° and 1.0 s counting per step. Single-crystal X-ray dif-
fraction (SCXRD) was performed on an Oxford Diffraction Xcalibur
6.68 (m, 2H, HPh-2,6), 7.13–7.31 (m, 5H, HBz), 7.62–7.67 (m, 2H,
H
Ph-3,5); 13C NMR (300 MHz, CDCl3) d/ppm: 14.27 (CEt), 38.29
(Cb), 40.21 (CNMe), 53.51 (C ), 61.55 (CEt), 120.67 (CPh-1), 111.15
a
(CPh-3,5), 126.99 (CBz-p), 128.46, 128.55 (CBz-o,m), 129.49 (CPh-2,6
136.34 (CBz-i), 152.74 (CPh-4), 166.78 (COamide), 172.08 (COester); IR
(KBr) N–H); 1747 (sharp, C=O); 1634,
/cmꢁ1: 1m: 3371 (sharp,
1608, 1504 (all s, mixed C@O, C@C); 1o: 3370 (sharp, N–H);
1750 (sharp, C=O); 1631, 1610, 1507 (all s, mixed C@O, C@C);
)
CCD diffractometer with graphite-monochromated Mo K
a radia-
m
m
m
tion in a nitrogen vapor stream at 120 K using -scans. Mechano-
x
m
m
chemical routine: samples were neat ground in steel jars using
m
m
MS (ES+), m/z: 148.1 ([M–Phe]+, 100%), 341.2 ([M + H]+), 363.2
([M + Na]+). Single crystals suitable for X-ray diffraction were ob-
tained by slow evaporation of solvent mixtures Et2O/MeOH
(100:1) for 1m and EtOAc/hexane (4:1) for 1o.
Table 1
Crystallographic data for 1m (monoclinic) and 1o (orthorhombic) polymorphs.
1m
1o
Empirical formula
Formula weight (g molꢁ1
Crystal system
C
20H24N2O3
C20H24N2O3
340.41
Orthorhombic
0.07 ꢂ 0.1 ꢂ 0.3
Prism
)
340.41
2.3. Crystallization experiments
Monoclinic
0.03 ꢂ 0.03 ꢂ 0.5
Needle
Crystal size (mm3)
Crystal habitus
For each crystallization experiment, 5 mg of the sample was
dissolved in 1 mL of an assorted solvent in a test tube and the solu-
tions were left for slow evaporation at room temperature. Solvents
(methanol, ethanol, n-propanol, acetone, acetonitrile, chloroform,
dichloromethane and diethyl ether) were selected by their protic
and dielectric properties. Pseudo-peptide 1 proved insoluble in
diethyl ether. In alcohols, 1 would crystallize after 20 h standing
and the samples were filtrated and dried at air. From other sol-
vents, no crystallization occurred until the solvent completely
evaporated. In the second set of experiments, 3 mL of diethyl ether
was added to the respective solutions, which was followed by al-
most immediate precipitation of the product. PXRD spectra were
collected for all obtained samples.
Crystal color
Space group
Unit cell dimensions (Å, °)
a
b
c
a
b
Colorless
P21
Colorless
P212121
11.2142(6)
5.4222(3)
14.597(1)
90.00
94.440(5)
90.00
884.92(9)
2
1.278
5.3521(2)
14.5692(4)
22.9904(7)
90.00
90.00
90.00
1792.70(10)
4
1.261
0.085
728
24242/4162
4162/0/233
1.068
0.0332/0.0816
0.172/ꢁ0.140
c
Volume (Å3)
Z
Dcalc (g cmꢁ3
)
l
(mmꢁ1
F(000)
)
0.086
364
Reflections collected/unique
Data/restraints/parameters
Goodness-of-fit on F2, Sa
8089/4802
4802/1/233
0.922
0.0652/0.1029
0.257/ꢁ0.240
2.4. X-ray single-crystal diffraction
R/wR [I > 2
r
(I)]b
Largest diff. peak/hole (eÅꢁ3
)
Details of data collection and crystal structure refinement are
given in Table 1. Program CrysAlisPro [17] was used for data collec-
tion, cell refinement, and data reduction. Sample 1m was twinned,
consisting of two components where one was more dominant. The
2
1=2
½wðF2o ꢁ F2c Þ =ðNobs ꢁ NparamÞꢃ
.
a
S ¼
R ¼
R
R
2
2
2
wðF2o ꢁ F2c Þ =
R
ðFoÞ ꢃ1=2; w ¼ 1=½r2ðFo2Þ þ ðg1PÞ þ
b
kFoj ꢁ jFck=
R
jFoj; wR ¼ ½
R
g2Pꢃ where P ¼ ðF2o þ 2Fc2Þ=3.

ˇ
´
162
K. Uzarevic et al. / Journal of Molecular Structure 1031 (2013) 160–167
O
O
O
O
O
O
OH
H2N
NH
+
N
N
Scheme 1. Reaction conditions: (a) TBTU/HOBt/DIPEA, CH3CN, R.T., 16 h, 77%.
Fig. 1. Room temperature PXRD spectra for 1m and 1o forms.
observed intensities (‘‘hkl file’’) for this sample were produced by
extraction of reflections only from the more dominant component.
The structures were solved by direct methods using SIR97 program
[18]. The full-matrix least-squares refinements based on F2 against
all reflections were performed using SHELXL97 program [19]. The
refinements included anisotropic displacement parameters for all
non-H atoms and isotropic displacement parameters for all hydro-
gen atoms. The hydrogen atoms, except H(9) (involved in the
hydrogen bond), were positioned geometrically and refined in
the riding model [CAH = 0.93–0.98 Å; Uiso(H) = 1.2 or 1.5 ꢂ Ueq(C)].
Torsion angles of methyl groups around the bonds they form with
other non-H atoms were refined. Position of H(9) atom in both
polymorphs was determined from the difference Fourier maps, it
was included in the refinements unconstrained and with unre-
strained isotropic thermal parameter. Calculations were performed
using programs within WinGX program package [20]. Geometry
calculations were done by PLATON program [21] and the molecular
graphics were done by PLATON, ORTEPIII [22] and Mercury [23]
programs.
2.5. UV–Vis spectrophotometric measurements
The solution spectra of 1 (c ꢄ 3.5 ꢂ 10ꢁ5 mol dmꢁ3) were re-
corded in methanol and chloroform (600–240 nm), using quartz
cells with 1 cm optical path. In order to obtain solid-state spectra
of both polymorphs, pellets with ꢄ0.1 mg of sample (1m or 1o)
dispersed in KBr (m ꢄ 50 mg) were recorded against pellets of pure
KBr used as reference sample. To investigate the influence of high
temperature on the solid state UV–Vis spectrum of 1m, the sample
pellet was heated to 125 °C (3 °C minꢁ1) in the laboratory oven,
held at that temperature for 45 min, and the spectrum was re-
corded after cooling.
2.6. Competitive slurry experiments
15 mg of the sample mixture containing 1m (7 mg) and 1o
(8 mg) was added to n-propanol/diethyl ether solvent system
(2 mL, 1:1). Resulting slurry was stirred for 5 h at the room temper-
ature. Solid sample was immediately filtrated and analyzed by
PXRD.
3. Results and discussion
Fig. 2. (a) ORTEPIII [22] view of molecule of the title compound in 1o form.
Ellipsoids are drawn at the 50% probability level. Hydrogen atoms are presented as
spheres of arbitrary small radii. (b) Overlapped molecular structures of 1m
(colored), and 1o (violet).
3.1. Synthesis and identification
The title compound Me2N-pC6H4C(O)-Phe-OEt (1) was synthe-
sized by standard peptide coupling in solution using the TBTU/
HOBt/DIPEA protocol and obtained in good yield (77%) after purifi-
cation by column chromatography on silica, Scheme 1. Pseudo-
peptide 1 was characterized by 1H, 13C NMR and IR spectroscopy
and positive-ion electrospray mass spectrometry, ES + MS.
3.2. Crystallization experiments
Compound 1 was crystallized from different solvent mixtures in
order to prepare single crystals suitable for crystallographic

ˇ
´
163
K. Uzarevic et al. / Journal of Molecular Structure 1031 (2013) 160–167
analysis. Two polymorphs of 1 were obtained depending on the
solvent of crystallization. Slow evaporation of an Et2O/MeOH
(100:1) solution of 1 at room temperature yielded single crystals
of monoclinic polymorph 1m (colorless needles), while colorless
prism of orthorhombic polymorph 1o were obtained from EtOAc/
hexane (4:1) solution. Experimental PXRD spectra, Fig. 1, compared
to those calculated from single-crystals and refined in TOPAS [24],
showed that bulk samples from both solutions were mixtures of
1m (dominant) and 1o phases (see Supplementary Material).
Screening crystallization experiments revealed the following: con-
comitant mixture of 1m and 1o crystals (dominant) was obtained
from alcohols (methanol, ethanol, n-propanol), while the crystalli-
zation from dichloromethane yielded concomitant mixture of 1m
(dominant) and 1o. In all cases, addition of diethyl ether as preci-
pitant [25] to a solution resulted in pure 1m microcrystalline prod-
uct. Pure 1m was also obtained from acetone or chloroform, while
crystallization from acetonitrile yielded pure 1o phase.
(iii) Torsion angle C(9)AN(9)AC(10)AC(18) (
u
angle in peptides)
[26], having values of ꢁ83.7(3)° and ꢁ79.89(13)° in 1m or
1o forms, respectively, is not representative for any second-
ary peptide structure (e.g. for
N(9)AC(10)AC(11)AC(12) (
a
-helix or b-sheet). The angle
v
angle in peptides) [26] with
value of ꢁ65.5(2)° and ꢁ69.75(12)° in 1m and 1o forms,
respectively, indicate gaucheꢁ conformation of the phenylal-
anine side chain.
(iv) The torsion angle N(9)AC(10)AC(18)AO(18) exhibit a cis
conformation of the ester’s C(18)AO(18) carbonyl group
with value of ꢁ16.7(3)° or ꢁ19.39(16)° in 1m or 1o form,
respectively.
Intermolecular interactions in 1m and 1o polymorph forms are
listed in Table 2. The most important intermolecular interaction is
the hydrogen bond formed between two amide groups from neigh-
boring molecules and is common for both polymorphs, Fig. 3a. In
addition, structure of both polymorphic forms is stabilized by
CAHꢀ ꢀ ꢀ
group from Ph(2) ring from one molecule (A in Fig. 3b) with the
system of the Ph(2)ii ring from the neighboring molecule (Aii in
Fig. 3b) is found. And second, the contact between the C(8)AH(8)
group of Ph(1) ring from one molecule (A in Fig. 3b) and system
of Ph(1)iii ring from another neighboring molecule (Aiii in Fig. 2b)
can be considered as a weak CAHꢀ ꢀ ꢀ interaction, Table 2.
The three common intermolecular contacts described above,
interactions, are responsible
for the aggregation of molecules in two-dimensional layers equiv-
alent for both 1m and 1o forms, Table 2 and Fig. 3b. Basic repeti-
tion motif in these layers lies in the bmcm plane of monoclinic
unit cell, or in the aobo plane of orthorhombic unit cell.
p interactions, Fig. 3b. First, a contact of the C(14)AH(14)
4. Single crystal structure
p
Single crystal structures were determined for both 1m (needles,
monoclinic, P21) and 1o (prisms, orthorhombic, P212121); the
structure of 1o together with the atom numbering scheme is de-
picted in Fig. 2a. Molecular structures of both 1m and 1o have
identical conformation, the comparison of the molecular structures
in 1m and 1o forms is presented in Fig. 2b, the differences in anal-
ogous bond lengths are less than their 4 standard deviations and
analogous bond or torsion angles differ for less than 1° or 5o,
respectively. Several common features are found in molecular
structures of both polymorph forms:
p
p
one hydrogen bond and two CAHꢀ ꢀ ꢀꢀ
p
Intermolecular interactions specific to each polymorph form are
(i) The (CH3)2N-group contains sp2 hybridized N(1) atom and is
planar. The (CH3)2N-group is twisted with respect to the
central phenyl Ph(1) ring with dihedral angle between the
respective planes equal to 12.7(3)° and 9.24(17)° in 1m
and 1o form, respectively.
(ii) Twisting of amide plane defined by atoms C(9), O(9) and
N(9) with respect to the central phenyl ring Ph(1) is identical
in both polymorphs, 25.5(3)° and 25.64(13)° for 1m or 1o
forms, respectively.
CAHꢀ ꢀ ꢀ
p interactions with an aliphatic donor CAH group. In partic-
ular, a C(19)AH(19B)ꢀ ꢀ ꢀPh(2)iv contact was found in 1m, while a
C(20)AH(20B)ꢀ ꢀ ꢀPh(2)iv interaction is present in 1o, Table 2. These
distinct intermolecular interactions are clearly visible in the Hirsh-
feld surface fingerprint plots [27,28], Fig. 4. They are responsible
for the different packing of the identical two-dimensional layers
in the crystals of 1m and 1o. In 1m the layers are simply stacked
(translated) in a parallel fashion one above the other along direc-
tion of the monoclinic unit cell axis am, Fig. 5a, while in 1o the
Table 2
Intermolecular interactions in 1m and 1o forms.
Interactions common for both polymorphs (Fig. 3):
Hydrogen bonds
Form
DAHꢀ ꢀ ꢀA
DAH (Å)
Hꢀ ꢀ ꢀA (Å)
2.44(3)
Dꢀ ꢀ ꢀA (Å)
3.265(3)
DAHꢀ ꢀ ꢀA (o)
166(2)
1m
1o
N(9)AH(9)ꢀ ꢀ ꢀO(9)i,a
0.84(3)
0.852(16)
N(9)AH(9)ꢀ ꢀ ꢀO(9)i,b
2.380(16)
3.2072(14)
163.9(13)
CAHꢀ ꢀ ꢀ
Form
p interactions
CAHꢀ ꢀ ꢀ
p
CAH (Å)
Hꢀ ꢀ ꢀp
(Å)
Cꢀ ꢀ ꢀp
(Å)
CAHꢀ ꢀ ꢀp
(o)
1m
1o
1m
1o
C(14)AH(14)ꢀ ꢀ ꢀPh(2)ii,a
C(14)AH(14)ꢀ ꢀ ꢀPh(2)ii,b
C(8)AH(8)ꢀ ꢀ ꢀPh(1)iii,a
C(8)AH(8)ꢀ ꢀ ꢀPh(1)iii,b
0.93
0.93
0.93
0.93
2.90
2.94
3.06
3.10
3.636(2)
3.6923(15)
3.715(3)
137
138
128
125
3.7157(15)
Interactions specific for each polymorph (Fig. 5):
CAHꢀ ꢀ ꢀ
Form
p interactions
CAHꢀ ꢀ ꢀ
p
CAH (Å)
Hꢀ ꢀ ꢀ
2.54
2.87
p
(Å)
Cꢀ ꢀ ꢀ
3.425(3)
p
(Å)
CAHꢀ ꢀ ꢀp
(o)
151
137
1m
1o
C(19)AH(19B)ꢀ ꢀ ꢀPh(2)iv,a
0.97
0.96
C(20)AH(20B)ꢀ ꢀ ꢀPh(2)iv,b
3.6290(18)
a
Symmetry codes: (i) x, ꢁ1 + y, z; (ii) ꢁx, ꢁ1/2 + y, 1 ꢁ z; (iii) ꢁx, 1/2 + y, ꢁz; and (iv) 1 ꢁ x, 1/2 + y, 1 ꢁ z.
Symmetry codes: (i) ꢁ1 + x, y, z; (ii) ꢁ1/2 + x, 3/2 ꢁ y, ꢁz; (iii) 1/2 + x, 1/2 ꢁ y, ꢁz; and (iv) 1 ꢁ x, ꢁ1/2 + y, 1/2 ꢁ z.
b

ˇ
´
K. Uzarevic et al. / Journal of Molecular Structure 1031 (2013) 160–167
164
Fig. 4. Hirshfeld surfaces fingerprint plots for polymorph forms 1m and 1o. While
the NAHꢀ ꢀ ꢀO interactions are similar in both forms (presented by sharp spikes), the
intermolecular contacts are more pronounced in the structure of 1m (side-
CAHꢀ ꢀ ꢀ
p
wings). (For interpretation to colours in this figure, the reader is referred to the web
version of this paper.)
Exploring only organic compounds from Cambridge Structural
database (CSD) [29], a number of biologically interesting com-
pounds including carcinogenic toxin Aflatoxin [30], antidiabetic
drug Chlorpropamide [31,32], and naturally occurring plant qui-
nine b-lapachone [33] revealed a layer polymorphism with fea-
tures similar to 1m/1o: (a) the two polymorphs differ only in the
relative stacking of identical two-dimensional layers, (b) the two
polymorphs crystallize in monoclinic P21 and orthorhombic
P212121 groups, respectively (although in some literature cases
additional polymorphs are known as well), and (c) in both poly-
morphs two axis are of equal length, while the third axis is twice
as long in the orthorhombic polymorph.
5. Spectrophotometric investigations
Fig. 3. Common intermolecular interactions responsible for formation of two-
dimensional isostructural layers in both polymorphs: (a) Hydrogen bond
N(9)AH(9)ꢀ ꢀ ꢀO(9)i, (b) CAHꢀ ꢀ ꢀ
p
interactions [C(14)AH(14)ꢀ ꢀ ꢀPh(2)ii and
Since the crystallization product varied depending on the used
solvent, UV–Vis spectra of 1 in protic and aprotic solvents of differ-
ent polarities were recorded to gain more insight into the solvent
effect, Fig. 6a. Slight increase in absorbance of the band at
306 nm was observed for methanol solution. The corresponding
band can be assigned to the excitation of the benzene rings. The
similarity of the obtained UV–Vis spectra indicates that there is
no significant impact of the solvent proticity on the specific sol-
vent-pseudo-peptide 1 interactions [34]. No direct correlation
C(8)AH(8)ꢀ ꢀ ꢀPh(1)iii]. Symmetry codes are given in Table 2. Unit cells for
orthorhombic and monoclinic forms are shown by full and dotted black lines,
respectively. Interactions are denoted by gray dashed lines. View on (b) is along bm
axis of 1m form or along ao axis of 1o form.
layers are stacked in an antiparallel fashion (rotated for 180°) one
above the other around co, the axis perpendicular to the layers,
Fig. 5b.

ˇ
´
165
K. Uzarevic et al. / Journal of Molecular Structure 1031 (2013) 160–167
between the solution behavior and crystallization outcomes can be
established.
Transparency of the material in visible spectral region is one of
6. Thermal and mechanochemical reactivity
The relative thermodynamic relations of two polymorph forms
1m and 1o and the possibility of their thermally or mechanochem-
ically induced solid-state interconversions were investigated by
means of differential scanning calorimetry (DSC), thermogravimet-
ric analysis (TGA) and neat grinding. DSC measurements of form
1m in nitrogen atmosphere revealed a weak endothermic peak at
110 °C followed by strong endothermic peak at 137 °C (onset),
Fig. 7. To establish whether the first thermal event corresponds
to a transformation of 1m to a new phase, 1m was heated to
120 °C and allowed to cool to room temperature. PXRD identified
this sample as pure 1o, i.e. the endotherm at 110 °C corresponds
to a phase change of 1m ? 1o. Consequently, the strong endo-
therm at 137 °C corresponds to melting of pure 1o; melting point
of 1m was thus not possible to determine. No mass loss was ob-
served during both thermal events (TGA). Thermally induced
1m ? 1o phase transition is irreversible. When 1m or 1o samples
were heated to 145 °C and the melt cooled to room temperature,
the prerequisites for their potential optical application. Solid state
UV spectra of both polymorphs showed similar characteristics;
both 1m and 1o being transparent in the visible spectral region,
from 800 to 370 nm, Fig. 6b. In the UV spectral range two absorp-
tion bands appeared. One band was positioned at 210 nm for both
forms, while the other occurred at 290 nm for 1m or at 285 nm in
the spectrum of 1o. It should also be noted that a significant corre-
lation between spectra in the solid state and solution was ob-
served, with slight hypsochromic shift of the characteristic
maximum in solution. Since the absorption cut-off wavelength of
1 is in UV region, both polymorphs would be adequate for optical
studies. Although both polymorphs are non-centrosymmetric and
thus sustainable for the second order non-linear optical (NLO)
behavior [35], different modes of layers staking could induce larger
differences in NLO characteristics [36] than in spectrophotometric
properties presented here.
Fig. 5. Distinct intermolecular interactions: (a) C(19)AH(19B)ꢀ ꢀ ꢀPh(2)iv specific for 1m form and (b) C(20)AH(20B)ꢀ ꢀ ꢀPh(2)iv specific for 1o form. Symmetry codes are given in
Table 2. Interactions are denoted as gray dashed lines on the left part of the figures. Twofold symmetry axes characteristic for these interactions are also shown. Right parts of
the figures are perspective views of crystal structures where opposite orientations of successive layers are denoted in black and white color for 1o form (b). In 1m form all
layers have the same orientation (black). Views are same as in Fig. 3b.

ˇ
´
K. Uzarevic et al. / Journal of Molecular Structure 1031 (2013) 160–167
166
sults imply that same polymorph, 1o, is thermodynamically stable
both at high temperature (DSC) and at low temperature (grinding).
However, there were some examples reported where the grinding
yielded metastable polymorph [42]. Also, one should not exclude
possibility that, during the grinding process, localized parts of sam-
ple would be heated by friction and by impact with the ball inside
the jar [16,43]. Therefore, further experiments needed to be em-
ployed to unambiguously determine thermodynamic relation in
this system.
7. Relative thermodynamic stability
In this system, we were not able to determine melting points for
both polymorphs, due to 1m ? 1o solid phase transition and con-
sequently, the ‘‘heat of fusion’’ rule to distinguish between a mono-
tropic or an enantiotropic system [3,8] could not be employed. At
first glance, the fact that 1m ? 1o phase change is endothermic
suggested that the 1m/1o system is enantiotropic with 1o as the
high-temperature polymorph, while 1m would be thermodynami-
cally stable phase at low temperatures [12,44]. However, distin-
guishing thermodynamic relation between the polymorphs is not
always straightforward. In the cases where the solid–solid phase
transition is of complicated nature, DSC traces can depend on many
factors and the identification of the relation became difficult to
determine [45]. To undoubtedly determine which polymorph is
thermodynamically stable at room temperature, competitive slur-
ry experiment in n-propanol/diethylether was performed. PXRD of
the sample collected after 5 h of stirring confirmed that, from 1m/
1o mixture, 1m transforms completely to 1o. Hence, 1o is thermo-
dynamically stable phase between room temperature and melting
point and the system is thus monotropic. Additionally, kinetic
experiments, like addition of antisolvent (diethyl ether) and crys-
tallization from the melt confirmed that 1m is kinetically favored
polymorph [25].
Fig. 6. (a) Electronic absorption spectra of 1 in chloroform (—) and methanol (– –)
at 25 °C. (b) Solid-state UV–Vis spectra of 1o (—) and 1m (– –) polymorphs in KBr
pellet (w = 2‰) at 25 °C.
8. Conclusions
Pseudo-peptide 1 crystallizes in two polymorphic forms (1m
and 1o), concomitantly or as a pure phase depending on the sol-
vent of crystallization. In both polymorphs, molecule 1 retains
the same molecular conformation. The polymorphism of 1m and
1o is characterized by different stacking of identical two-dimen-
sional layers in respective non-centrosymmetric space groups. In
the lower UV region the crystalline polymorphs 1m and 1o show
slight absorbance differences. The work presented here clearly
defined thermodynamic relation between two forms in this mono-
tropic polymorph system. 1m is kinetically stable form and it
transfers to the thermodynamically stable 1o by heating or by
application of various solvent-free methods at room temperature.
These methodologies can be used for purification of the concomi-
tant mixture and controllable synthesis of target phase. Since both
polymorphs crystallize in non-centrosymmetric groups, polar P21
and nonpolar P212121, in our future work we will aim to grow crys-
tals of suitable size for study of their non-linear optical properties.
Fig. 7. DSC trace of 1m form. The inset represents the thermal event corresponding
to the transformation of 1m to 1o. The sharp endothermic peak corresponds to
melting of 1o form.
Acknowledgements
the resulting solid was pure 1m, independent of the cooling rate
(30 or 1 °C minꢁ1) (see ESI).
These materials are based on work financed by the Unity
Through Knowledge Fund (Grant UKF-36/08), the Croatian Science
Foundation (Grant HrZZ-02.04/23) and the Ministry of Science,
Education and Sports of Croatia (Grant 098-0982904-2946). The
Mechanochemical methods have recently proved as a viable
alternative for the synthesis of new materials which were hardly
obtainable from solution [37,38], and also for the resolution of
polymorphs in various systems [39–41]. Neat grinding (25 Hz,
30 min, room temperature) in a ball mill induced irreversible
1m ? 1o transformation, while 1o was stable during grinding. Re-
ˇ
´
´
authors thank Professor Dubravka Matkovic-Calogovic (Faculty of
Science, University of Zagreb) for X-ray single crystal data collec-
tion and Dr. Ivan Halasz (Faculty of Science, University of Zagreb)
for invaluable intellectual contributions to this work.

ˇ
´
167
K. Uzarevic et al. / Journal of Molecular Structure 1031 (2013) 160–167
[18] A. Altomare, M.C. Burla, M. Camalli, G.L. Cascarano, C. Giacovazzo, A.
Guagliardi, A.G.G. Moliterni, G. Polidori, R. Spagna, J. Appl. Crystallogr. 32
(1999) 115.
[19] G.M. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr. 64 (2008) 112.
[20] L.J. Farrugia, J. Appl. Crystallogr. 32 (1999) 837.
Appendix A. Supplementary material
CCDC 870669 and 870670 contain the supplementary crystallo-
graphic data for 1m and 1o, respectively. These data can be ob-
tained free of charge via http://www.ccdc.cam.ac.uk/conts/
retrieving.html, or from the Cambridge Crystallographic Data Cen-
tre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-
033; or e-mail: deposit@ccdc.cam.ac.uk. Supplementary data asso-
ciated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.molstruc.2012.07.026.
[21] A.L. Spek, PLATON, Utrecht University, Utrecht, The Netherlands,
Multipurpose Crystallographic Tool, 2005.
A
[22] L.J. Farrugia, J. Appl. Crystallogr. 30 (1997) 565.
[23] C.F. Macrae, P.R. Edgington, P. McCabe, E. Pidcock, G.P. Shields, R. Taylor, M.
Towler, J. van de Streek, J. Appl. Crystallogr. 39 (2006) 453.
[24] Topas, version 4.1. Bruker-AXS, Karlsruhe, Germany, Single-crystal diffraction
experiments for both polymorphs were conducted at 120 K and their
calculated powder diffractograms differ from experimental diffractograms,
measured by PXRD at room temperature. To obtain comparable data, both
calculated spectra were refined by Pawley method in Topas.
[25] G.P. Stahly, Cryst. Growth. Des. 7 (2007) 1007.
References
[26] IUPAC-IUB Commission on Biochemical Nomenclature, J. Mol. Biol. 52 (1970)
1.
[1] W.C. McCrone, in: D. Fox, M.M. Labes, A. Weissberger (Eds.), Physics and
Chemistry of the Organic Solid State, vol. 2, Interscience Publishers, New York,
1965, p. 725.
[2] G.R. Desiraju, Science 278 (1997) 404.
[3] J. Bernstein, Polymorphism in Molecular Crystals, first ed., Oxford University
Press Inc., New York, 2002. p. 29.
[27] J.J. McKinnon, M.A. Spackman, A.S. Mitchell, Acta Crystallogr., Sect. B: Struct.
Sci. 60 (2004) 627.
[28] M.A. Spackman, D. Jayatilaka, CrystEngComm 11 (2009) 19.
[29] F.H. Allen, Acta Crystallogr., Sect. B: Struct. Sci. 58 (2002) 380.
[30] T.C. van Soest, A.F. Peerdeman, Acta Crystallogr., Sect. B: Struct. Sci. 26 (1970)
1947.
[4] J. Bernstein, Cryst. Growth. Des. 11 (2011) 632.
[5] G.R. Desiraju, Crystal Engineering: The Design of Organic Solids, Elsevier,
Amsterdam, 1989.
[6] N. Blagden, R.J. Davey, Cryst. Growth Des. 3 (2003) 873.
[7] J. Aaltonen, M. Alleso, S. Mirza, V. Koradia, K.C. Gordon, J. Rantanen, Eur. J.
Pharm. Biopharm. 71 (2009) 23.
[31] T.N. Drebushchak, N.V. Chukanov, E.V. Boldyreva, Acta Crystallogr., Sect. C:
Cryst. Struct. Commun. 63 (2007) o355.
[32] T.N. Drebushchak, Y.A. Chesalov, E.V. Boldyreva, Acta Crystallogr., Sect. B:
Struct. Sci. 65 (2009) 770.
[33] E. Sugiono, M. Bolte, Acta Crystallogr., Sect. C: Cryst. Struct. Commun. 64
(2008) o84.
[8] H.G. Brittain, Polymorphism in Pharmaceutical Solids, second ed., Informa
Healthcare USA, Inc., New York, 2009.
[9] P.N. Prasad, D.J. William, Introduction to Nonlinear Optical Effects in Molecules
and Polymers, J. Wiley & Sons, New York, 1991.
[10] T. Watanabe, H. Yamammoto, T. Hosomi, S. Miyata, in: J. Messier, F. Kajzar, P.
Prasad (Eds.), Organic Molecules for Nonlinear Optics and Photonics, Kluwer
Academic Publishers, Dordrecht, 1991, p. 151.
ˇ ´
ˇ
´
´
´
[34] M. Rubcic, K. Uzarevic, I. Halasz, N. Bregovic, M. Mališ, I. Ðilovic, Z. Kokan, R.S.
Stein, R.E. Dinnebier, V. Tomišic´, Chem. Eur. J. 18 (2012) 5620.
[35] P.S. Halasyamani, K.R. Poeppelmeier, Chem. Mater. 10 (1998) 2753.
[36] K.T. Holman, A.M. Pivovar, M.D. Ward, Science 294 (2001) 1907.
[37] D. Braga, D. D’Addario, S.L. Giaffreda, L. Maini, M. Polito, F. Grepioni, Top. Curr.
Chem. 254 (2005) 71.
ˇ ´
[38] T. Frišcic, J. Mater. Chem. 20 (2010) 7599.
[11] K-S. Huang, D. Britton, M.C. Etter, S.R. Byrn, J. Mater. Chem. 6 (1996) 123.
[12] J. Bernstein, R.J. Davey, J.-O. Henck, Angew. Chem. Int. Ed. 38 (1999) 3440.
[13] Z. Kokan, S.I. Kirin, RSC Adv. 2 (2012) 5729.
[39] W.J. Belcher, C.A. Longstaff, M.R. Neckenig, J.W. Steed, Chem. Commun. (2002)
1602.
ˇ ´
[40] D. Cincic, I. Brekalo, B. Kaitner, Cryst. Growth Des. 12 (2012) 44.
ˇ
[14] R.A. Storey, I. Ymén, Solid State Characterization of Pharmaceuticals, Blackwell
ˇ
´
ˇ ´
´
´
´
´
[41] K. Uzarevic, M. Rubcic, I. Ðilovic, Z. Kokan, D. Matkovic-Calogovic, M. Cindric,
Cryst. Growth Des. 9 (2009) 5327.
Publishing, John Wiley & Sons, Chichester, UK, 2011.
ˇ ´
´
´
´
[15] M. Rubcic, D. Milic, G. Pavlovic, M. Cindric, Cryst. Growth Des. 11 (2011) 5227.
[42] M. Descamps, J.F. Willart, E. Dudognon, V. Caron, J. Pharm. Sci. 96 (2007) 1398.
ˇ ´
[16] S.L. James, C.J. Adams, C. Bolm, D. Braga, P. Collier, T. Frišcic, F. Grepioni, K.D.M.
ˇ ´
[43] T. Frišcic, Chem. Soc. Rev. 41 (2012) 3493.
Harris, G. Hyett, W. Jones, A. Krebs, J. Mack, L. Maini, A.G. Orpen, I.P. Parkin,
W.C. Shearouse, J.W. Steed, D.C. Waddell, Chem. Soc. Rev. 41 (2012) 413.
[17] Oxford Diffraction, Oxford Diffraction Ltd., Xcalibur CCD system, CRYSALIS
Software system, Version 1.170.33.32, 2009.
[44] F.H. Herbstein, Acta Crystallogr., Sect. B: Struct. Sci. 62 (2006) 341.
[45] D. Khamar, I.J. Bradshaw, G.A. Hutcheon, L. Seton, Cryst. Growth. Des. 12
(2012) 109.