Pseudopeptides designed to form supramolecular helixes: The role of the stereogenic centers

Article abstract of DOI:10.1021/cg9012558

The two epimers Boc-L-Phe-D-Oxd-(S)-β³-hPhg-OBn (1) and Boc-L-Phe-D-Oxd-(R)-β³-hPhg-OBn (2) have been prepared by standard methods in solution, and their conformation was analyzed both in solution and in the solid state. While in sol

Full text of DOI:10.1021/cg9012558

DOI: 10.1021/cg9012558
Pseudopeptides Designed to Form Supramolecular Helixes: The Role of
the Stereogenic Centers
2010, Vol. 10
923–929
Gaetano Angelici,† Nicola Castellucci,‡ Giuseppe Falini,*,‡ Daniel Huster,#
Magda Monari,‡ and Claudia Tomasini*,‡
†Department of Chemistry, University of Basel, St. Johanns-Ring 19 CH-4056 Basel, Switzerland,
‡
ꢀ
Dipartimento di Chimica “G. Ciamician” - Alma Mater Studiorum Universita di Bologna - Via Selmi 2,
I-40126 Bologna, Italy, and #Institute of Medical Physics and Biophysics - University of
Leipzig - Ha€rtelstrasse 16-18, D-04107 Leipzig, Germany
Received October 9, 2009; Revised Manuscript Received November 16, 2009
ABSTRACT: The two epimers Boc-L-Phe-D-Oxd-(S)-β3-hPhg-OBn (1) and Boc-L-Phe-D-Oxd-(R)-β3-hPhg-OBn (2) have been
prepared by standard methods in solution, and their conformation was analyzed both in solution and in the solid state. While in
solution 1 shows a random coil structure, 2 tends to assume a γ-turn conformation that is nearly retained in the solid state. On the
other hand, in the solid state molecules of 1 associate generating a helix that involves the formation of elongated crystals with
hexagonal cross-section. This effect is not observed in the crystals formed by Boc-L-Phe-D-Oxd-(R)-β3-hPhg-OBn 2.
Introduction
as white solids after purification by flash chromatography
(cyclohexane/ethyl acetate 8/2 as eluants).
In recent years, the self-assembly of small molecules is
receiving a great deal of interest due to the possibility to form
complex structures in a very simple and sustainable way.1
Applications in all fields of chemistry, materials science, and
even pharmacological applications have already been demon-
strated. In this context, the self-assembly of small peptides
made of only 2-3 amino acids has been recently studied by
Banerjee and co-workers, who demonstrated that one can
obtain stable β-sheet or helix-like structures by interaction of
smallpeptides thatare held together by 2-3 hydrogenbonds.2
We have recently demonstrated that stable fibers can be
obtained through self-assembly of small pseudopeptides that
are connected by a single hydrogen bond, in a parallel or
antiparallel structure.3 Moreover, other researchers demon-
strated that very simple compounds such as L-Phe-L-Phe may
produce complex structures such as helices or carbon nano-
tubes.4
Here we describe our recent results on the self-assembly of
two epimers, Boc-L-Phe-D-Oxd-(S)-β3-hPhg-OBn (1) and
Boc-L-Phe-D-Oxd-(R)-β3-hPhg-OBn (2). Both of these mole-
cules contain the scaffold L-Phe-D-Oxd that we have recently
demonstrated to be the basic pattern for the formation of
fibers in the solid state, but differ for the introduction of the
two enantiomers of β3-homophenylglycine. The reversal of
the stereogenic center contained in this moiety has important
effects on the preferred molecular conformation and, as a
result, on the crystal morphology.
The preferential conformation assumed by the two com-
pounds was analyzed by FT-IR spectroscopy in diluted solu-
tion (3 mM) Figure 1. At this concentration, intermolecular
interactions are negligible, so that the presence of intramole-
cular hydrogen bonds can be checked. Interestingly, 2 shows
the presence of a chelate NH hydrogen bond, as demonstrated
by the peaks centered at3434and 1670cm-1, while 1 does not.
This outcome suggests that only 2 forms a γ-turn structure.
This outcome is confirmed by solution NMR spectroscopy,
where we investigated the DMSO-d6 dependence of the NH
proton chemical shifts. DMSO is a strong hydrogen bond
acceptor and, if bound to a free NH proton, a considerable
downfield shift of the proton signal can be expected. Both
compounds have two NH groups, belonging to a carbamate
group and an amide group, respectively. The results are
reported in Figure S7, Supporting Information. While the
chelation of the carbamate NH is uncertain for both com-
pounds, we can notice that the Phe-NH group is poorly
chelated for 1 and much more chelated for 2, thus confirming
the outcome of the FT-IR spectra.
The FT-IR spectra of 1 and 2 in the solid state are very
similar and show thatall the NH hydrogens are chelated, as all
the stretching signals are below 3400 cm-1 (Figure 2).
- þ
Scheme 1. (i) CF3CO2 3HN-(S)-β3-hPhg-OBn (1 equiv),
HBTU (1.1 equiv), Et3N (3 equiv), dry CH3CN, 40 min, r.t.; (ii)
CF3CO2 3HN-(R)-β3-hPhg-OBn (1 equiv), HBTU (1.1 equiv),
- þ
Et3N (3 equiv), dry CH3CN, 40 min, r.t.
Results and Discussion
Compounds 1 and 2 are easily prepared by coupling of the
already described Boc-L-Phe-D-Oxd-OH moieties with (R)-
β3-hPhg-OBn or (S)-β3-hPhg-OBn, respectively, in high yield
by standard methods in solution. The coupling steps are
reported in Scheme 1. The compounds were obtained pure
*To whom correspondence should be addressed. E-mail: claudia.tomasini@
unibo.it (C.T.), giuseppe.falini@unibo.it (G.F.).
r
2009 American Chemical Society
Published on Web 12/10/2009
pubs.acs.org/crystal

924 Crystal Growth & Design, Vol. 10, No. 2, 2010
Angelici et al.
Figure 1. FT-IR absorption spectra in the N-H (left) and CdO (right) stretching regions for 3 mM concentration samples of oligomers 1 and 2
in pure CH2Cl2 at room temperature.
Figure 2. FT-IR absorption spectra in the N-H (left) and CdO (right) stretching regions for a 1% solid mixture with KBr of oligomers 1 and 2
at room temperature.
Table 1. Comparison of the 13C NMR Results for the Phe Residue in
Compounds 1 and 2 in Solution and in the Solid State
Further interesting results are obtained by comparison of
the 13C NMR spectra of 1 and 2 in solution and in the solid
state (Table1). As a reference, the chemical shifts of CR and Cβ
of the phenylalanine residue in the peptide were measured in
CDCl3 solution and furnished very similar outcomes (40.7
and 54.1 for 1 and 40.7 and 53.9 for 2), while significantly
different chemical shifts were observed in the solid state. Two
samples of each peptide, 10% 13C enriched for Phe, were
prepared andanalyzedby solid-stateNMR. Both1 and 2 have
been evaporated under different conditions (1:1 mixture of
cyclohexane/ethyl acetate and protonated solvents, methanol
or ethanol).
sample δ Phe CR/ppm δ Phe Cβ/ppm
condition
CDCl3 solution
evaporated from cyclohexane/
ethyl acetate (1:1)
evaporated from methanol
CDCl3 solution
evaporated from cyclohexane/
ethyl acetate (1:1)
evaporated from methanol
1
54.1
56.8
40.7
37.9
56.8
53.9
56.3
37.8
40.7
39.7
2
56.3
39.6
The samples were precipitated by overnight evaporation of
A significant difference in the chemical shifts of Phe in the
tripeptides between solution and the solid state is observed. In
the solid state, the chemical shifts of the Phe are indicative of
random coil structures if compared to the standard secondary
chemical shifts known from solution NMR data of proteins
containing L-amino acids.5 The chemical shifts in solution
indicate a tendency for β-sheet. However, having compounds
featuring L- and D- as well as nonstandard amino acids, it is
not straightforward torelate secondary chemical shift changes
to secondary structure.
From the analysis of the two compounds in solution and in
the solid state, we can infer only that both compounds tend to
aggregate in the solid state, without any information about
how they do it. As 2 tends to form a γ-turn structure in
solution, while 1 does not, we expect a different aggregation
in the solid state. For this purpose, an analysis of the two
compounds was carried out by means of microscopy and
X-ray diffraction.
aliquots (20.0 mg each) from several solvents (methanol,
ethanol, acetonitrile, cyclohexane/ethyl acetate, 1:1 mixture,
or diethyl ether, 1.0 mL each). A fast precipitation was also
achieved by rotary evaporation. The obtained solids showed
different morphologies depending on the kind of solvent and
the solvent evaporation rate. In any case, the precipitation of1
provoked the formation of a solid material made of filamen-
tous aggregates, in which the crystals showed a preferential
direction of growth and had a strong tendency to aggregate
laterally. This material always showed birefringence suggest-
ing a high crystallinity (Figure 3a,c). Only when 1 was
exsiccated by means of a rotary evaporator a powder-like
material was obtained. Crystals of 1 suitable for a single
crystal X-ray diffraction investigation were obtained only
from methanol evaporation. An image of these crystals by
scanning electron microscopy is reported in Figure 3c. They
appear elongated in one direction with a hexagonal cross-
section. Their length varied from one crystal to another in the
range of several hundred of micrometers, while their thickness
was almost constant around 20 μm. On the contrary, com-
pound 2 did not always precipitate forming a filamentous
A wide morphological variety of samples based on com-
pounds 1 and 2 obtained by evaporation from different
solvents were analyzed by optical and electronic microscopy.

Article
Crystal Growth & Design, Vol. 10, No. 2, 2010 925
Figure 3. (a) Optical micrographs of 1 precipitated from cyclohexane/ethyl acetate. The elongated growth of the crystal is observable together
with their high lateral aggregation. (c) Single crystals of 1 precipitated from ethanol, they are birefringent and have a hexagonal cross-section.
(b, d) crystals of 2 precipitated from ethanol observed by means of an optical and an electron microscope, respectively.
Figure 4. (Left) X-ray powder diffraction patterns of 1 precipitated from different solvents and exsiccated using a rotary evaporator. The
calculated diffraction pattern from the structure of 1 obtained from crystals precipitated from methanol is shown as well. (Right) X-ray powder
diffraction patterns of 2 precipitated from different solvents and exsiccated using a rotary evaporator. The calculated diffraction pattern from
the structure of 2 obtained from crystals precipitated from ethanol is shown as well. Rotar indicate a material exsiccated by means of a rotary
evaporator; MeOH, EtOH, MeCN, cHex/AcOEt, and Et2O indicate a material precipitated from methanol, ethanol, acetonitrile, cyclohexane/
ethyl acetate, and diethyl ether respectively; calc indicates the calculated diffraction pattern.
structure (data not shown). It precipitated as a mixture of
powder and small (around 1 μm) elongated crystal aggregates
from acetonitrile, cyclohexane/ethyl acetate, or diethyl ether,
while by evaporation from protonated solvents, methanol or
ethanol, elongated single crystals were obtained (Figure 3b,d).
Ofthese, only the onesprecipitated from ethanol weresuitable
for single crystal X-ray diffraction studies (see later). These
crystals were also observed by scanning electron microscopy
that showed an irregular morphology in which plate-like
shapes are visible.
The crystals of 1 and 2 that precipitated from different
solvents were analyzed by X-ray powder diffraction. Com-
pound 1 gave only one kind of crystals, irrespective of the
solvent used (methanol, ethanol, acetonitrile, cyclohexane/
ethyl acetate, or diethyl ether) in the crystallization trials.
Interestingly, the same kind of crystals precipitated even
when the process was carried out in very harsh conditions,
such as rotary evaporator (Figure 4a). This tendency to
precipitate in a unique conformation may indicate a
high stability of this form, which may be already present
in solution, before the precipitation process starts. Com-
pound 2 showed a different behavior with respect to
compound 1. It precipitated as a crystalline polymorph
from methanol or acetonitrile, as a different polymorph
from ethanol, and as a third polymorph from cyclohexane/
ethyl acetate or diethyl ether (Figure 4b). The exsiccation
of 2 using a rotary evaporator provoked the formation of a
low crystallinity material, which could be a new polymorph
or a mixture of polymorphs already precipitated from
other solvents. It is conceivable to consider that the pre-
cipitation of this latter polymorph is strongly affected by
the polarity and protonation of the solvent, as well as
by the rate of crystallization. It is also interesting to note
that the material precipitated from ethanol contained
traces of the polymorphs obtained after precipitation from
methanol or acetonitrile (Figure 5b).

926 Crystal Growth & Design, Vol. 10, No. 2, 2010
Angelici et al.
Figure 5. X-ray molecular structures of the two epimers 1 (a) and 2 (b).
Table 3. Most Relevant H Bond Parameters for 1 and 2
Table 2. Comparison of the Backbone Torsional Angles (°) for Molecules
1 and 2
˚
A, A
˚
A, A
˚
D-H
A
H
D
D-H A, A
3 3 3
3 3 3
3 3 3
3 3 3
torsion angles
1
2
1
2.03
2.16
C31-N2-C23-C22 (L-Phe1) (φ1)
N2-C23-C22-N1 (ψ1)
C22-N1-C3-C5 (φ2)
N1-C3-C5-N3 (ψ2)
C5-N3-C6-C13 (φ3)
N3-C6-C13-C14 (ψ3)
-130.0(2)
154.6(2)
60.0(3)
-126.5(2)
150.9
-132.6(2)
169.3(2)
74.2(3)
-118.0(3)
-105.9(3)
68.3(4)
a
a
N3-H3N O10
2.902(3)
2.964(3)
165(3)
151(3)
3 3 3
N2-H2N O30
3 3 3
2
b
b
N2-H2N O70
2.29(2)
2.44(2)
3.131(3)
3.230(4)
160(3)
149(3)
3 3 3
-67.6(3)
N3-H3N O40
3 3 3
a Symmetry operation for compound 1: (I) x þ y, -x, z þ 1/3.
b Symmetry operation for compound 2: (I) x - 1, y, z.
Crystals suitable for an X-ray diffraction study were grown
only by slow evaporation of a solution of 1 in methanol and of
2 in ethanol, both at room temperature. Any effort to grow
from methanol crystals of 2, suitable for X-ray studies, was
unsuccessful.
The X-ray molecular structures of 1 and 2 are reported
together in Figure 5, panels a and b, respectively, and relevant
torsionangles for both arelisted in Table2. The two molecules
are epimers and differ only for the configuration of the
stereogenic center C6 which is S in 1 and R in 2. As a
consequence of this different stereogenic center, 2 has a higher
˚
degree of folding than 1 (closer contact O8 H15A 3.05 A),
3 3 3
but no classical intramolecular hydrogen bond is observed in
both epimers, in contrast with the behavior of 2 in solution
(Table 3).
Interestingly, the crystal packing of 1 is completely different
from those observed in the recently reported Boc-L-Phe-D-
Oxd-OBn3a and Boc-(L-Phe-D-Oxd)2-OBn3b where single in-
termolecular NH CO hydrogen bonds between neighbor
3 3 3
units generate infinite parallel or antiparallel one-dimensional
(1-D) H-bonded polymers, respectively.
Each molecule of 1 is engaged in four intermolecular
Figure 6. View down the b axis of the crystal packing of 1 showing
the N-H OdC H bonds. Only the hydrogen atoms involved in H
bonding are shown.
N-H OdC hydrogen bonds of two types with the adjacent
3 3 3
3 3 3
neighbors. Two interactions connect the amidic hydrogens of
the side chain bearing the Boc group and the carbonylic
oxygens of the amidic unit of the other side arm
beside the already mentioned N-H CO hydrogen bonds
3 3 3
[N2-H2N O30, symm. op.: (I) -x þ y, -x, z þ 1/3;
generating the 1-D network [Figure S1, Supporting In-
formation]. Inparticular, two of theseH bonds areestablished
between the carbonylic oxygen O1and the acidic proton of the
3003 3
O3 H2N -N200, symm. op.: (II) -y, x - y, -1/3 þ z],
3 3 3
while the ones of the second type involve the amidic hydrogen
H3N and the carbonylic oxygen of the D-Oxd ring
[N3-H3N O10, O1 H3N00-N300]. The crystal packing
D-Oxd ring of an adjacent molecule [O1 H300 2.76 A,
˚
3 3 3
O1 H300-C300 136°]. Other two weak H bonds are observed
3 3 3
3 3 3
3 3 3
of 1 (Figure 6) therefore consists of parallel chains with a
helical arrangement (Figure 7) running along the c axis. The
formation of a ternary helix6 in the solid state is in agreement
with the SEM analysis as the crystals of 1 from methanol
appear elongated in one direction with a hexagonal cross-
section (Figure 3c).
between the endocyclic oxygen of the D-Oxd ring and one
˚
proton of
a
phenyl ring [O2 H1200 2.65 A,
3 3 3
O2 H1200-C1200 128.5°]. All these intermolecular interac-
tions further stabilize the 1-D network, being oriented almost
3 3 3
parallel to the stronger N-H CO hydrogen bonds.
A completely different packing is observed for epimer
3 3 3
A closer inspection of the crystal packing of 1 reveals the
presence of weaker intermolecular non classical H bonds
2 (Figure 8) which maintains one intermolecular NH
CO hydrogen bond between equivalent amidic groups
3 3 3

Article
Crystal Growth & Design, Vol. 10, No. 2, 2010 927
[N2-H2N O70, symm. op.: x - 1, y, z] as observed in Boc-
The supramolecular assembly of 2 is completed by other
hydrogen bonding interactions. In detail the 1-D supramole-
cular polymer of 2 is further stabilized by CH/π bonding
between the acidic hydrogen (H6) of one molecule and one
3 3 3
L-Phe-D-Oxd-OBn3a and Boc-(L-Phe-D-Oxd)2-OBn.3b In ad-
dition, a second weaker hydrogen bond is observed between
the amidic hydrogen of the other amide group and the
uncoordinated carboxylic oxygen bearing the Bn group
˚
phenyl ring of the neighbors being 2.33 A distance from the
[N3-H3N O40]. As a result, epimer 2 is engaged in four
C7-C8-C9-C10-C11-C12 phenyl ring. As a difference
with the crystal packing of 1, there are also weak interchain H
bonds involving the endocyclic oxygen O2 of the D-Oxd ring
3 3 3
interactions that are weak in comparison with those found in
Boc-L-Phe-D-Oxd-OBn3a and Boc-(L-Phe-D-Oxd)2-OBn,3b
but the hydrogen bonding pattern in the crystal lattice shows
the formation of infinite 1-D H-bonded polymer of tape
running along the a axis.
and an aromatic hydrogen H19 [H19 O200 2.71 A,
˚
3 3 3
C19-H19 O200 ca. 150°].
3 3 3
To further investigate the effect of the absolute configura-
tion variation of the β3-hPhg moiety on the properties of 1 and
2 in the solid state, single crystals of 1 and 2, obtained
respectively, from methanol and ethanol, were also analyzed
in their thermal properties by differential scanning calori-
metry and thermogravimetry. Compound 1 undergoes an
endothermic transition at a temperature of 170 °C, where it
fuses converting to an amorphous material. After this first
thermal event, 1 shows a successive broad endothermic peak
having a temperature of onset at about 206 °C and maximum
at about 222 °C (Figure 9). At 206 °C 1 starts to decompose, as
shown by thermogravimetric analysis (TGA) (Figure S12,
Supporting Information). Compound 2 shows a differential
scanning calorimetry (DSC) profile similar to that of 1. First
an endothermic peak appears at 167 °C, which is associated
with a transition from the crystalline to an amorphous state
Figure 7. Space filling model showing the helical arrangement of
one of the chains of 1: (a) top view, (b) view along the c axis. The
molecules are represented in different colors for clarity. The hydro-
gen atoms involved in N-H CO hydrogen bonds are represented
3 3 3
as white cups.
Figure 10. FT-IR absorption spectra in the CdO (right) stretching
regions for oligomers 1 and 2 under different conditions. Black line:
1 in 3 mM solution in CH2Cl2; magenta line: 1 in 1% mixture with
KBr; blue line: 1 crystallized from methanol, after heating at 180 °C
(polymorph 1-II); red line: 2 in 3 mM solution of CH2Cl2; yellow
line: 2 in 1% mixture with KBr; green line: 2 crystallized from
ethanol, after heating at 180 °C (polymorph 2-II).
Figure 8. View down the c axis of the crystal packing of 2 showing
the N-H CO hydrogen bonds. Only the hydrogen atoms in-
3 3 3
volved in N-H CO hydrogen bonding are shown.
3 3 3
Figure 9. (Left) Differential scanning calorimetry profiles from single crystals of 1 and 2. (Right) X-ray powder diffraction patterns
corresponding to different regions in the thermal profiles of 1 and 2.

928 Crystal Growth & Design, Vol. 10, No. 2, 2010
Angelici et al.
(Figure 9), and then a broad endothermic peak follows with
an onset and maximum temperature of about 218 and 240 °C,
respectively.
4.32 (d, 1H, 3J(H,H) = 2.8 Hz, CHN-Oxd), 4.66 (dq, 1H, 3J(H,H)
= 2.8, 7.2 Hz, CHO-Oxd), 5.03 (bs, 1H, NH-Boc), 5.05 (AB, 2H,
2J(H,H) = 12.0 Hz, OCH2Ph), 5.46 (dt, 1H, 3J(H,H) = 7.2, 7.2 Hz,
CHN-Phe), 5.64-5.67 (m, 1H, CHN-hPhg), 7.21-7.38 ppm (m,
16H, NH-hPhg þ 3 x Ph); 13C NMR (CDCl3, 100 MHz, 25 °C): δ =
21.1, 28.5, 40.7, 50.6, 54.1, 62.9, 66.8, 80.8, 126.8, 127.6, 128.0,
128.4, 128.5, 128.7, 129.0, 129.6, 151.83, 166.69 ppm; IR (CH2Cl2,
10 mM): ν = 3437 (N-H), 3366 (N-H), 1789 (CdO), 1716 (CdO),
1696 (CdO) cm-1; IR (1% in dry KBr): ν = 3358 (N-H), 3318
(N-H), 1785 (CdO), 1763 (CdO), 1735 (CdO), 1719 (CdO), 1701
(CdO), 1687 (CdO) cm-1; elemental analysis calcd. (%) for
The 1-II FT-IR spectrum in the CdO bands region presents
a band at 1787 cm-1 and a broadband centered at about
1700 cm-1. A similar FT-IR spectral region is observed when
1 is present in a dilute solution of CH2Cl2 (Figure 10). This
suggests that, as a consequence of the thermal treatment, after
the first endothermic peak, compound 1 goes in an amor-
phous state, in which it assumes conformations similar to
those in solution. In contrast, the IR analysis of a sample of 2
C
N 6.66.
35H39N3O8: C, 66.76; H, 6.24; N, 6.67; found: C 66.71, H 6.25,
after thermal treatment does not show any peak at 1780 cm-1
;
Boc-L-Phe-D-Oxd-(R)-β3-hPhg-OBn 2. A solution of Boc-L-Phe-
D-Oxd-OH6 (0.27 mmol, 0.10 g) and HBTU (0.3 mmol, 0.12 g) in dry
acetonitrile (25 mL) was stirred under inert atmosphere for 10 min at
thus, its amorphous state somehow still retains an ordered
structure.
room temperature. Then a mixture of H-(R)-β3-hPhg-OBn TFA
3
(0.27 mmol, 0.10 g) and Et3N (0.81 mmol, 0.12 mL) in dry
acetonitrile (10 mL) was added at room temperature. The solution
was stirred for 40 min under inert atmosphere, and then acetonitrile
was removed under reduced pressure and replaced with ethyl
acetate. The mixture was washed with brine, 1 N aqueous HCl
(3 ꢀ 30 mL), and 5% aqueous NaHCO3 (1 ꢀ 30 mL), dried over
sodium sulfate, and concentrated in vacuo. The product was
obtained pure after silica gel chromatography (cyclohexane/ethyl
acetate 8:2 as eluant) in 81% yield. mp = 160 °C; [R]D þ35.9 (c 1.0,
CH2Cl2); 1H NMR (CDCl3, 400 MHz): δ = 1.23 (s, 9H, t-Bu), 1.31
(d, 3H, 3J(H,H) = 6.8 Hz, Me-Oxd), 2.85-2.92 (m, 2H, CHH-Phe
þ CHH-hPhg), 2.99 (dd, 1H, 3J(H,H) = 8.4, 15.6 Hz, CHH-Phe),
3J(H,H) = 4.0 Hz, CHN-Oxd), 4.672 (dq, 1H, 3J(H,H) = 4.0, 6.8
Hz, CHO-Oxd), 5.00 (d, 1H, 3J(H,H) = 4 Hz, NH-Boc), 5.07 (s, 2H,
OCH2Ph), 5.46 (dt, 1H, 3J(H,H) = 6.0, 8.4 Hz, CHN-Phe),
5.67-5.5.75 (m, 1H, CHN-hPhg), 7.20-7.36 (m, 15H, 3 x Ph),
7.54 ppm (d, 1H, 3J(H,H) = 7.6 Hz, NH-hPhg); 13C NMR (CDCl3,
100 MHz): δ = 21.1, 28.4, 29.9, 31.1, 37.6, 40.7, 50.9, 53.9, 62.7,
66.8, 75.3, 81.0, 126.4, 127.6, 127.7, 128.4, 128.5, 128.7, 128.8, 129.1,
129.6, 135.4, 135.7, 135.8, 140.5, 151.9, 167.1, 170.5, 173.6 ppm; IR
(CH2Cl2, 10 mM): ν = 3436 (N-H), 3350 (N-H), 1788 (CdO),
1716 (CdO), 1693 (CdO), 1679 (CdO) cm-1; IR (1% in dry KBr): ν
= 3381 (N-H), 3323 (N-H), 1764 (CdO), 1742 (CdO), 1719
(CdO), 1701 (CdO), 1691 (CdO), 1684 (CdO) cm-1; elemental
analysis calcd. (%) for C35H39N3O8: C, 66.76; H, 6.24; N, 6.67;
found: C 66.75, H 6.25, N 6.65.
Crystal Precipitation. Several portions (20 mg each) of the
purified compounds 1 and 2 dissolved in different solvents
(ethanol, methanol, diethyl ether, acetonitrile, a 1:1 mixture of
cyclohexane/ethyl acetate, 1 mL each) were crystallized by slow
evaporation at room temperature. Samples suitable for single
crystal X-ray diffraction were obtained from methanol (com-
pound 1) and ethanol (compound 2).
Microscopy. The precipitates were systematically observed by
optical microscopy (OM) and scanning electron microscopy (SEM).
The OM images were collected using a Leica optical microscope
equipped with a CCD camera. Samples SEM images were collected
on glass coverslip after coating with gold and observed using a
Philips XL20 scanning electron microscope. The images were
recorded using a CCD digital camera.
Conclusions
We reported the synthesis and the conformational analysis
in solution and in the solid state of two pseudopeptides of the
general formula Boc-L-Phe-D-Oxd-(S,R)-β3-hPhg-OBn.
These two compounds form crystals after evaporation from
protonated solvents, but the reversal of the absolute confi-
guration of the stereogenic center of the hPhg moiety ends in a
dramatic variation of the preferential conformation of the two
compounds, that in turn induces a different crystal packing
and consequently a different crystal morphology. Indeed,
Boc-L-Phe-D-Oxd-(S)-β3-hPhg-OBn forms a ternary helixthat
crystallizes in hexagonal elongated crystals, while Boc-L-Phe-
D-Oxd-(R)-β3-hPhg-OBn forms a 1-D H-bonded polymer of
tape that crystallizes in different polymorphs, depending on
the evaporation solvent.
3
3.15 (dd, 1H, J(H,H) = 6.0, 13.6 Hz, CHH-hPhg), 4.32 (d, 1H,
Experimental Section
Synthesis. The melting points of the compounds were determined
in open capillaries and are uncorrected. High quality infrared
spectra (64 scans) were obtained at 2 cm-1 resolution using a
1 mm NaCl solution cell and a Nicolet 210 FT-infrared spectro-
meter. All spectra were obtained in 3 mM solutions in dry CH2Cl2 at
297 K or as a 1% solid mixture with dry KBr. All compounds were
dried in vacuo, and all the sample preparations were performed in a
nitrogen atmosphere. Routine NMR spectra were recorded with
spectrometers at 400 or 300 MHz (1H NMR) and at 100 or 75 MHz
1
(
13C NMR). High quality H NMR spectra were recorded with a
Varian Inova 600. The measurements were carried out in CDCl3 and
in CD3OD. The proton signals were assigned by gCOSY spectra.
Chemical shifts are reported in δ values relative to the solvent
(CDCl3 or CD3OD) peak. The β-amino acids (S)-β3-hPhg (D-β3-
homophenylglycine) and (R)-β3-hPhg (L-β3-homophenylglycine)
were prepared at DSM Research as described in PCT Patent Appl.
WO 01/42173 and Eur. Patent Appl. No. 04075597.7 (patent
pending), respectively.
Boc-L-Phe-D-Oxd-(S)-β3-hPhg-OBn 1. A solution of Boc-L-Phe-
D-Oxd-OH7 (0.27 mmol, 0.10 g) and HBTU (0.3 mmol, 0.12 g) in dry
acetonitrile (25 mL) was stirred under inert atmosphere for 10 min at
Single Crystal X-ray Diffraction for 1 and 2. The X-ray intensity
data for 1 and 2 were measured on a Bruker SMART Apex II CCD
area detector diffractometer. Cell dimensions and the orientation
matrix were initially determined from a least-squares refinement on
reflections measured in three sets of 20 exposures, collected in three
different ω regions, and eventually refined against all data. A full
sphere of reciprocal space was scanned by 0.3° ω steps. The software
SMART8 was used for collecting frames of data, indexing reflec-
tions, and determination of lattice parameters. The collected frames
were then processed for integration by the SAINT program,9 and an
empirical absorption correction was applied using SADABS.11 The
structure was solved by direct methods (SIR 97)10 and subsequent
Fourier syntheses and refined by full-matrix least-squares on F2
(SHELXTL),11 using anisotropic thermal parameters for all non-
hydrogen atoms. All hydrogen atoms, except the amidic protons
room temperature. Then a mixture of H-(S)-β3-hPhg-OBn TFA
3
(0.27 mmol, 0.10 g) and Et3N (0.81 mmol, 0.12 mL) in dry
acetonitrile (10 mL) was added at room temperature. The solution
was stirred for 40 min under inert atmosphere, and then acetonitrile
was removed under reduced pressure and replaced with ethyl
acetate. The mixture was washed with brine, 1 N aqueous HCl (3
ꢀ 30 mL), and 5% aqueous NaHCO3 (1 ꢀ 30 mL), dried over
sodium sulfate, and concentrated in vacuo. The product was
obtained pure after silica gel chromatography (cyclohexane/ethyl
acetate 8:2 as eluant) in 80% yield. mp = 168 °C; [R]D = þ14.0 (c =
1.0 in CH2Cl2); 1H NMR (CDCl3, 400 MHz; 25 °C): δ = 1.32 (d,
3H, 3J(H,H) = 7.2 Hz, Me-Oxd), 1.33 (s, 9H, t-Bu), 2.84-2.92 (m,
3
2H, CHH-Phe þ CHH-hPhg), 2.97 (dd, 1H, J(H,H) = 7.2, 15.6
Hz, CHH-Phe), 3.12 (dd, 1H, 3J(H,H) = 6.0, 13.6 Hz, CHH-hPhg),

Article
Crystal Growth & Design, Vol. 10, No. 2, 2010 929
and methine hydrogens, were added in calculated positions, in-
cluded in the final stage of refinement with isotropic thermal
parameters, U(H) = 1.2 Ueq(C) [U(H) = 1.5 Ueq(C-Me)], and
allowed to ride on their carrier carbons. The absolute structure
configuration was not determined from X-ray data, but was known
from the synthetic route. Crystal data and details of the data
collection for 1 and 2 are reported in Table S2, Supporting Informa-
tion.
X-ray Powder Diffraction Analysis. Powder X-ray diffraction
patterns were collected using a PanAnalytical X’Pert Pro equipped
with X’Celerator detector powder diffractometer using Cu KR
radiation generated at 40 kV and 40 mA. The instrument was
configured with a 1/32° divergence and 1/32° antiscattering slits.
A standard quartz sample holder 1 mm deep, 20 mm high and
15 mm wide was used. The diffraction patterns were collected within
the 2θ range from 3° to 40° with a step size (Δ 2θ) of 0.02° and a
counting time of 300 s.
Thermal Analysis. Calorimetric measurements were performed
using a Perkin-Elmer DSC-7. Temperature and enthalphy calibra-
tions were performed by using high purity standards (n-decane,
benzene, and indium). The material (about 1.5 mg of sample) was
sealed in aluminum pans. Heating was carried out at 3 °C min-1 in
the temperature range 20-170 °C. Transition, denaturation, or
desolvation temperature (TD) was determined as the maximum
peak value of the corresponding endothermic phenomena. The
value of denaturation enthalpy was calculated with respect to the
mass of the sample. Weight loss during heating was evaluated by
TGA using a calorimeter DSC-Q100 from TA Instruments Waters
(USA). The temperature range was from 23 to 350 °C at a heating
rate of 5 °C/min under dry nitrogen atmosphere.
Solid-State NMR. For the solid-state NMR studies, powdered
samples were filled into 4-mm MAS rotors with a Teflon insert
providing a volume of approximately 50 μL. All 13C magic angle
spinning (MAS) NMR experiments were carried out on a Bruker
Avance 750 NMR (Bruker Biospin, Rheinstetten, Germany) spec-
trometer operating at a resonance frequency of 749.7 MHz for 1H
and 188.5 MHz for 13C. A double-resonance MAS probe equipped
with a 4 mm spinning module was used with a MAS frequency of
7 kHz at 30 °C. The 13C 90° pulse length was typically 5 μs. Cross-
polarization (CP) MAS spectra were recorded with a 1H 90° pulse
length of 4 μs and a CP contact time of 700 μs. The 1H radio
frequency field strength during heteronuclear decoupling using
TPPM12 was about 65 kHz. The 13C chemical shifts were referenced
to the 13CdO signal of 13C-labeled Gly at 176.45 ppm as external
standard. Thus, all ppm values are relative to TMS.13
Physics Institutes of the University of Leipzig for measuring
time the Avance 750 MHz NMR spectrometer. Furthermore,
we thank DSM Research for a generous gift of (S)-β3-hPhg
(D-β3-homophenylglycine) and (R)-β3-hPhg (L-β3-homo-
phenylglycine).
Supporting Information Available: Crystallographic data (cif
files) for 1 and 2. IR and NMR spectra, supplemental crystallo-
graphic figures and thermogravimetric analysis of 1 and 2. This
material is available free of charge via the Internet at http://pubs.
acs.org.
References
(1) (a) Ulijn, R. V.; Smith, A. M. Chem. Soc. Rev. 2008, 37, 664–675. (b)
Khakshoor, O.; Nowick, J. S. Curr. Opin. Chem. Biol. 2008, 12, 722–
729. (c) Aggeli, A.; Nyrkova, I. A.; Bell, M.; Harding, R.; Carrick, L.;
McLeish, T. C. B.; Semenov, A. N.; Boden, N. Proc. Nat. Acad. Sci.
2001, 98, 11857–11862.
(2) (a) Samit, G.; Drew, M. G. B.; Banerjee, A. Chem. Mater. 2008, 20,
2282–2290. (b) Bose, P. P.; Das, A. K.; Hedge, R. P.; Shamala, N.;
Banerjee, A. Chem. Mater. 2007, 19, 6150–6157. (c) Guta, S.; Drew,
M. G. B.; Banerjee, A. Org. Lett. 2007, 9, 1347–1350. (d) Das, A. K.;
Manna, S.; Drew, M. G. B.; Malik, S.; Nandi, A. K.; Banerjee, A.
Supramol. Chem. 2006, 18, 645–655. (e) Ray, S.; Das, A. K.; Drew, M.
G. B.; Banerjee, A. Chem. Commun. 2006, 4230–4232. (f) Das, A. K.;
Haldar, D.; Hedge, R. P.; Shamala, N.; Banerjee, A. Chem. Commun.
2005, 1836–1838.
(3) (a) Angelici, G.; Falini, G.; Hofmann, H.-J.; Huster, D.; Monari,
M.; Tomasini, C. Angew. Chem., Int. Ed. 2008, 47, 8075–8078. (b)
Angelici, G.; Falini, G.; Hofmann, H.-J.; Huster, D.; Monari, M.;
Tomasini, C. Chem.;Eur. J. 2009, 15, 8037–8048.
(4) (a) Wang, Y.; Lingenfelder, M.; Classen, T.; Costantini, G. J. Am.
Chem. Soc. 2007, 129, 15742–15743. (b) Smith, A. M.; Williams, R. J.;
Tang, C.; Coppo, P.; Collins, R. F.; Turner, M. L.; Saiani, A.; Ulijn, R. V.
Adv. Mater. 2008, 20, 37–41. (c) Ryu, J.; Park, C. B. Angew. Chem.,
Int. Ed. 2009, 48, 4820–4823.
(5) Wang, Y.; Jardetzky, O. Protein Sci. 2002, 11, 852–861.
(6) Kodama, K.; Kobayashi, Y.; Saigo, K. Cryst. Growth Des. 2007, 7,
935–939.
(7) For the preparation, see ref 3b.
(8) Sheldrick, G. M. SADABS, Program for Empirical Absorption
€
Correction; University of Gottingen: Germany, 1996.
(9) Altomare, A.; Burla, M. C.; Camalli, M.; Cascarano, G. L.;
Giacovazzo, C.; Guagliardi, A.; Moliterni, A. G. G.; Polidori,
G. R. Spagna, SIR97: a new tool for crystal structure determina-
tion and refinement. J. Appl. Crystallogr. 1999, 32, 115.
(10) Sheldrick, G. M. SHELXTLplus Version 5.1 (Windows NT
version)-Structure Determination Package; Bruker Analytical X-ray
Instruments Inc.: Madison, WI, 1998.
(11) Angelici, G.; Contaldi, S.; Green, S. L.; Tomasini, C. Org. Biomol.
Chem. 2008, 6, 1849–1852.
(12) Bennett, A. E.; Rienstra, C. M.; Auger, M.; Lakshmi, K. V.;
Griffin, R. G. J. Chem. Phys. 1995, 103, 6951–6958.
Acknowledgment. G.A., N.C., G.F., M.M., and C.T. are
ꢀ
grateful to Ministero dell’Universita e della Ricerca Scientifi-
ca(PRIN2006)andUniversita diBologna (Funds for selected
ꢀ
topics) for financial support. C.T. is grateful to Fondazione
del Monte di Bologna e di Ravenna for financial support.
D.H. thanks the DFG (SFB 610, A14) and the Experimental
(13) Morcombe, C. R.; Zilm, K. W. J. Magn. Reson. 2003, 162, 479–486.