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A 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 positions.

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 [1].

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) [5] 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 [6]. 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.

b I-turn

Phb 7 Pro

( f , y ) = (–46,–31)

l -Phe

( 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 [7]. 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 [8]. 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 [9]. 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 side chain.

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.

Principal publication

A.M. Gil, E. Buñuel, A.I. Jiménez, C. Cativiela, Tetrahedron Lett. 2003, 44 , 5999.

Acknowledgements

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.

References

[1] M. Marraud, A. Aubry, Biopolymers 1996, 40 , 45.

[2] A.I. Jiménez, C. Cativiela, A. Aubry, M. Marraud, J. Am. Chem. Soc. 1998, 120 , 9452.

[3] A.I. Jiménez, C. Cativiela, J. Gómez-Catalán, J.J. Pérez, A. Aubry, M. París, M. Marraud, J. Am. Chem. Soc. 2000, 122 , 5811.

[4] A.I. Jiménez, C. Cativiela, M. Marraud, Tetrahedron Lett. 2000, 41 , 5353.

[5] A.M. Gil, E. Buñuel, P. López, C. Cativiela, Tetrahedron: Asymmetry 2004, 15 , 811.

[6] A. Aubry, M.T. Cung, M. Marraud, J. Am. Chem. Soc. 1985, 107 , 7640.

[7] Y. Otani, O. Nagae, Y. Naruse, S. Inagaki, M. Ohno, K. Yamaguchi, G. Yamamoto, M. Uchiyama, T. Ohwada, J. Am. Chem. Soc. 2003, 125 , 15191.

[8] G. Fischer, Chem. Soc. Rev. 2000, 29 , 119. [9] T. Steiner, G. Koellner, J. Mol. Biol. 2001, 305 , 535.

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