β-Phenylproline: The high β-turn forming propensity of proline combined with an aromatic side chain

Article abstract of DOI:10.1039/c1ob06561k

The conformational propensities of the proline analogue bearing a phenyl substituent attached to the β carbon, in either a cis or a trans configuration relative to the carbonyl group, have been investigated. The behaviour of cis- and trans(βPh)Pro has bee

Full text of DOI:10.1039/c1ob06561k

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2012, 10, 640
www.rsc.org/obc
PAPER
b-Phenylproline: the high b-turn forming propensity of proline combined with
an aromatic side chain†‡
Paola Fata´s, Ana I. Jime´nez,* M. Isabel Calaza and Carlos Cativiela*
Received 12th September 2011, Accepted 5th October 2011
DOI: 10.1039/c1ob06561k
The conformational propensities of the proline analogue bearing a phenyl substituent attached to the b
carbon, in either a cis or a trans configuration relative to the carbonyl group, have been investigated.
The behaviour of cis- and trans(bPh)Pro has been compared with that of proline in homochiral and
heterochiral dipeptide sequences. NMR and IR studies as well as X-ray diffraction analysis provide
evidence that the b-phenyl substituent does not disrupt the tendency of proline to occupy the i+1
position of a b-turn. The puckering of the pyrrolidine ring is significantly affected by the presence of the
aromatic substituent, which tends to occupy positions that minimize steric repulsions. As a
consequence, this substituent adopts specific well-defined orientations, which are more restricted for the
cis derivative. Interactions between this aromatic group and that in the adjacent phenylalanine residue
may be responsible for some of the conformational differences observed among the different peptides
studied.
conformational properties of the parent amino acid or at the
addition of new side-chain functionalities. Among the latter is the
attachment of a substituent to the pyrrolidine b carbon to generate
an analogue bearing the side chain of another proteinogenic
amino acid. This is the case with b-phenylproline, (bPh)Pro (Fig.
1), which can be regarded as a proline–phenylalanine hybrid
in which the orientation of the aromatic substituent is dictated
by the conformation of the five-membered ring and the cis or
Introduction
The applications of peptides in medicine and other fields rely
on the three-dimensional arrangement of their main-chain and
side-chain functionalities. Great efforts have been devoted in the
last decades to developing methods to efficiently control the
folding mode of the peptide backbone.1 Among them is the
incorporation of non-proteinogenic residues whose structure has
been designed to stabilize certain conformations among those
energetically accessible to the coded amino acids.1,2
Proline is the only proteinogenic amino acid that exhibits limited
trans configuration of the phenyl group relative to the carbonyl
moiety. Accordingly, cis(bPh)Pro and trans(bPh)Pro combine the
conformational properties of proline with an aromatic side-chain
conformational flexibility and a marked propensity to explore
functionality that is rigidly oriented with respect to the peptide
only a few regions of the Ramachandran map.3,4 Due to its cyclic
backbone, and this may be useful in the design of biologically
structure, rotation around the Ca–N bond is forbidden and, as a
active peptides and other applications relying on specifically-
consequence, proline can only adopt a narrow range of f values
(ª -60◦). This is at the basis of the well-known tendency of
proline to act as a turn inductor and, in particular, to occupy
the i+1 position of b-turns.4,5 The unique structure of proline
may serve as inspiration for the construction of novel amino acids
with well-defined conformational propensities. Such proline-based
residues may incorporate modifications aimed at fine-tuning the
oriented side-chain moieties.
Departamento de Qu´ımica Orga´nica, ISQCH, Universidad de
Zaragoza–CSIC, 50009 Zaragoza, Spain. E-mail: anisjim@unizar.
es, cativiela@unizar.es
† This article is part of an Organic & Biomolecular Chemistry web theme
issue on Foldamer Chemistry.
‡ Electronic supplementary information (ESI) available: Complete list of
geometrical parameters for the X-ray structures, copies of 1H-NMR, 13C-
NMR and NOESY spectra of 1–4, and characterization data for the
precursors 1a–4a. CCDC reference numbers 842881–842885. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c1ob06561k
Fig. 1 Comparison of the structures of proline and phenylalanine with
those of the hybrid amino acid b-phenylproline, (bPh)Pro. The aromatic
side chain in the latter may exhibit a cis or a trans configuration relative to
the carboxylic acid group.
(bPh)Pro has been inserted into a number of bioactive peptides6
as a replacement for either proline or phenylalanine and has led to
640 | Org. Biomol. Chem., 2012, 10, 640–651
This journal is
The Royal Society of Chemistry 2012
©

View Article Online
enhanced peptide-receptor affinity and improved stability in some
cases. Although some of these studies have used computational
methods to try to rationalise the biological activity observed
as a function of the structural modification performed,6a,d the
conformational propensities of (bPh)Pro have not been established
to date. This can be best achieved through the in-depth analysis of
small model peptides that incorporate this amino acid. In the
present work, the ability of cis(bPh)Pro and trans(bPh)Pro to
induce a b-turn when occupying the i+1 position of a dipeptide
sequence has been evaluated and compared to that of the natural
amino acid. Although several b-substituted proline analogues
other than (bPh)Pro have been the subject of structural studies
(mainly, as diamide derivatives),7 the formation of b-turns has
been addressed only in a few cases,8 in which other amino acids
with restricted conformational properties either coded (proline)8c,d
or not (N-methyl or cyclopropaneamino acids)8a,b were present. In
this work, (bPh)Pro is combined with phenylalanine, a residue
with no specific conformational bias. This allows the evaluation
of very subtle structural aspects and a precise comparison of
the behaviour of (bPh)Pro and proline as b-turn inductors. The
conformation adopted by the (bPh)Pro-containing model peptides
has been established in solution and in the solid state using
different spectroscopic techniques and X-ray diffraction studies.
Accordingly, proline is overwhelmingly found in the (-60,-30) and
(-60,140) regions of the Ramachandran map.3,4 These (f,y) angles
are very close to the ideal values corresponding to the i+1 position
of a b-turn of type I or II,5,9 respectively, which explains the high
propensity of proline to induce these folding modes. The presence
of an L or D residue in the i+2 position (as L-Phe or D-Phe in the
sequences selected for study) entails a preference for the type I or
II b-turn in the absence of intermolecular interactions,5,10 as seen
below. The two b-turn types differ mainly in the orientation of the
central peptide bond (that formed by the Pro-C¢O and Phe-NH
moieties), which is rotated by nearly 180◦.5,9
Enantiomerically pure L-cis(bPh)Pro bearing the amino func-
tion protected with a tert-butoxycarbonyl (Boc) group was coupled
with L- or D-phenylalanine-N¢-methylamide (H-L-Phe-NHMe or
H-D-Phe-NHMe) through activation with BOP11 [(benzotriazol-
1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate].
The resulting dipeptides 1a and 2a were treated with a solution
of hydrogen chloride in ethyl acetate to remove the Boc group
and subsequently acylated with pivaloyl chloride to afford, respec-
tively, Piv-L-cis(bPh)Pro-L-Phe-NHMe (1) and Piv-L-cis(bPh)Pro-
D-Phe-NHMe (2) (Piv = pivaloyl, tBuCO) (Fig. 2). Following an
identical procedure, enantiomerically pure L-trans(bPh)Pro was
transformed into Piv-L-trans(bPh)Pro-L-Phe-NHMe (3) and Piv-
L-trans(bPh)Pro-D-Phe-NHMe (4) (Fig. 2) through the N-Boc-
protected intermediates 3a and 4a.
Compounds 1 and 3 are characterized by a homochiral sequence
(L-L) and their behaviour is to be compared with that of the
analogous dipeptide containing the natural amino acid, Piv-L-
Pro-L-Phe-NHMe (5). In a similar way, Piv-L-Pro-D-Phe-NHMe
(6) serves as model for the heterochiral (L-D) dipeptides 2 and
4. The conformational propensities of the two L-Pro-containing
peptides have previously been established in detail, both in the
solid state10,12 and in solution.10,13
The four dipeptides 1–4 and the N-Boc precursor 4a provided
single crystals suitable for X-ray diffraction analysis.‡ Their
molecular structures are shown in Fig. 3–7. Two independent
molecules (A, B) were found in the asymmetric unit of 3, 4 and
4a. All the compounds crystallised adopt a b-turn stabilised by an
intramolecular hydrogenbondthatlinks theterminalmethylamide
NH and pivaloyl or Boc C¢O group and closes a ten-membered
cycle. The distances and angles for this intramolecular hydrogen-
Results and discussion
To explore the ability of (bPh)Pro to induce a b-turn conformation,
both the cis and trans stereoisomers of this amino acid were
inserted in the i+1 position of two peptide sequences differing
in the chirality of the i+2 residue, namely RCO-L-Pro*-L-Phe-
NHR¢ and RCO-L-Pro*-D-Phe-NHR¢, where Pro* stands for cis
and trans (bPh)Pro as well as for the natural amino acid, L-Pro. In
all cases, the (bPh)Pro residues exhibiting an S stereochemistry at
the a carbon (equivalent to an L configuration) were considered,
that is, (2S,3S)(bPh)Pro for the cis derivative and (2S,3R)(bPh)Pro
for the trans isomer (note that the a and b carbons correspond
to positions 2 and 3 of the pyrrolidine ring, respectively). To
help comparison with the natural amino acid, these residues
will be termed L-cis(bPh)Pro and L-trans(bPh)Pro, respectively,
all through the text.
The terminally blocked dipeptides RCO-L-Pro*-L-Phe-NHR¢
and RCO-L-Pro*-D-Phe-NHR¢ are adequate models to study the
formation of b-turns of types I and II. As stated in the Introduction
section, the f dihedral of proline (defining rotation about the N–
Ca bond) is confined to around -60◦. The y torsion a◦ngle (Ca–C¢
torsion) has been shown to prefer values around -30 or 140◦.3,4
˚
bond interaction are within the range 2.86–2.95 A (N ◊ ◊ ◊ O) and
151–161◦ (N–H ◊ ◊ ◊ O).14 Table 1 lists the main backbone torsion
angles. Those corresponding to the pyrrolidine ring and the phenyl
substituent in (bPh)Pro as well as to the benzyl side chain of L/D-
Phe are given in Table 2. A complete list of geometrical parameters
is provided as ESI.‡ The data in Tables 1 and 2 show that the
Fig. 2 Structure of dipeptides 1–4 that incorporate the cis or trans stereoisomer of L-(bPh)Pro in position i+1. Abbreviations: Boc = tert-butoxycarbonyl;
Piv = pivaloyl (tert-butylcarbonyl).
This journal is
The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 640–651 | 641
©

View Article Online
Fig. 3 Crystal molecular structure of Piv-L-cis(bPh)Pro-L-Phe-NHMe
(1). Only the hydrogens linked to nitrogen or chiral carbon atoms are
shown. The intramolecular hydrogen bond is indicated by a dashed line.
Fig. 5 Crystal molecular structure of Piv-L-trans(bPh)Pro-L-Phe-NHMe
(3) (two independent molecules: A, B). Only the hydrogens linked to
nitrogen or chiral carbon atoms are shown. The intramolecular hydrogen
bond is indicated by a dashed line.
All the amide bonds are found14 in the usual trans conformation
(w ª 180◦), including the urethane in 4a, with only slight deviations
from planarity except for that involving the pivaloyl group in 2 (w =
171◦). Not surprisingly, the torsion angles in Table 1 correspond
in all cases to a b-turn of type II, as previously observed in
the solid state for the reference compounds 5 and 6.10,12 In this
disposition, the (bPh)Pro Ca–H and Phe N–H bonds are parallel
and point in the same direction (Fig. 3–7). All the (bPh)Pro
Fig. 4 Crystal molecular structure of Piv-L-cis(bPh)Pro-D-Phe-NHMe
(2). Only the hydrogens linked to nitrogen or chiral carbon atoms are
shown. The intramolecular hydrogen bond is indicated by a dashed line.
two independent molecules in 3, 4 and 4a exhibit quite similar
backbone conformations while differing in the arrangement of the
side chain moieties.
Table 1 Main backbone torsion anglesa (deg) in the X-ray diffraction structures of dipeptides 1–4 and 4a.b Those reported for the analogous compounds
containing L-Pro (5, 6) are included for comparison
Pro*c
Phe
Peptide
f
y
f
y
Piv-L-cis(bPh)Pro-L-Phe-NHMe
Piv-L-cis(bPh)Pro-D-Phe-NHMe
Piv-L-trans(bPh)Pro-L-Phe-NHMe
1
2
3
-60
-62
-53
-56
-58
-60
-56
-55
-64
-57
130
134
132
135
128
128
127
131
139
138
52
62
64
60
72
91
86
81
62
72
35
26
19
25
5
-11
4
0
23
12
mol. A
mol. B
mol. A
mol. B
mol. A
mol. B
Piv-L-trans(bPh)Pro-D-Phe-NHMe
Boc-L-trans(bPh)Pro-D-Phe-NHMe
4
4a
Piv-L-Pro-L-Phe-NHMed
Piv-L-Pro-D-Phe-NHMee
5
6
a For standard deviations, see the ESI.‡ b The asymmetric unit of 3, 4 and 4a contains two independent molecules (A, B). c Pro* refers to Pro or the
corresponding (bPh)Pro stereoisomer. d From ref. 10. e From ref. 12.
642 | Org. Biomol. Chem., 2012, 10, 640–651
This journal is
The Royal Society of Chemistry 2012
©

View Article Online
Fig. 6 Crystal molecular structure of Piv-L-trans(bPh)Pro-D-Phe-NHMe
(4) (two independent molecules: A, B). Only the hydrogens linked to
nitrogen or chiral carbon atoms are shown. The intramolecular hydrogen
bond is indicated by a dashed line.
Fig.
7
Crystal molecular structure of Boc-L-trans(bPh)Pro-D-
Phe-NHMe (4a) (two independent molecules: A, B). Only the hydrogens
linked to nitrogen or chiral carbon atoms are shown. The intramolecular
hydrogen bond is indicated by a dashed line.
residues in the crystallised molecules, whatever the cis or trans
stereochemistry, display (f,y) values near (-60,130) (Table 1),
that is, very close to those considered ideal for the i+1 position
of a bII-turn,5,9 (-60,120). This suggests that the additional b-
phenyl substituent in the pyrrolidine ring does not hamper the
orientation of the adjacent carbonyl moiety leading to a y angle
in this region of the conformational map. On the contrary, all
(bPh)Pro residues adopt y values about 10◦ inferior to those
exhibited by L-Pro in the reference compounds (y ª 140◦) and
therefore closer to those considered optimal for bII-folding. As for
the i+2 residue, significant deviations are observed with respect to
the (f,y) values of (80,0) in an ideal bII-turn, and the distortion
is more pronounced for L-Phe than for D-Phe (Table 1). The latter
has been shown to be a general trend when comparing the crystal
molecular structures of bII-folded L-Pro-L-Xaa and L-Pro-D-Xaa
dipeptides.10 Indeed, the L-Phe residue in 1 and 3 (as in 5) is forced
to assume positive f values when occupying the i+2 position of a
bII-turn, that is, to behave as a D residue.
Table 2 Side-chain torsion anglesa (deg) in the X-ray diffraction structures of dipeptides 1–4 and 4ab
(bPh)Pro–pyrrolidinec
(bPh)Pro–phenyld
Phe
1
2
3
4
1¢
2¢
1
2
Peptide
q
c
c
c
c
Puckering
c
c
c
c
Piv-L-cis(bPh)Pro-L-Phe-NHMe
Piv-L-cis(bPh)Pro-D-Phe-NHMe
Piv-L-trans(bPh)Pro-L-Phe-NHMe
1
2
3
-9
30 -40
31 -39
34 -16 Cg -endo/Cb-exo 157
31 -12 Cg -endo/Cb-exo 160
70/-111
78/-101
73/-109
31/-152
95/-81
-77
61
-90
-74
72
61
57
-6/174
-12
8
56/-126
94/-88
mol. A
mol. B
mol. A
mol. B
-31
-31
-34
43
43
44
-38
-37
-37
18 Cg -exo/Cb-endo -158
18 Cg -exo/Cb-endo -157
16 Cg -exo/Cb-endo -164
9
11
0
8
2
90/-94
Piv-L-trans(bPh)Pro-D-Phe-NHMe
4
73/-109
100/-82
26/-156
33/-154
19 -32
31 -19 Cg -endo
-107
82/-99
Boc-L-trans(bPh)Pro-D-Phe-NHMe 4a mol. A
-28
-23
39
35
-33
-33
16 Cg -exo/Cb-endo -156
24/-158
113/-65
mol. B
19 Cg -exoe
-151
71
1
a For standard deviations, see the ESI.‡ b The asymmetric unit of 3, 4 and 4a contains two independent molecules (A, B). c q (Cd–N–Ca–Cb dihedral), c ,
2
3
4
1¢
2¢
c , c , and c correspond to the pyrrolidine ring in (bPh)Pro. d
c
and c correspond to the phenyl side chain in (bPh)Pro. e Cb deviates slightly from the
plane leading to some Cb-endo character.
This journal is
The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 640–651 | 643
©

View Article Online
Table 3 Amide NH and C¢O stretching frequenciesa (cm-1) for Piv-L-Pro*-L/D-Phe-NHMeb in CH2Cl2 solution (c = 5 mM)
Comp.
Peptide sequence
NH(Me)
Phe-NH
Piv-C¢O
Pro*-C¢Ob
Phe-C¢O
1
3
5
L-cis(bPh)Pro-L-Phe
L-trans(bPh)Pro-L-Phe
L-Pro-L-Phe
3450w/3359s
3448w/3358s
3448w/3356s
3436m/3425m/3416s
3431m/3412s
1620vw/1615s/1604w
1620vw/1614s/1603w
1620vw/1612s/1604vw
1691
1667
1666
1666
1692w/1686s
1684
3432m/3414s
2
4
6
L-cis(bPh)Pro-D-Phe
L-trans(bPh)Pro-D-Phe
L-Pro-D-Phe
3354
3417
3414
3420
1614w/1602s
1614w/1601s
1614w/1602s
1689
1691
1691
1665
1665
1665
3349
3449vw/3345s
a When the same vibrator gives rise to several absorption bands, their relative intensity is indicated as strong (s), medium (m), weak (w) or very weak
(vw). b Pro* refers to Pro or the corresponding (bPh)Pro stereoisomer.
Table 4 Chemical shift and solvent sensitivity (ppm) of the amide NH
proton resonances for Piv-L-Pro*-L/D-Phe-NHMea (c = 10 mM)
The L-Pro-L-Xaa and L-Pro-D-Xaa dipeptide sequences are
known to prefer b-turns of type I and II, respectively, in
environments where intermolecular interactions do not play a
significant role.5,10 In the bI-turn, the i+2 L-Xaa residue adopts
values near (-90,0), more favourable to its L stereochemistry than
those corresponding to the bII-turn, (80,0). However, in a bI-
folded L-Pro-L-Xaa dipeptide, the L-Xaa N–H bond is oriented
towards the side chains of the two adjacent L-residues and is
inaccessible to intermolecular hydrogen bonding,10,13a whereas in
the bII form this N–H bond points in the opposite direction (as
in Fig. 3–7) and can interact with the surrounding molecules.
This is the reason why most L-Pro-L-Xaa sequences experience
a bI-to-bII transition on going from low-polarity solvents to
strongly solvating media10,13 or the solid state.5,10,13 In comparison,
L-Pro-D-Xaa dipeptides are more propitious to bII-folding in all
environments.5,10,13 The observation of a bII-turn in the solid state
for all the dipeptides crystallised in this work (1–4, 4a), irrespective
of their homochiral or heterochiral sequence, follows this trend.
As expected, in all cases, the NH of the L/D-Phe residue is engaged
in an intermolecular hydrogen bond with the carbonyl group of
a neighbouring molecule. The acceptor site is (bPh)Pro-C¢O in
compounds 3 and 4, and Phe-C¢O in 1, 2, and 4a.14
In solution, the conformation adopted by compounds 1–4 was
investigated by FT-IR and 1H-NMR spectroscopies. Studies were
performed in dichloromethane and deuterochloroform, respec-
tively, on 5–10 mM peptide solutions after having confirmed that
no aggregation occurred at these concentrations. For comparative
purposes, new spectra were also registered for the reference
compounds 5 and 6 under identical conditions to those used for the
(bPh)Pro derivatives. The pivaloyl group linked to the pyrrolidine
nitrogen presents a two-fold advantage for solution-phase studies:
it prevents the cis state of the acyl-proline bond for steric reasons
and shifts the Piv-C¢O frequency out of the IR region typical for
peptide carbonyls, thus enabling easier interpretation of spectra.
The IR and NMR studies confirmed that compounds 1–4
mainly or exclusively adopt a b-turn conformation in chlorinated
solvents, as previously observed for the L-Pro counterparts 5
and 6.10,13 Thus, the terminal NH(Me) and Piv-C¢O groups are
hydrogen-bonded, as evidenced by their low IR stretching fre-
quencies in comparison with those expected for free groups (about
3450 and 1620 cm-1, respectively),10,13 and this interaction must be
intramolecular because intermolecular contacts are discarded at
the concentration used. In all cases, the NH(Me) site gives rise
to an intense IR band around 3350 cm-1 (Table 3) and its proton
resonance is only moderately affected by the addition of DMSO-
d6 (Table 4), suggesting that it is engaged in a hydrogen bond.
NH(Me)
Phe-NH
b
b
Comp. Peptide sequence
CDCl3
Dd
CDCl3
Dd
1
3
5
L-cis(bPh)Pro-L-Phe
L-trans(bPh)Pro-L-Phe
L-Pro-L-Phe
5.66
6.49
6.68
1.08
1.08
0.99
5.55
6.12
5.87
1.92
1.65
1.74
2
4
6
L-cis(bPh)Pro-D-Phe
L-trans(bPh)Pro-D-Phe
L-Pro-D-Phe
7.11
7.56
7.33
0.55
0.44
0.56
5.17
5.42
5.93
2.85
3.02
2.33
a Pro* refers to Pro or the corresponding (bPh)Pro stereoisomer. b Shift of
the NH proton resonance on going from CDCl3 to DMSO-d6 solution.
In comparison, the Phe-NH stretching absorption appears above
3400 cm-1 (Table 3) and its proton chemical shift shows a higher
sensitivity to DMSO-d6 solvation (Table 4), as expected for a free
NH amide moiety.
In spite of this common behaviour small, yet significant,
differences are observed between the homochiral and heterochiral
sequences. Thus, the L-L dipeptides 1, 3 and 5 present a weak
absorption near 3450 cm-1 (Table 3) that corresponds to a free
NH(Me) site and denotes the presence of a small percentage of
molecules exhibiting an open conformation devoid of intramolec-
ular hydrogen bonds. This band also appears in the L-Pro-D-Phe
derivative (6), although with a lower intensity, and cannot be
distinguished in the IR spectra of 2 and 4 (Table 3). This suggests
that the higher b-turn forming tendency exhibited by the L-Pro-
D-Phe sequence in comparison to L-Pro-L-Phe10,13 (in general, L-
Pro-D-Xaa vs. L-Pro-L-Xaa dipeptides10) is not only maintained
but even increased when the natural Pro residue is replaced by
(bPh)Pro independently of the trans or cis stereochemistry of the
b-phenyl substituent. The existence of a superior percentage of
open conformers in the homochiral peptides is in agreement with
the larger variation induced by DMSO-d6 on their NH(Me) amide
proton resonance (Dd ª 1.0 and 0.5 ppm for the L-L and L-D
dipeptides, respectively; Table 4).
However, the main difference observed in solution does not
concern the extent to which the homochiral and heterochiral
dipeptides under study adopt a b-turn conformation but the
type of b-turn accommodated, which can be unambiguously
distinguished by the IR frequency of the Piv-C¢O group.13 The
three L-D dipeptides (2, 4, 6) present an identical profile in this
region of the IR spectrum, with a major contribution at 1602
cm-1 and a weak absorption near 1612 cm-1 (Table 3), which have
644 | Org. Biomol. Chem., 2012, 10, 640–651
This journal is
The Royal Society of Chemistry 2012
©

View Article Online
been shown to reflect two different hydrogen-bonded states of the
Piv-C¢O group in Piv-L-Pro-Xaa-NHR dipeptides, namely those
corresponding to a bII- and a bI-turn, respectively.13 Thus, the
bII-turn observed in the solid state for 2 and 4 is also preferred
in solution, indicating that the incorporation of a b-phenyl group
into L-Pro does not alter the behaviour typically observed for L-
Pro-D-Xaa sequences.
In comparison, the L-Pro-L-Phe derivative (5) shows two very
weak contributions at about 1620 and 1602 cm-1, corresponding,
respectively, to a free and a bII-hydrogen-bonded Piv-C¢O group,
and an intense absorption at 1612 cm-1 (Table 3). This indicates
the predominance of the bI-turn for 5 in chlorinated solvents,
as already established.10,13 The band near 1602 cm-1 becomes
slightly more intense in the analogous sequence incorporating L-
cis(bPh)Pro (1) and even more in the L-trans(bPh)Pro derivative
(3) (Table 3), which shows the highest percentage of bII-folded
molecules among the three homochiral dipeptides investigated.
Yet, the bI-turn remains the preferred conformation for all three
compounds in chlorinated solvents.
60◦ value (in molecule B of 4, it is precluded by the arrangement of
the pyrrolidine moiety). The optimisation of such intramolecular
aromatic–aromatic interactions also seems to be the basis of the
2
uncommon c values observed for the L/D-Phe residue in some of
2
the crystallised molecules (Table 2). The c angle (Cb–Cg torsion)
reflects the orientation of the phenyl plane with respect to the
peptide backbone and is known to prefer values near 90/-90◦,
for which steric repulsions with the main chain are minimal, with
deviations within 30◦ being frequent.15 One notes, however, that
2
some of the c values assumed by the L/D-Phe residue in the
(bPh)Pro-containing dipeptides in Table 2 are severely dist◦orted
2
from this optimal position, as is the case of 4a (c = 26/-156 and
33/-154◦ in molecules A and B, respectively) and, particularly,
◦
2
2
of 1 (c = -6/174 ). In comparison, the corresponding c values
in the reference compounds 5 and 6 are -68/110◦ and 63/-122◦,
respectively. In fact, the variety of orientations adopted by the
2
phenyl plane of the L/D-Phe residue in 1–4 and 4a, reflected in c
(Table 2), seem to be the consequence of the interaction established
with the b-phenyl substituent in (bPh)Pro.
In the discussion above, the conformation adopted by the
peptide backbone in the (bPh)Pro derivatives under study has
been considered both in solution and in the solid state. The phenyl
substituent attached to the pyrrolidine moiety has been shown not
to perturb the conformational properties of L-Pro and, indeed, the
b-turn forming propensities of (bPh)Pro resemble closely those of
the parent amino acid. However, the arrangement of the side-
chain moieties in the compounds investigated and their possible
influence on the subtle conformational changes observed upon
replacement of L-Pro by cis or trans L-(bPh)Pro have not been
considered yet. They are discussed in the following.
The intramolecular aromatic–aromatic interactions observed in
the solid state for all the (bPh)Pro derivatives crystallised (with the
exception of one of the independent molecules in 4) seem to be
retained in solution only for the sequences incorporating D-Phe, as
inferred from the following observations. A striking feature in the
1H-NMR spectra of compounds 2 and 4 in CDCl3, which is also
present for the Boc precursors 2a and 4a,14 is the appearance of
an aromatic proton near 6.70 ppm. This signal has been assigned
to the ortho hydrogens of the D-Phe residue. In comparison, all
the aromatic protons in the parent D-Phe derivative 6, that lacks
the phenyl ring in the i+1 position, appear above 7.10 ppm, as
do those in the three L-Phe-containing peptides (1, 3, 5). The
strongly upfield shifted aromatic proton resonance of 2 and 4
can be attributed to the proximity of the phenyl group of the
contiguous (bPh)Pro residue. The relative orientation of the two
aromatic rings is to be perpendicular, with an ortho D-Phe hydrogen
pointing to the centre of the phenyl unit in (bPh)Pro.
In the crystal, the side chain of the L-Phe residue in dipept◦ides
1
1 and 3 (Table 2) exhibits a gauche(-) orientation (c = -74 to
-90◦) while tha◦t of D-Phe in 2, 4 and 4a corresponds to gauche(+)
◦
1
(c = 57 to 72 ), as respec◦tively found for the parent peptides 5
◦
1
1
(c = -42 )10 and 6 (c = 74 ).12 It should be noted that gauche(-)
1
is a sterically favoured c disposition (rotational state of the
Ca–Cb bond) for the L-Phe side chain and is, indeed, the one most
Such a relative disposition between the two aromatic rings in
2 and 4 differs from that observed in the solid state (Fig. 4 and
6). Molecular models show that it can be achieved if the aromatic
moiety of (bPh)Pro is roughly kept as in their respective crystalline
structures (in the case of 4, that in molecule A) while the Ca–Cb
commonly encountered in crystallised peptides and proteins.15
A
similar situation corresponds to the gauche(+) conformation for
D-Phe.
1
The most remarkable feature in Table 2 regarding c for the
bond of D-Phe is rotated by about 120◦ (from c ª 60◦ in the
1
i+2 residue (L/D-Phe) is the significantly more negative values
attained by this dihedral angle when L-Phe is attached to L-
(bPh)Pro, either cis (1) or trans (3), in relation to L-Pro (5).
crystals to ª -60◦) and the phenyl plane is allowed to assume the
◦
2
usual15 c values ª 90/-90 ( 30). Fig. 8 illustrates the relative
arrangement of the two aromatic rings when the orientation of the
D-Phe side chain in the crystalline structure of 4 (molecule A) is
changed in this way.
1
Actually, the c angle of L-Phe deviates from the standard value
corresponding to a canonical gauche(-) orientation (-60◦) in
opposite directions in 1 and 3 with reference to 5. In the latter
◦
1
compound, c = -42 makes the phenyl ring of L-Phe approach
the contiguous NH group probably to allow a weak attractive
NH ◊ ◊ ◊ p interaction. In the (bPh)Pro derivatives 1 and 3, th◦e
The modification proposed above for the orientation of the D-
Phe side chain in 2 and 4 with respect to that observed in the
X-ray diffraction structures is by no means surprising. Actually, it
has been proposed to occur for the parent compound containing
D-Phe (6) on going from the solid state to CDCl3 solution.10
As previously established and commented above, dipeptide 6
shows a preference for the same bII-turn conformation in both
environments. However, the aromatic side chain changes from the
1
1
L-Phe c moves in the opposite direction (c ranges from -74
to -90◦) and this distinct orientation may be attributed to the
interaction established between the two aromatic rings in the
(bPh)Pro-containing compounds, which are seen to adopt a
parallel arrangement in the cis derivative (1, Fig. 3) and to be
almost perpendicular in both independent molecules of the trans
compound (3, Fig. 5). When D-Phe is present (2, 4, 4a), the
adequate geometry for the two phenyl rings to interact is attained
◦
1
gauche(+) disposition in the crystal12 (c = 74 ) to a predominant
◦
gauche(-) arrangement (c ª -60 ) in CDCl3 or CH2Cl2.10 The
1
latter places the phenyl ring in the least sterically favoured among
1
1
without the need for a significant deviation of c from the standard
the three staggered positions available to c , namely flanked by
This journal is
The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 640–651 | 645
©

View Article Online
have been recognized as an important stabilizing factor in the
conformation of peptides and proteins.18
The situation is different for the three L-Phe-containing peptides
(1, 3, 5). There is no evidence that the aromatic contacts observed
in the X-ray diffraction structures of 1 and 3 (Fig. 3 and 5) are
kept in solution. The orientation of the benzyl side chain of L-Phe
1
in 5◦has been shown to change from gauche(-) in the cr◦ystal10 (c =
1
-42 ) to a preferential gauche(+) arrangement (c ª 60 ) in CDCl3
solution,10 thereby bringing the NH and phenyl moieties of L-Phe
into close proximity, in an analogous way to that described above
for 6 [note that gauche(+) for L-Phe is equivalent to gauche(-) for D-
Phe]. The same modification seems to occur for 1 and 3, as reflected
by the L-Phe-NH stretching frequency that denotes the existence
of a similar NH ◊ ◊ ◊ p interaction in the three homochiral peptides
(Table 3). However, it should be noted that in the L-L sequences,
this side chain rotation is associated with the bII-to-bI transition
experienced by the peptide backbone on going from the solid
to the solute state and the combination of both changes makes
the phenyl ring of L-Phe move far away from the (bPh)Pro side
chain, thus precluding the existence of intramolecular aromatic–
aromatic interactions. Not surprisingly, the population of L-Phe
gauche(+) species estimated10,16 for 5 in chloroform (54%)17 is not
increased in 1 (56%) and 3 (46%). This fact, together with the
observation of a similar percentage of non-b-turn-folded species
in solution for the three homochiral dipeptides, suggests that no
additional stabilising interaction operates in 1 and 3 with respect to
5. Obviously, this applies to the major conformer in solution, but
does not discard the existence of minor species with a bII-folded
backbone in which the L-Phe side chain retains the gauche(-)
arrangement and interacts with the (bPh)Pro aromatic substituent
as in the crystal structures.
The postulated interaction between the two aromatic rings
in the D-Phe derivatives 2 and 4 in solution also requires an
adequate orientation of the phenyl group in the contiguous
(bPh)Pro residue, which should be similar in both compounds
and is proposed not to differ much from that observed in the X-
ray structures (molecule A for 4). Such an orientation is given by
the cis/trans stereochemistry at the b carbon and the puckering of
the pyrrolidine moiety, as discussed below.
The five-membered ring in all (bPh)Pro derivatives crystallised
(Table 2) assumes a puckered conformation, with the g carbon
deviating from the plane towards the same (Cg -endo or down) or
the opposite (Cg -exo or up) side of the ring to where the carbonyl
group lies.19 In most of the cases, also the b carbon protrudes from
the plane, in the opposite direction to Cg , so that the conformation
is twist (half-chair) instead of envelope-like. The twisted character
is highly marked, as indicated by the magnitude of the q angle
(Cd–N–Ca–Cb dihedral) (Table 2), which gives the extent to which
Cb deviates from the plane defined ◦by the atoms involved in the
peptide bond (Cd, N, Ca) and is 0 for an ideal envelope with
Cg at the flap. The frequent occurrence of twist conformations
in Table 2 and the strong out-of-plane displacement observed for
Cb in them are certainly related to the b-substituted character
of (bPh)Pro. Deviation of Cb from planarity alleviates the steric
hindrance introduced by the bulky aromatic ring attached to it,
which is particularly severe in the cis derivative.
Fig. 8 Molecular model of 4 showing an ortho aromatic proton of D-Phe
pointing to the centre of the phenyl ring in L-trans(bPh)Pro. It has been
built from the X-ray diffraction structure of 4 (molecule A) by changing
the D-Phe c torsion from 72◦ to ª -60◦ and keeping c in the sterically
1
2
favourable region ª 90/-90◦ ( 30◦).
the NH and CO substituents, but provides the optimal geometry
for an attractive interaction with the adjacent NH moiety. The
existence of this NH ◊ ◊ ◊ p interaction is reflected10,13 in the lower
IR frequency observed for this NH in 6 (3420 cm-1; Table 3)
with respect to the analogous compound incorporating D-Ala in
place of D-Phe, in which no aromatic-NH interaction can occur
(3430 cm-1).10 In the crystal, the D-Phe NH moiety is involved
in a hydrogen bond with a carbonyl group of a neighbouring
molecule12 and the benzyl side chain adopts the sterically more
favourable gauche(+) arrangement.
It is highly probable that the D-Phe side chain in 2 and 4
experiences a similar rotation about the Ca–Cb bond on going
from the solid state to chlorinated solvents. In fact, the stretching
frequency of the D-Phe-NH site in these compounds also indicates
the existence of an NH ◊ ◊ ◊ p interaction (3417 cm-1 in 2 and 3414
cm-1 in 4; Table 3) and, therefore, of a high population of the D-Phe
1
c conformer providing the optimal geometry for it, gauche(-).
As shown in Fig. 8 for 4, this arrangement of the D-Phe side
chain places an ortho hydrogen of this residue pointing to the
centre of the phenyl ring in (bPh)Pro, thereby accounting for
1
the upfield aromatic signal observed in the H-NMR spectra of
2 and 4. The population of the gauche(-) D-Phe rotamer in 6
was estimated10,16 to be 52%17 from the vicinal coupling constants
between the a and two b protons measured for this residue in
CDCl3 solution. Notably enough, the same method16 provides
1
an increased percentage of this c conformer for both 2 (74%)
and 4 (66%). The stabilizing interaction established between the
two aromatic rings in the latter compounds could contribute
to increase the population of gauche(-) conformer for D-Phe in
spite of being the most sterically hindered staggered arrangement
1
available to c , thus operating in the same sense as does the
NH ◊ ◊ ◊ p interaction. The disappearance of the small amount
of open conformers detected for 6 upon replacement of L-Pro
by cis or trans L-(bPh)Pro (to yield 2 and 4, respectively) could
also be related to this additional stabilising interaction. Actually,
interactions between the side chain groups of aromatic residues
In the crystalline state, the two compounds incorporating L-
cis(bPh)Pro (1, 2) exhibit the same Cg -endo/Cb-exo puckering,
1
characterized by positive c and negative q values (Table 2; note
646 | Org. Biomol. Chem., 2012, 10, 640–651
This journal is
The Royal Society of Chemistry 2012
©

View Article Online
n
Table 5 Comparison of the vicinal coupling constants (Hz) of the b
hydrogen of L-cis(bPh)Pro measured in 1 and 2 with those estimated for
different arrangements of the pyrrolidine ring
that the c values given for (bPh)Pro correspond to the pyrrolidine
moiety while those describing the orientation of the phenyl side
1¢
2¢
chain in this residue are termed c and c ). This arrangement
moves the b-phenyl ring away from the vicinal carbonyl substituent
(Fig. 9a), which would otherwise be eclipsed. Thus, the C¢–Ca–
Cb–Cipso torsion angle, l [C¢ is the carbonyl carbon and Cipso is
the substituted aromatic carbon in (bPh)Pro], is as much as 40◦
in 1 and 43◦ in 2.14 In this Cg -endo/Cb-exo half-chair, the phenyl
substituent occupies an equatorial position, which is known to
minimize steric repulsions with the five-membered ring. The other
possible twist arrangement (Cg -exo/Cb-endo) (Fig. 9b) does not
seem to introduce any advantage because the phenyl ring would
separate from the contiguous carbonyl substituent in a similar
extent (although in the opposite direction, l ª -40◦) but would
be oriented axially, thus producing severe steric hindrance with
some pyrrolidine protons. The latter disposition should then be
strongly disfavoured. The five-membered ring in L-cis(bPh)Pro
is not likely to adopt a Cg -envelope conformation either, since
keeping Cb in the plane would make the b-phenyl and a-carbonyl
substituents lie closer to the eclipsed disposition associated with
their cis stereochemistry.
Calculatedb
Observedc
Cg -endo/
Cg -exo/
Cb-endod
3JH–H
Cb-exod,e
1
2
a
Ha–Hb
Hb–Hg
Hb–Hg
7.5
12.5
5.7
6.4
1.5
4.4
8.6
8.3
11.9
6.1
13.9
6.0
endo
exo
a The pyrrolidine g hydrogens are labelled as endo or exo as indicated in note
20. b Using the Haasnoot–Altona equation (ref. 21,22). c CDCl3 solution.
d Half-chair (twist) conformation. e Estimated from the geometry observed
in the crystalline structure of 1 (similar results are obtained from that of
2).
Fig. 9 Newman projection through the Ca–Cb bond for the two possible
half-chair conformations of the pyrrolidine ring in L-cis(bPh)Pro (a,b) and
L-trans(bPh)Pro (c,d). Those observed in the X-ray structures of the cis (1,
2) and trans peptides (3, 4, 4a) are marked. The equatorial (eq) or axial
(ax) orientation of the phenyl substituent is indicated.
The Cg -endo/Cb-exo arrangement observed in the solid state
seems therefore to be the most favourable puckering mode of L-
cis(bPh)Pro. There is strong evidence that this conformation also
predominates in solution. The vicinal coupling constants between
the pyrrolidine b hydrogen and those in the a (JHa–Hb ª 8.5 Hz)
and g (JHb–Hgendo ª 13 Hz, JHb–Hgexo ª 6 Hz) positions20 measured
in CDCl3 are similar for 1 and 2 (Table 5) and are in excellent
agreement with those calculated from their X-ray diffraction
geometries (Table 5). The latter have been estimated21,22 using the
modification of the Karplus equation developed by Haasnoot et
al., which has been shown to provide more reliable estimates for
the proline system.23 In contrast, a very small JHb–Hgendo value (<2
Hz) is expected for the other possible half-chair arrangement (Cg -
exo/Cb-endo) (Table 5). The largeJHb–Hgendo measured for both 1 and
2 allows us to discard the presence of a significant population of
species exhibiting the latter pyrrolidine shape. Another argument
supporting the assignment of a highly predominant Cg -endo/Cb-
exo conformation for L-cis(bPh)Pro in solution is the observation
in 1 and 2 (shown in Fig. 10 for 1) of a NOE cross-peak between
Hb and Hdexo, denoting their proximity, along with the absence of
Fig. 10 Section of the NOESY spectrum of compound 1 in CDCl3
solution (10 mM), showing the pyrrolidine Hb–Hd cross-peak. The
exo
absence of Ha–Hg correlation is also noteworthy. The hydrogen atoms
exo
labelled20 correspond to the (bPh)Pro residue.
correlation between Ha and Hg exo. The two latter protons should
become close for a g carbon deviating in the opposite direction to
the carbonyl substituent (Cg -exo), that is, towards the a hydrogen
(Fig. 9b).
The situation for the trans isomer is more complex. In
most of the crystallised molecules, the five-membered ring of
L-trans(bPh)Pro adopts a half-chair conformation (Table 2).
Displacement of the b carbon occurs in the direction that places
the phenyl substituent in an equatorial position (Cb-endo), with q
values around 10◦, and the g carbon moves towards the opposite
side (Cg -exo). However, this Cg -exo/Cb-endo arrangement (Fig. 9c)
makes the phenyl and carbonyl substituents approach (note that
they are trans here), with l angles about 80◦ being observed in the
X-ray structures.14 Accordingly, deviation of Cb from the plane
to place the aromatic ring equatorially is not as advantageous as
This journal is
The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 640–651 | 647
©

View Article Online
Table 6 Comparison of the vicinal coupling constants (Hz) of the b
hydrogen of L-trans(bPh)Pro measured in 3 and 4 with those estimated
for different arrangements of the pyrrolidine ring
seen before for the cis isomer, in which case it also provided the
maximum separation between the vicinal substituents.
This probably explains why other pyrrolidine arrangements
other than the most abundant Cg -exo/Cb-endo half-chair are
seen among the crystallised trans(bPh)Pro-containing peptides
(Table 2). Thus, molecule B of 4a retains the Cg -exo disposition
found for most trans compounds but Cb remains near the ring
plane (q = 2◦) so that the puckering is close to a Cg -exo
envelope. As a result, the phenyl substituent is pseudoequatorial
(an intermediate position between that corresponding to a planar
ring and the equatorial one in a half-chair), but this less favourable
orientation is somehow compensated ◦for by the lower proximity
to the carbonyl substituent (l = 90 ).14 It appears, therefore,
that conformations with a geometry intermediate between a Cg -
exo/Cb-endo half-chair and a Cg -exo envelope are also accessible
to L-trans(bPh)Pro, although they are expected to be less stable
than the half-chair.
Calculatedb
Observedc
Cg -exo/
Cg -endo/
Cg -exof,g Cg -endof,h Cb-exod
a
3JH–H
Cb-endod,e
3
4
Ha–Hb
Hb–Hg
Hb–Hg
11.1
5.1
12.7
9.9
6.8
11.9
2.1
9.3
0.5
0.6
5.6
1.0
5.5 9.3
ª 6.5i 6.3
ª 7.5i 11.8
endo
exo
a The pyrrolidine g hydrogens are labelled as endo or exo as indicated in note
20. b Using the Haasnoot–Altona equation (ref. 21,22). c CDCl3 solution.
d Half-chair (twist) conformation. e Estimated from the geometry observed
in the crystalline structure of mol. A of 3. f Envelope or envelope-like
conformation. g Estimated from the geometry observed in the crystalline
structure of mol. B of 4a (there is some Cb-endo character, see Table 2).
h Estimated from the geometry observed in the crystalline structure of mol.
B of 4. i Not determined with higher precision due to signal overlapping.
Additionally, a third type of pyrrolidine puckering is seen among
the X-ray structures of the trans(bPh)Pro derivatives, namely that
in molecule B of 4 (Table 2). In this case, Cb lies in◦the plane formed
by the atoms involved in the peptide bond (q = 0 ) and a pure Cg -
endo envelope is seen. This arrangement provides the maximum
distance with the carbonyl moiety (l = 134◦)14 while making the
aromatic substituent pseudoaxial.
The other possible half-chair of L-trans(bPh)Pro (Cg -endo/Cb-
exo) is not observed in any of the compounds crystallised. It would
introduce the maximum distance between the a and b substituents
(l ª 150◦, Fig. 9d) but also orientate the phenyl substituent axially
and therefore introduce severe steric repulsions. Indeed, when Cg
is endo, there seems to be no benefit in Cb also deviating from the
plane, that is, in changing from the Cg -endo envelope observed in
the molecule B of 4 to the Cg -endo/Cb-exo half-chair, since l is
slightly superior (Dl ª 15◦) but at the cost of increasing the axial
character of the aromatic substituent. Accordingly, the Cg -endo
envelope but not the Cg -endo/Cb-exo twist form is seen among the
trans compounds crystallised. Yet, the stability difference between
the two half-chairs in trans(bPh)Pro is expected to be less marked
than in the cis derivative because, in the latter, the most stable
half-chair combines an equatorial phenyl group and the maximum
possible separation between the bulky vicinal substituents whereas
this is not the case for the trans compound.
The vicinal coupling constants measured for the L-
trans(bPh)Pro Hb in CDCl3 solution for 3 and 4 are substantially
different (Table 6), indicating that the pyrrolidine puckering differs
in the two compounds. Comparison of the experimental values
with those predicted21,22 for the different geometries exhibited by
the five-membered ring in the crystalline structures of the trans
compounds (Table 6) suggests that dipeptide 4 adopts with high
preference a Cg -exo arrangement. Whether it corresponds to a Cg -
exo/Cb-endo half-chair, a Cg -exo envelope, a mixture of both or a
single conformation with an intermediate geometry is difficult to
ascertain on the basis of the J values only. However, the greater
stability expected for the half-chair form, which correlates with its
more frequent occurrence in the X-ray structures, together with
importantly, the large magnitude of JHa–Hb (9.3 Hz) and JHb–Hgexo
(11.8 Hz) measured for this compound allows us to discard the
presence of a significant ratio of Cg -endo conformers, for which
very small JHa–Hb (0.6–2.1 Hz) and JHb–Hgexo (<1 Hz) values are
expected (Table 6). Conversely, the coupling constants observed
for Hb in 3 can only be explained if a non-negligible population of
Cg -endo-puckered species is present (40–60%). In such conformers,
an ortho proton of the (pseudo)axial phenyl ring becomes close
to Hd and, accordingly, a NOE cross-peak appears.14 This fact
exo
together with the NOE correlation detected between Ha and
Hg exo, typical of the Cg -exo arrangement, is consistent with the
coexistence of Cg -endo and Cg -exo shapes in 3. Again, it is difficult
to discern from these results the extent to which they are envelope-
like or twist, that is, the degree of non-planarity at Cb. Yet, they
are most probably of the Cg -endo envelope and Cg -exo/Cb-endo
half-chair types, respectively, according to the X-ray diffraction
results and the comments thereof. It is worth noting that the higher
flexibility observed for the pyrrolidine ring in trans(bPh)Pro with
respect to the cis isomer is in agreement with the results from
computational analysis on diamide derivatives of the b-isopropyl
counterparts.24
According to the above discussion, the pyrrolidine ring in the
cis derivative 2 and the trans compound 4 adopts in solution a
conformation close to that observed in the solid state for each
compound (molecule A in the latter case). It is interesting to
note that the proposed intramolecular interaction between the
aromatic moieties in these peptides is based on an orientation
of the phenyl ring in (bPh)Pro similar to those in the crystals
(molecule A for 4). Even if the b-phenyl substituent is cis in 2
and trans in 4, their respective Cg -endo/Cb-exo and Cg -exo/Cb-
endo arrangements, observed in the solid state and believed to
be highly predominant in solution, place this group in positions
that are much closer than a priori expected from their different
1¢
stereochemistry (compare Fig. 9a and 9c). Thus, the c values
in Table 2, giving the orientation of the phenyl substituent in
(bPh)Pro with respect to the peptide backbone (N–Ca–Cb–Cipso),
are of 160◦ in 2 and -164◦ in 4 (molecule A), which means
a difference of only 36◦. When the relative orientation of the
aromatic substituent is compared taking as a reference the position
of◦the carbonyl group, a similar result is obtained: l is 43◦ in 2 and
75 in 4 (molecule A).14 Therefore, the (bPh)Pro aromatic group
the detection14 of NOE cross-peaks between Hb and Hd
(that
endo
get close for an endo Cb) and the Ha–Hg protons (denoting the
exo
exo arrangement of Cg ) point to a situation proximal to the Cg -
exo/Cb-endo half-chair, as observed in the crystalline state for
most L-trans(bPh)Pro derivatives (Table 2). In any case, and most
648 | Org. Biomol. Chem., 2012, 10, 640–651
This journal is
The Royal Society of Chemistry 2012
©

View Article Online
in 2 and 4 are not so differently oriented in solution, and this is
consistent with the existence, in both cases, of a similar interaction
with the D-Phe residue exhibiting the same gauche(-) side-chain
cis(bPh)Pro-OH and Boc-L-trans(bPh)Pro-OH were obtained by
chromatographic resolution of a racemic precursor; details will be
reported elsewhere. Thin-layer chromatography (TLC) was per-
formed on Macherey-Nagel PolygramꢀR SIL G/UV254 precoated
silica gel polyester plates. The products were visualized by exposure
to UV light or submersion in ninhydrin or cerium molybdate stain
[aqueous solution of phosphomolybdic acid (2%), CeSO4·4H2O
(1%) and H2SO4 (6%)]. Column chromatography was performed
using 60 M (0.04–0.063 mm) silica gel from Macherey-Nagel.
Melting points were determined on a Gallenkamp apparatus. IR
spectra in the solid state were recorded on a Nicolet Avatar 360
FTIR spectrophotometer; nmax is given for the main absorption
1
2
arrangement. In each case, small c /c variations in the latter
residue could allow for the optimal geometry. In turn, the fact that
this orientation of the (bPh)Pro phenyl group in 4 is necessary for
this interaction to occur could be the reason why the Cg -exo/Cb-
endo pyrrolidine conformation is overwhelmingly populated in
solution for this compound whereas the same does not hold true
for 3. In other words, the existence of an interaction with the
aromatic ring of D-Phe could shift the conformational equilibrium
of the pyrrolidine ring in 4 towards the puckering mode providing
the adequate geometry for it. In 3, the presence of L-Phe instead of
D-Phe does not allow for this interaction to occur and, accordingly,
such equilibrium is not shifted to any particular conformer.
1
bands. H- and 13C-NMR spectra were registered at room tem-
perature on a Bruker AV-500 or AV-400 instrument, respectively,
using the residual solvent signal as the internal standard; chemical
shifts (d) are expressed in ppm and coupling constants (J) in
Hertz. Peptide solutions at 10 mM concentration were used for the
1H-NMR spectra. Complete assignment of all proton resonances
was performed on the basis of COSY, NOESY (500 ms mixing
time), and HSQC experiments registered on the Bruker AV-500
instrument on 10 mM peptide solutions. High-resolution mass
spectra were recorded on a Bruker Microtof-Q spectrometer.
Conclusions
The structural propensities of homochiral and heterochiral dipep-
tide sequences that incorporate in the i+1 position the L-Pro ana-
logues L-cis(bPh)Pro and L-trans(bPh)Pro have been determined
in solution and the solid state and compared to those induced by
the natural amino acid. The phenyl substituent attached to the
pyrrolidine b carbon has been shown to be fully compatible with
the conformational properties of L-Pro and, thus, (bPh)Pro essen-
tially retains the b-turn forming propensities of the parent amino
acid. In the solid state, all the (bPh)Pro derivatives crystallised
adopt a bII-turn, as do the analogous compounds containing L-
Pro. In solution, L-trans(bPh)Pro induces a higher b-turn folding
ratio while the preference for the bI or bII folding modes parallels
that of the natural sequences, with only subtle differences.
General procedure for the synthesis of peptides 1–4
Boc-L-cis(bPh)Pro-OH or Boc-L-trans(bPh)Pro-OH (500
mg, 1.72 mmol) was suspended in acetonitrile (20 mL) and
BOP [(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate] (912 mg, 2.06 mmol) was added. After
10 min, N,N-diisopropylethylamine (0.66 mL, 3.78 mmol) was
added followed by L- or D-Phe-NHMe hydrochloride (552 mg,
2.58 mmol) and the stirring was continued for 4 days. The solvent
was evaporated and the remaining residue was redissolved in
dichloromethane (30 mL). The solution was successively washed
with 5% aqueous potassium bisulfate (15 mL), 5% aqueous
sodium bicarbonate (15 mL), and brine (15 mL). The organic
layer was dried over anhydrous MgSO4 and filtered. Evaporation
of the solvent followed by column chromatography (eluent:
ethyl acetate–dichloromethane–hexanes 6 : 3 : 1) furnished the
corresponding N-Boc protected dipeptide 1a–4a (644–690 mg,
1.43–1.53 mmol, 83–89% yield; full characterization is given in
the ESI‡). A 3N solution of hydrogen chloride in ethyl acetate
(5 mL) was then added to 1a–4a (500 mg, 1.11 mmol) and the
suspension formed was stirred at room temperature for 2 h. After
evaporation of the solvent, the residue was taken up in water
and lyophilized. The resulting solid was suspended in chloroform
(10 mL) and N-methylmorpholine (0.27 mL, 2.44 mmol) and
pivaloyl chloride (0.16 mL, 1.33 mmol) were added. The reaction
mixture was stirred at room temperature for 24 h. Evaporation
of the solvent followed by column chromatography (eluent:
hexanes–ethyl acetate 1 : 9) provided the desired dipeptide 1–4 in
the yield indicated in each case.
The pyrrolidine conformation is significantly affected by the
presence of the b-phenyl group. The puckering modes that alleviate
most the steric hindrance introduced by this substituent are
preferred. The cis(bPh)Pro residue shows a marked propensity
for the Cg -endo/Cb-exo arrangement both in the solid state
and in solution whereas the trans compound exhibits a higher
flexibility, with different pyrrolidine shapes being accessible. As
1
a consequence, the phenyl group in cis(bPh)Pro is fixed at c
near 160◦, while in trans(bPh)Pro it may explore the -160◦
(for a Cg -exo/Cb-endo pyrrolidine half-chair) or -110◦ (Cg -endo
envelope) regions. This issue is essential when a particular spatial
arrangement is required for interaction with the receptor binding
pocket if (bPh)Pro is inserted into a biologically active peptide or
for other applications requiring well-oriented substituents.
Interactions between the aromatic ring of (bPh)Pro and the
contiguous L- or D-Phe residue are observed in all the dipeptides
crystallised. In solution, they seem to occur only for the heterochi-
ral sequences. This intramolecular aromatic–aromatic interaction
may be responsible for the higher b-turn ratio observed for such
sequences and may also affect the conformational preferences of
the pyrrolidine ring.
Piv-L-cis(bPh)Pro-L-Phe-NHMe (1). (459 mg, 1.05 mmol,
◦
95% yield); mp 199 C; [a]2D0 = +64.1 (c = 0.40, MeOH); IR
Experimental section
1
(nujol) nmax/cm-1 3341, 3306, 1667, 1652, 1604; H-NMR (500
MHz, CDCl3, c = 10 mM) dH 1.20 (9H, s, Piv), 2.21 (1H, m, Pro-
Hg exo), 2.55 (3H, d, J = 4.5 Hz, NHMe) overlapped with 2.47–2.67
(1H, m, Pro-Hg endo), 2.79 (1H, dd, J = 13.8, 6.3 Hz, Phe-Hb), 3.19
(1H, dd, J = 13.8, 5.4 Hz, Phe-Hb), 3.53 (1H, ddd, J = 11.9, 8.6,
General
All reagents were used as received from commercial suppli-
ers without further purification. Enantiomerically pure Boc-L-
This journal is
The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 640–651 | 649
©

View Article Online
6.1 Hz, Pro-Hb), 3.75 (1H, m, Pro-Hdexo), 4.05 (m, 1H, Pro-Hdendo),
4.35 (1H, ddd, J = 8.4, 6.3, 5.4 Hz, Phe-Ha), 4.60 (1H, d, J = 8.6
Hz, Pro-Ha), 5.55 (1H, br d, J = 8.4 Hz, Phe-NH), 5.66 (1H, m,
NHMe), 7.10–7.19 (4H, m, Ar), 7.21–7.34 (6H, m, Ar); 13C-NMR
(100 MHz, CDCl3) dC 26.23 (NHMe), 27.14 (Piv-Me3), 29.91 (Pro-
Cg ), 36.84 (Phe-Cb), 39.15 (Piv-C), 44.87 (Pro-Cb), 48.13 (Pro-Cd),
53.35 (Phe-Ca), 67.51 (Pro-Ca), 126.93 (Ar), 127.40 (Ar), 127.92
(Ar), 128.47 (Ar), 128.64 (Ar), 129.48 (Ar), 136.76 (Ar), 136.83
(Ar), 169.37 (CO), 170.65 (CO), 177.95 (Piv-CO); HRMS (ESI)
C26H33N3NaO3 [M+Na]+: calcd. 458.2414, found 458.2437.
Cg ), 36.08 (Phe-Cb), 38.75 (Piv-C), 46.78 (Pro-Cb), 49.15 (Pro-Cd),
52.77 (Phe-Ca), 71.13 (Pro-Ca), 126.79 (Ar), 127.26 (Ar), 127.57
(Ar), 128.56 (Ar), 128.95 (Ar), 129.29 (Ar), 135.95 (Ar), 139.15
(Ar), 170.94 (CO), 171.31 (CO), 177.65 (Piv-CO); HRMS (ESI)
C26H33N3NaO3 [M+Na]+: calcd. 458.2414, found 458.2396.
IR spectroscopy in solution
IR spectra were recorded in a Nicolet Avatar 360 FTIR spec-
trophotometer working at a resolution of 2 cm-1 and averaging
a total of 256 scans. A cell equipped with CaF2 windows and a
path length fixed at 0.5 mm was used. The sample chamber was
flushed continuously with dry air. Measurements were carried out
at room temperature in dichloromethane at peptide concentrations
of 5 mM. The absence of solute–solute interactions was confirmed
by the fact that unmodified spectra were obtained after further
dilution. Absorbance of samples was calculated by subtracting the
pure solvent spectrum scanned under the same conditions. Second-
derivative and curve-decomposition of spectra were carried out to
obtain the absorption maxima of overlapping bands.
Piv-L-cis(bPh)Pro-D-Phe-NHMe (2). (377 mg, 0.87 mmol,
◦
78% yield); mp 260 C; [a]2D0 = +98.4 (c = 0.34, MeOH); IR
1
(nujol) nmax/cm-1 3330, 3276, 1672, 1653, 1606; H-NMR (500
MHz, CDCl3, c = 10 mM) dH 1.26 (9H, s, Piv), 2.07 (1H, dd, J =
13.6, 6.0 Hz, Phe-Hb), 2.18 (1H, m, Pro-Hg exo), 2.68 (3H, d, J =
4.0 Hz, NHMe), 2.97 (1H, m, Pro-Hg endo), 3.33 (1H, dd, J = 13.6,
3.8 Hz, Phe-Hb), 3.47 (1H, ddd, J = 13.9, 8.3, 6.0 Hz, Pro-Hb),
3.72 (1H, m, Pro-Hdexo), 4.16–4.22 (1H, m, Pro-Hdendo) overlapped
with 4.20 (1H, d, J = 8.3 Hz, Pro-Ha), 4.40 (1H, ddd, J = 9.5, 6.0,
3.8 Hz, Phe-Ha), 5.17 (1H, br d, J = 9.5 Hz, Phe-NH), 6.70 (2H,
m, Ar), 7.11 (1H, m, NHMe), 7.13–7.21 (3H, m, Ar), 7.27–7.46
(5H, m, Ar); 13C-NMR (100 MHz, CDCl3) dC 26.37 (NHMe),
27.18 (Piv-Me3), 29.76 (Pro-Cg ), 35.60 (Phe-Cb), 38.87 (Piv-C),
45.49 (Pro-Cb), 48.39 (Pro-Cd), 52.40 (Phe-Ca), 68.44 (Pro-Ca),
126.94 (Ar), 127.82 (Ar), 128.21 (Ar), 128.56 (Ar), 128.62 (Ar),
129.50 (Ar), 135.85 (Ar), 136.24 (Ar), 170.11 (CO), 170.73 (CO),
178.15 (Piv-CO); HRMS (ESI) C26H33N3NaO3 [M+Na]+: calcd.
458.2414, found 458.2393.
X-ray diffraction structures
Single crystals were obtained by slow evaporation from
dichloromethane–ethyl acetate–hexanes (1), diethyl ether–ethyl
acetate (2) or diisopropyl ether–dichloromethane (3, 4, 4a) so-
lutions. The X-ray diffraction data were collected at 150 K on
an Oxford Diffraction Xcalibur diffractometer provided with a
Sapphire CCD detector, using graphite monochromated Mo-Ka
Piv-L-trans(bPh)Pro-L-Phe-NHMe (3). (343 mg, 0.79 mmol,
˚
◦
radiation (l = 0.71073 A). The structures were solved by direct
71% yield); mp 143 C; [a]2D5 = +53.4 (c = 0.47, MeOH); IR
methods using SHELXS-9725a and refinement was performed
using SHELXL-9725b by the full-matrix least-squares technique
with anisotropic thermal factors for heavy atoms. Hydrogen atoms
were located by calculation (except those at the nitrogen atoms,
which were found on the E-map) and affected by an isotropic
thermal factor fixed to 1.2 times the Ueq of the carrier atom (1.5
for the methyl protons). CCDC 842881–842885 contain the sup-
plementary crystallographic data for this paper.‡ These data can
be obtained free of charge from the Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
1
(nujol) nmax/cm-1 3331, 3315, 1671, 1660, 1607; H-NMR (500
MHz, CDCl3, c = 10 mM) dH 1.17 (9H, s, Piv), 2.03 (1H, m, Pro-
Hg exo), 2.29 (1H, m, Pro-Hg endo), 2.74 (3H, d, J = 4.6 Hz, NHMe),
3.10 (1H, dd, J = 14.0, 6.0 Hz, Phe-Hb), 3.34 (1H, dd, J = 14.0,
6.7 Hz, Phe-Hb), 3.44 (1H, ddd, J = ª 7.5, ª 6.5, 5.5 Hz, Pro-Hb),
3.60 (1H, m, Pro-Hdendo), 3.89 (1H, m, Pro-Hdexo), 4.53 (1H, d, J =
5.5 Hz, Pro-Ha), 4.61 (1H, ddd, J = 8.4, 6.7, 6.0 Hz, Phe-Ha), 6.12
(1H, br d, J = 8.4 Hz, Phe-NH), 6.49 (1H, m, NHMe), 7.10–7.33
(10H, m, Ar); 13C-NMR (100 MHz, CDCl3) dC 26.27 (NHMe),
27.09 (Piv-Me3), 34.01 (Pro-Cg ), 36.72 (Phe-Cb), 39.00 (Piv-C),
45.55 (Pro-Cb), 47.72 (Pro-Cd), 53.94 (Phe-Ca), 68.77 (Pro-Ca),
126.83 (Ar), 126.88 (Ar), 127.10 (Ar), 128.60 (Ar), 128.74 (Ar),
129.21 (Ar), 137.02 (Ar), 140.68 (Ar), 170.93 (CO), 170.99 (CO),
178.22 (Piv-CO); HRMS (ESI) C26H33N3NaO3 [M+Na]+: calcd.
458.2414, found 458.2398.
Summary of crystallographic data for 1 (C26H33N3O3): mono-
˚
˚
clinic, space group P21; a = 5.7243(3) A, b = 18.0071(8) A, c =
◦
11.1680(5) A, b = 96.429(4) ; Z = 2; dcalcd = 1.265 g cm-3; 11168
reflections collected, 4431 unique (Rint = 0.033); data/parameters:
˚
4431/291; final R indices (I > 2sI): R1 = 0.033, wR2 = 0.056;
-3
˚
highest residual electron density: 0.16 e A .
Piv-L-trans(bPh)Pro-D-Phe-NHMe (4). (396 mg, 0.91 mmol,
Summary of crystallographic data for 2 (C26H33N3O3): mono-
◦
82% yield); mp 208 C; [a]2D5 = +106.9 (c = 0.46, MeOH); IR
clinic, space group P21; a = 5.7726(6) A, b = 17.590(2) A, c =
˚
˚
◦
1
(nujol) nmax/cm-1 3332, 3297, 1657, 1623, 1609; H-NMR (500
11.5824(14) A, b = 96.205(10) ; Z = 2; dcalcd = 1.237 g cm-3; 20298
˚
MHz, CDCl3, c = 10 mM) dH 1.27 (9H, s, Piv), 2.15 (1H, m, Pro-
Hg exo), 2.37 (1H, m, Pro-Hg endo), 2.69 (1H, dd, J = 13.8, 5.5 Hz,
Phe-Hb), 2.74 (3H, d, J = 4.5 Hz, NHMe), 3.37 (1H, dd, J = 13.8,
5.1 Hz, Phe-Hb), 3.41 (1H, ddd, J = 11.8, 9.3, 6.3 Hz, Pro-Hb), 3.81
(1H, m, Pro-Hdendo), 3.93 (1H, d, J = 9.3 Hz, Pro-Ha), 4.08 (1H,
m, Pro-Hdexo), 4.76 (1H, ddd, J = 10.0, 5.5, 5.1 Hz, Phe-Ha), 5.42
(1H, br d, J = 10.0 Hz, Phe-NH), 6.69 (2H, m, Ar), 7.05–7.23 (5H,
m, Ar), 7.32–7.42 (3H, m, Ar), 7.56 (1H, m, NHMe); 13C-NMR
(100 MHz, CDCl3) dC 26.46 (NHMe), 27.09 (Piv-Me3), 34.31 (Pro-
reflections collected, 4111 unique (Rint = 0.104); data/parameters:
4111/290; final R indices (I > 2sI): R1 = 0.048, wR2 = 0.087;
highest residual electron density: 0.21 e A .
-3
˚
Summary of crystallographic data for 3 (C26H33N3O3): mono-
˚
˚
clinic, space group P21; a = ◦9.5504(3) A, b = 25.2883(7) A, c =
10.0653(3) A, b = 103.733(3) ; Z = 4; dcalcd = 1.225 g cm-3; 57501
˚
reflections collected, 10806 unique (Rint = 0.044); data/parameters:
10806/579; final R indices (I > 2sI): R1 = 0.031, wR2 = 0.057;
-3
˚
highest residual electron density: 0.14 e A .
650 | Org. Biomol. Chem., 2012, 10, 640–651
This journal is
The Royal Society of Chemistry 2012
©

View Article Online
Summary of crystallographic data for 4 (C26H33N3O3): mono-
Huby, G. L. Olson, R. Sarabu, J. Guenot, V. Madison, J. Hammer, F.
Sinigaglia, M. Steinmetz and Z. A. Nagy, Nat. Biotechnol., 1999, 17,
562–567; (i) Y. Tong, Y. M. Fobian, M. Wu, N. D. Boyd and K. D.
Moeller, Bioorg. Med. Chem. Lett., 1998, 8, 1679–l682.
˚
˚
clinic, space group P21; a = 9.6700(3) A, b = 12.4890(5) A, c =
◦
19.8078(7) A, b = 93.060(3) ; Z = 4; dcalcd = 1.211 g cm-3; 17815
reflections collected, 9060 unique (Rint = 0.038); data/parameters:
˚
7 (a) C. A. Thomas, E. R. Talaty and J. G. Bann, Chem. Commun., 2009,
3366–3368; (b) W. Kim, K. I. Hardcastle and V. P. Conticello, Angew.
Chem., Int. Ed., 2006, 45, 8141–8145; (c) C. M. Taylor, R. Hardre´ and P.
J. B. Edwards, J. Org. Chem., 2005, 70, 1306–1315; (d) C. L. Jenkins, L.
E. Bretscher, I. A. Guzei and R. T. Raines, J. Am. Chem. Soc., 2003, 125,
6422–6427; (e) L. Halab, F. Gosselin and W. D. Lubell, Biopolymers
(Pept. Sci.), 2000, 55, 101–122; (f) E. Beausoleil, R. Sharma, S. W.
Michnick and W. D. Lubell, J. Org. Chem., 1998, 63, 6572–6578; (g) J.
L. Flippen-Anderson, R. Gilardi, I. L. Karle, M. H. Frey, S. J. Opella,
L. M. Gierasch, M. Goodman, V. Madison and N. G. Delaney, J. Am.
Chem. Soc., 1983, 105, 6609–6614; (h) N. G. Delaney and V. Madison,
J. Am. Chem. Soc., 1982, 104, 6635–6641.
9060/580; final R indices (I > 2sI): R1 = 0.055, wR2 = 0.091;
-3
˚
highest residual electron density: 0.19 e A .
Summary of crystallographic data for 4a (C26H33N3O4): mon-
˚
˚
oclinic, space group P21; a = 13.539(2) A, b = 13.615(2) A, c =
◦
13.6874(17) A, b = 97.1390(12) ; Z = 4; dcalcd = 1.198 g cm-3; 14654
˚
reflections collected, 7271 unique (Rint = 0.077); data/parameters:
7271/597; final R indices (I > 2sI): R1 = 0.056, wR2 = 0.091;
-3
˚
highest residual electron density: 0.16 e A .
8 (a) K. Guitot, M. Larregola, T. K. Pradhan, J.-L. Vasse, S. Lavielle,
P. Bertus, J. Szymoniak, O. Lequin and P. Karoyan, ChemBioChem,
2011, 12, 1039–1042; (b) C. Mothes, M. Larregola, J. Quancard, N.
Goasdoue´, S. Lavielle, G. Chassaing, O. Lequin and P. Karoyan,
ChemBioChem, 2010, 11, 55–58; (c) J. Quancard, P. Karoyan, O.
Lequin, E. Wenger, A. Aubry, S. Lavielle and G. Chassaing, Tetrahedron
Lett., 2004, 45, 623–625; (d) P. W. Baures, W. H. Ojala, W. B. Gleason
and R. L. Johnson, J. Pept. Res., 1997, 50, 1–13.
Acknowledgements
The authors thank the Ministerio de Ciencia e Innovacio´n –
FEDER (project CTQ2010-17436; FPU fellowship to P.F.) and
Gobierno de Arago´n (research group E40) for financial support.
9 Torsion angles (f,y) for an ideal bI-turn: in i+1 (-60,-30), in i+2
(-90,0); for an ideal bII-turn: in i+1 (-60,120), in i+2 (80,0). Note that
they differ in the i+1 y and the i+2 f angles, as a consequence of a ª
180◦ flip of the plane defined by the central amide group.
References
1 (a) Peptide and Protein Design for Biopharmaceutical Applications, ed.
K. J. Jensen, John Wiley & Sons, Chichester, 2009; (b) Foldamers:
Structure, Properties, and Applications, ed. S. Hecht and I. Huc, Wiley-
VCH, Weinheim, 2007.
10 A. Aubry, M. T. Cung and M. Marraud, J. Am. Chem. Soc., 1985, 107,
7640–7647.
11 B. Castro, J. R. Dormoy, G. Evin and C. Selve, Tetrahedron Lett., 1975,
14, 1219–1222.
12 R. Bardi, A. M. Piazzesi, C. Toniolo, N. Sen, H. Balaram and P.
Balaram, Acta Crystallogr., Sect. C, 1988, 44, 1972–1976.
2 For reviews, see: (a) K. Estieu-Gionnet and G. Guichard, Expert Opin.
Drug Discovery, 2011, 6, 937–963; (b) C. Tomasini, G. Angelici and N.
Castellucci, Eur. J. Org. Chem., 2011, 3648–3669; (c) G. Guichard and
I. Huc, Chem. Commun., 2011, 47, 5933–5941; (d) W. S. Horne and S.
H. Gellman, Acc. Chem. Res., 2008, 41, 1399–1408; (e) D. Seebach and
J. Gardiner, Acc. Chem. Res., 2008, 41, 1366–1375; (f) J. Chatterjee, C.
Gilon, A. Hoffman and H. Kessler, Acc. Chem. Res., 2008, 41, 1331–
1342; (g) P. Maity and B. Ko¨nig, Biopolymers (Pept. Sci.), 2008, 90,
8–27; (h) D. Seebach, D. F. Hook and A. Gla¨ttli, Biopolymers (Pept.
Sci.), 2006, 84, 23–37; (i) P. Mathur, S. Ramakumar and V. S. Chauhan,
Biopolymers (Pept. Sci.), 2004, 76, 150–161; (j) C. Toniolo, M. Crisma,
F. Formaggio and C. Peggion, Biopolymers (Pept. Sci.), 2001, 60, 396–
419; (k) J. Venkatraman, S. C. Shankaramma and P. Balaram, Chem.
Rev., 2001, 101, 3131–3152; (l) R. P. Cheng, S. H. Gellman and W. F.
DeGrado, Chem. Rev., 2001, 101, 3219–3232; (m) D. J. Hill, M. J. Mio,
R. B. Prince, T. S. Hughes and J. S. Moore, Chem. Rev., 2001, 101,
3893–4011.
13 (a) A. I. Jime´nez, C. Cativiela, J. Go´mez-Catala´n, J. J. Pe´rez, A. Aubry,
M. Par´ıs and M. Marraud, J. Am. Chem. Soc., 2000, 122, 5811–5821;
(b) A. I. Jime´nez, C. Cativiela, A. Aubry and M. Marraud, J. Am.
Chem. Soc., 1998, 120, 9452–9459.
14 See the ESI‡.
15 (a) R. O. Gould, A. M. Gray, P. Taylor and M. D. Walkinshaw, J.
Am. Chem. Soc., 1985, 107, 5921–5927; (b) E. Benedetti, G. Morelli, G.
Ne´methy and H. A. Scheraga, Int. J. Pept. Protein Res., 1983, 22, 1–15.
16 M. T. Cung and M. Marraud, Biopolymers, 1982, 21, 953–967.
17 The JHa–Hb values measured in the present work (500 MHz) for the
L-Phe residue in 5 and D-Phe in 6 differ slightly from those reported
1
in ref. 10 (100 MHz). The predicted populations of c rotamers (using
the equation in ref. 16, as done in ref. 10) for the new J values are: 50%
gauche(-) for D-Phe in 6; 56% gauche(+) for L-Phe in 5.
3 P. Chakrabarti and D. Pal, Prog. Biophys. Mol. Biol., 2001, 76, 1–102.
4 M. W. MacArthur and J. M. Thornton, J. Mol. Biol., 1991, 218, 397–
412.
5 G. D. Rose, L. M. Gierasch and J. A. Smith, Adv. Protein Chem., 1985,
37, 1–109.
18 (a) R. Mahalakshmi, S. Raghothama and P. Balaram, J. Am. Chem.
Soc., 2006, 128, 1125–1138; (b) E. A. Meyer, R. K. Castellano and
F. Diederich, Angew. Chem., Int. Ed., 2003, 42, 1210–1250; (c) C. A.
Hunter, J. Singh and J. M. Thornton, J. Mol. Biol., 1991, 218, 837–
846; (d) S. K. Burley and G. A. Petsko, Adv. Protein Chem., 1988, 39,
125–189; (e) S. K. Burley and G. A. Petsko, Science, 1985, 229, 23–28.
19 T. Ashida and M. Kakudo, Bull. Chem. Soc. Jpn, 1974, 47, 1129–1133.
20 The g and d hydrogens of the pyrrolidine ring have been labelled as endo
or exo depending on whether they are on the same or the opposite side
of the ring as the carbonyl group, respectively.
6 (a) C. Armishaw, A. A. Jensen, T. Balle, R. J. Clark, K. Harpsøe, C.
Skonberg, T. Liljefors and K. Strømgaard, J. Biol. Chem., 2009, 284,
9498–9512; (b) J. A. Tran, F. C. Tucci, M. Arellano, W. Jiang, C. W.
Chen, D. Marinkovic, B. A. Fleck, J. Wen, A. C. Foster and C. Chen,
Bioorg. Med. Chem. Lett., 2008, 18, 1931–1938; (c) E. F. Rosloniec, T.
Brandstetter, S. Leyer, F.-W. Schwaiger and Z. A. Nagy, J. Autoimmun.,
2006, 27, 182–195; (d) M. Cai, C. Cai, A. V. Mayorov, C. Xiong, C. M.
Cabello, V. A. Soloshonok, J. R. Swift, D. Trivedi and V. J. Hruby, J.
Pept. Res., 2004, 63, 116–131; (e) I. J. McFadyen, K. Sobczyk-Kojiro,
M. J. Schaefer, J. C. Ho, J. R. Omnaas, H. I. Mosberg and J. R. Traynor,
J. Pharmacol. Exp. Ther., 2000, 295, 960–966; (f) D. R. Bolin, A. L.
Swain, R. Sarabu, S. J. Berthel, P. Gillespie, N. J. S. Huby, R. Makofske,
L. Orzechowski, A. Perrotta, K. Toth, J. P. Cooper, N. Jiang, F. Falcioni,
R. Campbell, D. Cox, D. Gaizband, C. J. Belunis, D. Vidovic, K. Ito, R.
Crowther, U. Kammlott, X. Zhang, R. Palermo, D. Weber, J. Guenot,
Z. Nagy and G. L. Olson, J. Med. Chem., 2000, 43, 2135–2148; (g) Y.
Tong, Y. M. Fobian, M. Wu, N. D. Boyd and K. D. Moeller, J. Org.
Chem., 2000, 65, 2484–2493; (h) F. Falcioni, K. Ito, D. Vidovic, C.
Belunis, R. Campbell, S. J. Berthel, D. R. Bolin, P. B. Gillespie, N.
21 C. A. G. Haasnoot, F. A. A. M. de Leeuw and C. Altona, Tetrahedron,
1980, 36, 2783–2792. The equation including the b-effect was used.
22 The Haasnoot–Altona equation from ref. 21 was used as implemented
in the free-access program MestReJ: A. Navarro-Va´zquez, J. C. Cobas,
F. J. Sardina, J. Casanueva and E. D´ıez, J. Chem. Inf. Comput. Sci.,
2004, 44, 1680–1685.
23 E. Aliev and D. Courtier-Murias, J. Phys. Chem. B, 2007, 111, 14034–
14042.
24 J. Quancard, P. Karoyan, S. Sagan, O. Convert, S. Lavielle, G. Chassaing
and O. Lequin, Eur. J. Biochem., 2003, 270, 2869–2878.
25 (a) G. M. Sheldrick, SHELXS-97, Program for the Solution of
Crystal Structures, University of Go¨ttingen: Go¨ttingen, 1997; (b) G.
M. Sheldrick, SHELXL-97. Program for the Refinement of Crystal
Structures, University of Go¨ttingen: Go¨ttingen, 1997.
This journal is
The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 640–651 | 651
©