Bicyclic Analogue of Proline
A bicyclic analogue of proline stabilises the b i-turn peptide conformation
A.M. Gil, E. Buñuel, A.I. Jiménez, C. Cativiela
Small and medium-sized peptides are usually characterised by
a high conformational freedom. This flexibility precludes their use as medicinal
agents since the various conformers available may interact with multiple receptors
and thus lead to undesired side effects. Reduction of the peptide backbone flexibility
through the stabilisation of secondary structure elements constitutes a major
approach in the design of therapeutically useful peptides as well as in the
investigation of structure-activity relationships.
Among the elements of secondary structure usually found in
peptides and proteins, b -turns are of enormous significance not only from a
structural viewpoint but also regarding biological activity. b -Turns involve
four consecutive residues and are classified according to the ( f ,
y ) dihedral angles of the central i +1 and i +2
positions. Types I and II b -turns, which are the most widely distributed, are
characterised by all trans peptide bonds and generally stabilised
by an intramolecular hydrogen bond between the i and i +3
Because of its cyclic nature, proline (Pro) is the most conformationally
constrained of the proteinogenic amino acids, and this property makes it a key
residue both in peptide conformation and biology. The N–C a torsion angle (
f ) in proline is intrinsically restricted to near –60° and, accordingly,
proline is most frequently found at the i +1 position of types I and
II b -turns. In the b I-turn the y angle of proline (C a
–C O torsion) lies in the –30° region, whereas the b II-turn
is characterised by y around 130°. Dipeptides of l -Pro- l -Xaa
sequence typically adopt a b I-turn in low-polarity solvents but, in general,
this conformation is not retained in the crystalline state, where the b II-turn
disposition is preferred because it allows intermolecular hydrogen-bonding of
the Xaa NH site .
Figure 1: Structure of proline (Pro) and
its bicyclic analogue studied in this work (Phb 7 Pro).
We have evaluated the relative stability of the b I- and b
II-turns in model peptides RCO- L -Pro- L -Phe-NHR' when
phenylalanine was replaced by different constrained derivatives [2–4]. Now,
we have undertaken the replacement of the proline residue in this sequence by
a bicyclic analogue of norbornane structure. In the proline surrogate considered,
that we denote as Phb 7 Pro (Fig. 1), the flexibility of the pyrrolidine ring
has been frozen by connecting the a - and d -carbons through an ethylene bridge.
Additionally, a phenyl substituent, which can interact with the backbone both
electronically and sterically, has been introduced on the b -carbon.
Figure 2: Structure of the dipeptide studied,
incorporating the bicyclic analogue of l -proline in position i+1.
The norbornane proline analogue Phb 7 Pro was incorporated
into the PhCO-Phb 7 Pro -L -Phe-NH i Pr sequence
(Fig. 2)  following standard methods of peptide synthesis. This dipeptide
yielded single crystals that were subjected to X-ray diffraction analysis. The
crystalline structure obtained is shown in Fig. 3. The molecule is folded in
a b -turn, with an intramolecular hydrogen bond between the benzoyl oxygen and
the isopropylamide hydrogen (N ... O distance 2.89 Å, N–H ... O angle
159°), and the backbone torsion angles correspond to a type I b -turn. This
is highly remarkable since the analogous dipeptide t BuCO- L
-Pro- L -Phe-NHMe is not able to retain the b I-turn conformation
in the solid state, where it has been shown to accommodate a b II-turn disposition
. It should be emphasised that in the crystal structure of PhCO-Phb
7 Pro -L -Phe-NH i Pr the phenylalanine
amide proton is not involved in any intermolecular contact, whereas in the L
-Pro-containing dipeptide this middle NH is hydrogen-bonded to a carbonyl group
of a neighbouring molecule.
Phb 7 Pro
( f , y ) = (–46,–31)
( f , y ) = (–73,–12)
Figure 3: X-ray diffraction structure of
the PhCO-Phb 7 Pro -L -Phe-NH i Pr dipeptide,
showing a b I-turn conformation with an intramolecular i+3
to i hydrogen bond.
This result evidences that Phb 7 Pro exhibits a
propensity for b I-folding higher than that of proline. Several factors can
contribute to it.
The three amide bonds in Fig. 3 present a trans disposition
with the torsion angles w close to the standard ±180°.
However, the nitrogen atom in the 7-position of the norbornane moiety exhibits
a significant distortion from planarity, lying at a distance of 0.38 Å
from the plane defined by the three carbon atoms bonded to it. In fact, the
pyramidal character of this nitrogen has been highlighted as an intrinsic feature
of the tensioned 7-azanorbornane system . In the case considered here, this
out-of-plane deviation is particularly strong, as denoted by the sum of the
valence angles around the nitrogen (339°) in comparison to the value expected
for a planar trigonal arrangement (360°).
The marked pyramidal geometry of this tertiary nitrogen confers
it a pronounced sp 3 character and hampers delocalization of the lone pair to
the adjacent carbonyl group. As a consequence, a significant lengthening of
the N–C O bond (1.38 Å) is observed with respect to the standard
amide value (1.33 Å). At the same time, the higher accessibility
of the lone pair allows the nitrogen to act as a weak hydrogen-bond acceptor.
Thus, in the crystalline structure shown in Fig. 3, the phenylalanine amide
hydrogen points towards the lone pair of the pyramidalised nitrogen (H ... N
distance 2.59 Å, N ... N distance 2.92 Å), giving rise
to an N–H ... N attractive interaction. Interestingly, an interaction of this
type has been proposed to promote the cis-trans isomerization of the
amide bond preceding proline by stabilising the lone pair of the pyramidalised
proline nitrogen in the transition state . It should be noted that y
values for the bicyclic proline surrogate in the 130° region (corresponding
to the i +1 position of a b II-turn) would not allow the establishment
of this N–H ... N interaction. In fact, a b II-turn disposition for this dipeptide
would place the Phb 7 Pro carbonyl oxygen in the neighbourhood of the pyramidalised
nitrogen lone pair, thus introducing a repulsive interaction.
An additional factor that can contribute to the stabilisation
of the b I-turn conformation encountered for PhCO-Phb 7 Pro -L
-Phe-NH i Pr is the presence of the extra b -phenyl substituent in
the bicyclic analogue of proline. In the structure shown in Fig. 3, this exo
-oriented phenyl ring lies in close proximity to the phenylalanine amide
hydrogen, giving rise to an attractive interaction of the N–H ... p type. Amide-aromatic
interactions have been frequently cited as stabilising factors in the structure
of peptides and proteins . Again, such an interaction would not occur in
a b II-turn disposition.
Another remarkable feature in Fig. 3 is the gauche (+)
disposition adopted by the phenylalanine side chain ( c 1 = 58°).
This is the least common of the three staggered rotamers available to this residue,
because of steric hindrance between the phenyl ring and both the amino and carbonyl
substituents. However, this orientation allows the existence of an additional
amide-aromatic interaction between the phenylalanine amide hydrogen and phenyl
The b I-turn conformation accommodated by the Phb 7
Pro-containing dipeptide in the crystalline state provides evidence that the
extra attractive intramolecular interactions involving the bicyclic proline
analogue and the middle amide hydrogen compensate for the intermolecular hydrogen
bond that stabilises the b II-turn conformation in the L -Pro- L
-Phe sequence. Proline analogues, as the one presented here, that are able to
stabilise the b I-turn disposition are extremely helpful in the design of peptide
analogues with well-defined conformational features.
A.M. Gil, E. Buñuel, A.I. Jiménez, C. Cativiela,
Tetrahedron Lett. 2003, 44 , 5999.
Financial support was provided by MCyT (PPQ2001-1834 and PPQ2002-819).
The Centro de Excelencia Bruker-ICMA is gratefully acknowledged for
collection and preliminary treatment of the X-ray diffraction data.
 M. Marraud, A. Aubry, Biopolymers 1996, 40
 A.I. Jiménez, C. Cativiela, A. Aubry, M. Marraud, J. Am. Chem.
Soc. 1998, 120 , 9452.
 A.I. Jiménez, C. Cativiela, J. Gómez-Catalán, J.J.
Pérez, A. Aubry, M. París, M. Marraud, J. Am. Chem.
Soc. 2000, 122 , 5811.
 A.I. Jiménez, C. Cativiela, M. Marraud, Tetrahedron Lett.
2000, 41 , 5353.
 A.M. Gil, E. Buñuel, P. López, C. Cativiela, Tetrahedron:
Asymmetry 2004, 15 , 811.
 A. Aubry, M.T. Cung, M. Marraud, J. Am. Chem. Soc. 1985, 107
 Y. Otani, O. Nagae, Y. Naruse, S. Inagaki, M. Ohno, K. Yamaguchi, G. Yamamoto,
M. Uchiyama, T. Ohwada, J. Am. Chem. Soc. 2003, 125 ,
 G. Fischer, Chem. Soc. Rev. 2000, 29 , 119. 
T. Steiner, G. Koellner, J. Mol. Biol. 2001, 305