Influence of side chain restriction and NH···π Interaction on the β-turn folding modes of dipeptides incorporating phenylalanine cyclohexane derivatives

Article abstract of DOI:10.1021/ja993568k

We have synthesized the model dipeptides Piv-L-Pro-c₆Phe-NH(i)pr, incorporating each of the two cis cyclohexane analogues of phenylalanine: (S,S)- and (R,R)-1-amino-2-phenylcyclohexanecarboxylic acid. Their structural analysis has been carried

Full text of DOI:10.1021/ja993568k

J. Am. Chem. Soc. 2000, 122, 5811-5821
5811
Influence of Side Chain Restriction and NH‚‚‚π Interaction on the
â-Turn Folding Modes of Dipeptides Incorporating Phenylalanine
Cyclohexane Derivatives
Ana I. Jime´nez,*,‡ Carlos Cativiela,‡ Jesu´s Go´mez-Catala´n,# Juan J. Pe´rez,# Andre´ Aubry,§
,¶
Miguel Par´ıs, and Michel Marraud
Contribution from the Department of Organic Chemistry, ICMA, CSIC-UniVersity of Zaragoza,
50009 Zaragoza, Spain, Department of Chemical Engineering, Technical UniVersity of Catalonia,
08028 Barcelona, Spain, Laboratory of Crystallography and Modeling of Mineral and Biological
Materials, ESA-7036, UniVersity Henri Poincare´ of Nancy, BP 236, 54509 VandoeuVre, France, and
Laboratory of Macromolecular Physical Chemistry, UMR-7568 CNRS-INPL, ENSIC, BP 451,
54001 Nancy, France
ReceiVed October 4, 1999. ReVised Manuscript ReceiVed March 21, 2000
Abstract: We have synthesized the model dipeptides Piv-L-Pro-c6Phe-NHiPr, incorporating each of the two
cis cyclohexane analogues of phenylalanine: (S,S)- and (R,R)-1-amino-2-phenylcyclohexanecarboxylic acid.
1
Their structural analysis has been carried out in solution by H NMR and FTIR absorption spectroscopy and
in the solid state by X-ray diffraction. In weakly polar chlorinated solvents, the (S,S)c6Phe-containing dipeptide
mainly accommodates a type I â-turn, whereas the (R,R) residue shows a greater propensity to âII-folding.
This behavior does not differ significantly from that exhibited by the analogous dipeptides containing L- and
D-Phe. However, the L-Pro-L-Phe sequence has been shown to undergo a âI-to-âII transition in the presence
of a strong solvating medium, such as DMSO, or in the crystalline state. Interestingly, Piv-L-Pro-(S,S)c6Phe-
NHiPr, incorporating its cyclohexane analogue with ø1 fixed at +60°, retains the âI-folded structure under
these conditions. Theoretical calculations, supported by the experimental data, indicate that a c6Phe-NH to
aromatic π-orbitals interaction has an important influence on the observed â-folding preferences.
Small and medium peptides are generally highly flexible
molecules that exist in solution in a dynamic equilibrium of
interchanging conformations. As a consequence, most natural
peptides with physiological activity cannot be used for thera-
peutic purposes, since the various conformers available may
activate different receptors, thus leading to undesired side effects.
The introduction of conformational constraints constitutes one
of the most promising approaches to overcome this difficulty.
In comparison with native peptides, restricted analogues usually
display more favorable pharmacological properties, such as
enhanced selectivity and increased metabolic stability. In
addition, this strategy offers a valuable means to investigate
the relationship between structure and function, the understand-
ing of which is essential for the rational design of peptide-based
drugs.
modification,1a,c,3 incorporation of specific amino acids,1,4 and
use of fragments of a nonpeptide nature that mimic secondary
structure elements1,5 (R-helices, â-sheets, turns).
Although recent years have seen enormous progress in the
design of peptides with well-defined backbone conformations,
insufficient attention has been paid to the geometry of the side
chain moieties. Side chains are, however, essential in peptide-
(2) (a) Gilon, C.; Halle, D.; Chorev, M.; Selinger, Z.; Byk, G. Biopoly-
mers 1991, 31, 745-750. (b) Toniolo, C. Int. J. Pept. Protein Res. 1990,
35, 287-300. (c) Hruby, V. J.; Al-Obeidi, F.; Kazmierski, W. Biochem. J.
1990, 268, 249-262. (d) Kessler, H. Angew. Chem., Int. Ed. Engl. 1982,
21, 512-523.
(3) (a) Spatola A. F. In Chemistry and Biochemistry of Amino Acids,
Peptides and Proteins; Weinstein, B., Ed.; Marcel Dekker: New York, 1983;
Vol. 7, pp 267-357. (b) Marraud, M.; Aubry, A. Biopolymers 1996, 40,
45-83. (c) Hruby, V. J. Biopolymers 1993, 33, 1073-1082. (d) Gante, J.
Synthesis 1989, 405-413.
(4) (a) Obrecht, D.; Abrecht, C.; Altorfer, M.; Bohdal, U.; Grieder, A.;
Kleber, M.; Pfyffer, P.; Mu¨ller, K. HelV. Chim. Acta 1996, 79, 1315-1337.
(b) Benedetti, E. Biopolymers 1996, 40, 3-44. (c) Toniolo, C.; Crisma,
M.; Formaggio, F.; Benedetti, E.; Santini, A.; Iacovino, R.; Saviano, M.;
Di Blasio, B.; Pedone, C.; Kamphuis, J. Biopolymers 1996, 40, 519-522.
(d) Toniolo, C.; Crisma, M.; Formaggio, F.; Valle, G.; Cavicchioni, G.;
Pre´cigoux, G.; Aubry, A.; Kamphuis, J. Biopolymers 1993, 33, 1061-1072.
(e) Toniolo, C.; Benedetti, E. Macromolecules 1991, 24, 4004-4009. (f)
Karle, I. L.; Balaram, P. Biochemistry 1990, 29, 6747-6756.
(5) (a) Hanessian, S.; McNaughton-Smith, G.; Lombart, H.-G.; Lubell,
W. D. Tetrahedron 1997, 53, 12789-12854. (b) Gillespie, P.; Cicariello,
J.; Olson, G. L. Biopolymers 1997, 43, 191-217. (c) Adang, A. E. P.;
Hermkens, P. H. H.; Linders, J. T. M.; Ottenheijm, H. C. J.; Van Staveren,
C. J. Recl. TraV. Chim. Pays-Bas 1994, 113, 63-78. (d) Kahn, M. Synlett
1993, 821-826. (e) Olson, G. L.; Bolin, D. R.; Bonner, M. P.; Bo¨s, M.;
Cook, C. M.; Fry, D. C.; Graves, B. J.; Hatada, M.; Hill, D. E.; Kahn, M.;
Madison, V. S.; Rusiecki, V. K.; Sarabu, R.; Sepinwall, J.; Vincent, G. P.;
Voss, M. E. J. Med. Chem. 1993, 36, 3039-3049. (f) Symposia-in-print
No. 50: Tetrahedron 1993, 49 (17).
A major approach in the development of conformationally
restricted peptides is to decrease the flexibility of the backbone.1
The different methodologies applied to limit the allowed
combinations of the φ,ψ torsion angles include short-, medium-
and long-range cyclization1b,c,2 (backbone to backbone, side
chain to backbone, side chain to side chain), peptide bond
‡ Department of Organic Chemistry, University of Zaragoza.
# Department of Chemical Engineering, Technical University of Cata-
lonia.
§ Laboratory of Crystallography and Modeling of Mineral and Biological
Materials, University Henri Poincare´ of Nancy.
Laboratory of Macromolecular Physical Chemistry, ENSIC-INPL.
¶ Current address: University of La Rioja, 26001 Logron˜o, Spain.
(1) (a) Gante, J. Angew. Chem., Int. Ed. Engl. 1994, 33, 1699-1720.
(b) Liskamp, R. M. J. Recl. TraV. Chim. Pays-Bas 1994, 113, 1-19. (c)
Giannis, A.; Kolter, T. Angew. Chem., Int. Ed. Engl. 1993, 32, 1244-1267.
10.1021/ja993568k CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/31/2000

5812 J. Am. Chem. Soc., Vol. 122, No. 24, 2000
Jime´nez et al.
receptor recognition processes, to the extent that their interaction
with the complementary receptor groups determines biological
specificity. Their three-dimensional arrangement is therefore
critical for bioactivity, and the elucidation of the precise spatial
disposition required for binding to the receptor is of the utmost
importance in establishing the so-called bioactive conformation.
The incorporation of side chain constrained amino acids
constitutes a powerful tool to explore this important aspect of
peptide structure.6 Restrictions on the ø torsion angles will affect
the conformational preferences of the side substituents and have
an impact on the rotameric distribution with respect to unmodi-
fied residues. The equilibrium among the three low-energy CR-
Câ staggered conformations assumed by proteinogenic amino
acids may be shifted to a particular rotamer. Moreover,
dispositions not accessible to these residues may be allowed,
preferred, or even fixed by adequately restricting the ø values.
Thus, the incorporation of amino acids with well-defined ø
tendencies into bioactive peptides may provide fundamental
insights into the precise conformational requirements of the side
chain groups to fit the receptor binding site.
Phenylalanine, in the same way as other aromatic residues,
seems to play an important role in recognition processes and is
often located in pharmacophoric regions.7 In addition, a wide
variety of constrained phenylalanine analogues with different
degrees of control over the benzylic chain orientation has
become synthetically available.8 This amino acid therefore
constitutes an appropriate candidate to investigate the structural
and biological consequences arising from side chain restriction.
The rotational freedom of the side chain can first be influenced
by methylation at the R- or â-positions, which has been
extensively studied in (RMe)Phe-4d,9 and (âMe)Phe-containing6,9f,10
peptides. The presence of more sterically demanding groups,
as in (REt)Phe,11 (âiPr)Phe,6,12 and Dip (diphenylalanine),13 will
further reduce the space available to the phenyl ring. In all these
cases, rotation about CR-Câ is limited by steric interactions
between the vicinal substituents. Rigidity can be increased
through the introduction of bridges of variable length between
different parts of the molecule, and this type of modification
has proven extremely useful in the case of Tic (1,2,3,4-
tetrahydroisoquinoline carboxylic acid).13b,d,14
An attractive series of phenylalanine cyclic derivatives can
be obtained by linking the R- and â-carbons through an
alkylidene bridge. Rotation about CR-Câ is then prohibited, the
side chain orientation being dictated by both the bridge length
(and subsequent ring size) and the stereochemistry at CR and
Câ. The smallest member of this family, in which an additional
methylene unit closes a cyclopropane and which we denote as
c3Phe, has been incorporated into several peptide sequences.13b,15,16
In this context, we have studied its behavior in the model
dipeptides RCO-L-Pro-c3Phe-NHMe16 and observed an influence
of c3Phe chirality on the â-turn type accommodated in weakly
polar solvents. Of special relevance is the finding that, among
the two cis derivatives, (S,S)c3Phe (analogous to L-Phe)
preferentially induces a type I â-turn, whereas (R,R)c3Phe
(analogous to D-Phe) tends more to âII-folding. It should be
noted that for both cis stereoisomers the rigid cyclopropane
moiety confines the amino and phenyl groups to an eclipsed
conformation (ø1 ≈ 0°, Figure 1). On the basis of these
observations we have proposed that an attractive interaction
between the free c3Phe-NH and the aromatic π-orbitals might
play an important role on the observed â-turn tendencies. It
appears that this stabilizing intramolecular interaction can be
overcompensated by solvation or molecular aggregation, since
in DMSO solution and in the solid state both dipeptides
incorporating cis-c3Phe accommodate the same âII-turn struc-
ture.
(12) Liao, S.; Shenderovich, M. D.; Lin, J.; Hruby, V. J. Tetrahedron
1997, 53, 16645-16662.
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G.; Couraud, J. Y.; Lavielle, S. Biopolymers 1996, 39, 67-74. (c) Cody,
W. L.; He, J. X.; DePue, P. L.; Waite, L. A.; Leonard, D. M.; Sefler, A.
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Beaujouan, J.-C.; Glowinski, J.; Chassaing, G. J. Med. Chem. 1994, 37,
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Chem. Soc. 1998, 120, 4763-4770. (b) Benedetti, E.; Gavuzzo, E.; Santini,
A.; Kent, D. R.; Zhu, Y.-F.; Zhu, Q.; Mahr, C.; Goodman, M. J. Pept. Sci.
1995, 1, 349-359. (c) Pantano, M.; Formaggio, F.; Crisma, M.; Bonora,
G. M.; Mammi, S.; Peggion, E.; Toniolo, C.; Boesten, W. H. J.; Broxterman,
Q. B.; Schoemaker, H. E.; Kamphuis, J. Macromolecules 1993, 26, 1980-
1984. (d) Valle, G.; Pantano, M.; Formaggio, F.; Crisma, M.; Toniolo, C.;
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Schoemaker, H. E.; Kamphuis, J. Biopolymers 1993, 33, 1617-1625. (e)
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Schoemaker, H. E.; Kamphuis, J.; Temussi, P. A.; Becker, E. L.; Pre´cigoux,
G. Tetrahedron 1993, 49, 3641-3653. (f) Huang, Z.; He, Y.-B.; Raynor,
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Jaspers, H.; To´th, G.; Pe´ter, A.; Kerte´sz, I.; To¨ro¨k, G.; Chung, N. N.;
Schiller, P. W. J. Med. Chem. 1998, 41, 5167-5176. (b) Misicka, A.;
Cavagnero, S.; Horvath, R.; Davis, P.; Porreca, F.; Yamamura, H. I.; Hruby,
V. J. J. Pept. Res. 1997, 50, 48-54. (c) Haskell-Luevano, C.; Toth, K.;
Boteju, L.; Job, C.; Castrucci, A. M. L.; Hadley, M. E.; Hruby, V. J. J.
Med. Chem. 1997, 40, 2740-2749. (d) Azizeh, B. Y.; Shenderovich, M.
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2449-2455. (e) Mosberg, H. I.; Omnaas, J. R.; Lomize, A.; Heyl, D. L.;
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7562.

Phenylalanine Cyclohexane Analogues
J. Am. Chem. Soc., Vol. 122, No. 24, 2000 5813
Figure 2. Synthesis of the two Piv-Pro-c6Phe-NHiPr dipeptides 1 and
2 from racemic H-c6Phe-OMe. Conditions: (a) Boc-L-Pro-OH/iBuO-
COCl/NMM/CH2Cl2, -15 °C 24 h; yield: 85%. (b) Eluent AcOEt/
hexanes 1/1; Rf 0.67 (S,S), 0.50 (R,R). (c) iPrNH2/AlMe3/toluene, 0 °C
1 h, 50 °C 36 h; yield 48-50%. (d) 1: TFA/CH2Cl2 2/3, room
temperature 2 h. 2: Piv2O/Et3N/DMAP/CH2Cl2, 0 °C 1 h, room
temperature overnight; yield 83-85%.
Figure 1. Newman projections of the two cis stereoisomers of c3Phe
and c6Phe (assuming a chair conformation for the cyclohexane ring
and an equatorial orientation for the phenyl substituent). Comparison
with the three staggered CR-Câ rotamers in L- and D-Phe.
anecarboxylic acid. The existence of an NH to phenyl ring
interaction as a discriminating factor between the âΙ and âΙΙ-
turns has also been investigated by theoretical calculations.
Preliminary results have already been published.18
To further investigate the existence of the aforementioned
NH‚‚‚π interaction and in an attempt to evaluate its influence
on the â-folding mode, we undertook the study of the cis
cyclohexane analogues of phenylalanine, which we denote as
c6Phe. Although, in principle, the cyclohexane ring containing
CR and Câ would be expected to exhibit a certain degree of
flexibility, its conformational freedom is in fact reduced by the
presence of the aromatic substituent. The six-membered cycle
is likely to adopt a chair conformation and the bulky phenyl
ring most probably accommodates an equatorial disposition.
Assuming that this is the case, the side chain will be confined
to be gauche (+) in (S,S)c6Phe (analogous to L-Phe) and gauche
(-) in the (R,R) residue (analogous to D-Phe). As depicted in
Figure 1, the aromatic substituent is, in both cases, flanked by
the amino and the carbonyl groups, and this disposition
corresponds to the sterically most disfavored conformation
among the three CR-Câ staggered rotamers accessible to L- and
D-Phe.
Experimental Section
Synthesis. Figure 2 shows the synthetic route for dipeptides 1 and
2. The preparation of cis-methyl 1-amino-2-phenylcyclohexanecar-
boxylate by Diels-Alder reaction of 1,3-butadiene and (Z)-2-phenyl-
4-benzylidene-5(4H)-oxazolone has already been reported.19 The
racemic amino ester was classically coupled to N-tert-butyloxycarbonyl-
L-proline by the mixed anhydride procedure using isobutyl chlorofor-
mate,20 and the resulting diastereoisomeric dipeptides were separated
by column chromatography on silica gel. Subsequent treatment with
isopropylamine in the presence of AlMe3 allowed the transformation
of the methyl esters into the corresponding amides.21 The Boc group
was then cleaved by TFA and the amino group was acylated with pivalic
anhydride to give enantiomerically pure 1 and 2. Taking L-proline as
a reference, the resolution of their crystal structures revealed the absolute
configuration of the c6Phe residue to be (2S,3S) in 1 and (2R,3R) in 2.
The Ac6c-containing dipeptide 3 was obtained from commercially
available 1-aminocyclohexanecarboxylic acid following standard pro-
cedures, using Boc as transitory protection and isobutyl chloroformate
as the coupling agent. The use of the Piv group in 1-3 was preferred
to Boc to avoid the cis-trans isomerization about the Boc-Pro tertiary
urethane bond.22 All intermediate derivatives gave satisfactory analytical
The present paper describes the synthesis and structure
elucidation of the model dipeptides Piv-L-Pro-(S,S)c6Phe-NHiPr
(1) and Piv-L-Pro-(R,R)c6Phe-NHiPr (2). Their conformational
properties have been studied in the solid state by X-ray
1
diffraction and in solution by H NMR and FTIR absorption
spectroscopy, and the results compared to those of the analogous
dipeptides incorporating L- and D-Phe.16a,17 With the aim of
evaluating separately the effects of the selective orientation of
the aromatic side chain and those arising from tetrasubstitution
at CR, we have carried out a parallel study on Piv-L-Pro-Ac6c-
NHiPr (3), which contains the unsubstituted 1-aminocyclohex-
(18) Jime´nez, A. I.; Cativiela, C.; Par´ıs, M.; Peregrina, J. M.; Avenoza,
A.; Aubry, A.; Marraud, M. Tetrahedron Lett. 1998, 39, 7841-7844.
(19) (a) Avenoza, A.; Cativiela, C.; Par´ıs, M.; Peregrina, J. M.
Tetrahedron 1996, 52, 4839-4848. (b) Cativiela, C.; D´ıaz-de-Villegas, M.
D.; Avenoza, A.; Peregrina, J. M. Tetrahedron 1993, 49, 10987-10996.
(20) (a) Bodanszky, M.; Bodanszky, A. The Practice of Peptide Synthesis;
Springer-Verlag: Berlin, Germany, 1994. (b) Vaughan, J. R., Jr.; Osato,
R. L. J. Am. Chem. Soc. 1952, 74, 676-678.
(17) Aubry, A.; Cung, M. T.; Marraud, M. J. Am. Chem. Soc. 1985,
107, 7640-7647.
(21) Cativiela, C.; D´ıaz-de-Villegas, M. D.; Ga´lvez, J. A. Synthesis 1990,
198-199.

5814 J. Am. Chem. Soc., Vol. 122, No. 24, 2000
Jime´nez et al.
and spectroscopic data. Elemental analyses were carried out on a Perkin-
Elmer 200 C,H,N,S analyzer. Specific rotations were measured in a
10 cm cell at 25 °C using a Perkin-Elmer 241-C polarimeter. Melting
points were determined on a Bu¨chi SMP-20 or Bu¨chi 510 apparatus
and were not corrected. Solid-state IR spectra were registered on a
Table 1. Crystal Data
1
2
crystal system
space group
unit cell dimensions
a, Å
triclinic
orthorhombic
P22121
P1
1
Mattson Genesis FTIR spectrophotometer. H and 13C NMR spectra
10.165(2)
10.536(2)
12.664(1)
86.27(1)
78.62(1)
87.52(1)
2
17.166(2)
23.613(3)
6.347(1)
were recorded on a Bruker ARX-300 apparatus using the solvent signal
as internal standard. CLâ/CDâ and CLγ/CγD denote the carbon atoms of the
cyclohexane ring that occupy positions â and γ, respectively, in an
L-/D-amino acid.
b, Å
c, Å
R, deg
â, deg
Piv-L-Pro-(2S,3S)c6Phe-NHiPr (1): mp 155 °C. Rf (AcOEt/hexanes
1/1) ) 0.22. [R]D -111.7 (c 1.33, CH2Cl2). IR (Nujol) 3402, 3373,
3338, 1678, 1641, 1605 cm-1. Anal. Calcd for C26H39N3O3: C, 70.71;
H, 8.90; N, 9.52. Found: C, 70.59; H, 8.94; N, 9.56. 1H NMR (CDCl3,
c 0.01 M, 300 MHz) δ 0.78 (d, 3H, J ) 6.6 Hz, iPr-CH3); 1.04 (d, 3H,
γ, deg
Z
4
densitycalcd, g‚cm-3
no. of reflcns collected
no. of independent reflcns
data/parameters
residual factors [I > 2 σ(I)]
1.151
4688
4688
1.140
2514
2514
i
4688/618
R1 ) 0.0392
wR2 ) 0.1117
1.038
2514/297
R1 ) 0.0391
wR2 ) 0.0869
1.125
J ) 6.6 Hz, Pr-CH3); 1.12 (s, 9H, Piv-(CH3)3); 0.80-2.20 (m, 11H,
c6Phe-Câ Hax
+
c6Phe-CγH2
+
c6Phe-Cγ H2
+
c6Phe-CδH2
+
D
L
D
â
Pro-CâH2 + Pro-CγH2); 3.06 (bd, 1H, J ) -14.7 Hz, c6Phe-CDHeq);
2
goodness-of-fit
3.33 (dd, 1H, J ) 12.9, 3.0 Hz, c6Phe-CâLH); 3.68 (m, 1H, Pro-CδH);
3.79 (m, 1H, Pro-CδH); 3.89 (m, 1H, Pr-CH); 4.36 (dd, 1H, J ) 6.3
i
Hz, J ) 5.7 Hz, Pro-CRH); 6.18 (bs, 1H, c6Phe-NH); 6.20 (bd, 1H,
J ) 7.8 Hz, NHiPr); 7.15-7.30 (m, 5H, c6Phe-C6H5). 13C NMR (CDCl3,
75 MHz) δ 21.3, 22.1, 22.2, 25.7, 26.4, 26.7, 27.5, 27.7, 30.3, 39.1,
41.3, 48.1, 48.7, 64.5, 64.8, 127.1, 128.3, 128.6, 140.5, 171.1, 171.3,
176.8.
located by calculation and affected by an isotropic thermal factor of 5
Å2. The NH hydrogens were replaced at a distance of 1.03 Å from
nitrogen in the direction found by refinement.25 The main crystal-
lographic data, together with the residual R factors, are given in Table
1. Crystal data, fractional coordinates for the hydrogen and non-
hydrogen atoms, equivalent thermal parameters and anisotropic tem-
perature parameters for the non-hydrogen atoms, interatomic bond
lengths and bond angles, and torsion angles have been deposited as
Supporting Information.
Piv-L-Pro-(2R,3R)c6Phe-NHiPr (2): mp 192 °C. Rf (AcOEt/hexanes
1/1) ) 0.29. [R]D +8.0 (c 1.8, CH2Cl2). IR (Nujol) 3373, 3323, 1691,
1631, 1616 cm-1. Anal. Calcd for C26H39N3O3: C, 70.71; H, 8.90; N,
9.52. Found: C, 70.80; H, 8.87; N, 9.49. 1H NMR (CDCl3, c 0.01 M,
i
1H NMR and IR Absorption Spectroscopy. Structural analysis of
dipeptides 1, 2, and 3 in solution was performed by NMR and IR
absorption techniques. IR spectra were run in the Fourier transform
mode on a Bruker IFS-25 apparatus at a resolution of 2 cm-1. A cell
equipped with CaF2 windows and with a path length fixed at 0.5 mm
was used, and a total of 256 scans were averaged. The sample chamber
was flushed continuously with dry air. Measurements were carried out
at room temperature in CH2Cl2 and DMSO with peptide concentrations
of 0.005 M. 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, and the contribution of
residual water was eliminated, if necessary, by correction in the 3500-
3600 cm-1 region, where the peptide does not absorb. Attention was
focused on the most informative frequency domains corresponding to
the NH (3200-3500 cm-1) and C′O (1550-1750 cm-1) stretching
vibrations. A free secondary amide group is expected to give an NH
300 MHz) δ 0.83 (d, 3H, J ) 6.6 Hz, Pr-CH3); 0.98 (d, 3H, J )
i
6.6 Hz, Pr-CH3); 1.18 (s, 9H, Piv-(CH3)3); 1.10-2.25 (m, 11H,
c6Phe-CâHax
+
c6Phe-CγH2
+
c6Phe-Cγ H2
+
c6Phe-CδH2
+
L
L
D
â
Pro-CâH2 + Pro-CγH2); 3.08 (bd, 1H, J ) -15.0 Hz, c6Phe-CLHeq);
2
3.41 (dd, 1H, J ) 13.2, 3.0 Hz, c6Phe-CâDH); 3.71 (m, 2H, Pro-CδH2);
3.93 (m, 1H, Pr-CH); 4.02 (dd, 1H, J ) 7.5, 5.1 Hz, Pro-CRH); 5.57
i
(bs, 1H, c6Phe-NH); 6.76 (bd, 1H, J ) 7.8 Hz, NHiPr); 7.18-7.33 (m,
5H, c6Phe-C6H5). 13C NMR (CDCl3, 75 MHz) δ 21.4, 21.7, 22.4, 25.8,
26.3, 26.6, 27.2, 28.3, 30.2, 38.5, 41.2, 47.8, 48.6, 63.7, 64.9, 127.4,
128.1, 128.7, 140.1, 170.9, 172.4, 177.2.
Piv-L-Pro-Ac6c-NHiPr (3): mp 240 °C. Rf (CH2Cl2/iPrOH 9/1) )
0.50. [R]D -14.3 (c 0.47, MeOH). IR (Nujol) 3351, 3297, 1691, 1639,
1604 cm-1. Anal. Calcd for C20H35N3O3: C, 65.72; H, 9.65; N, 11.50.
Found: C, 65.79; H, 9.67; N, 11.46. 1H NMR (CDCl3, c 0.01 M, 300
i
MHz) δ 1.06 (d, 3H, J ) 6.6 Hz, Pr-CH3); 1.13 (d, 3H, J ) 6.6 Hz,
iPr-CH3); 1.26 (s, 9H, Piv-(CH3)3); 1.18-2.23 (m, 14H, Ac6c-C6H10
+
Pro-CâH2 + Pro-CγH2); 3.73 (m, 2H, Pro-CδH2); 4.0 (m, 1H, iPr-CH);
4.29 (dd, 1H, J ) 6.8, 5.8 Hz, Pro-CRH); 5.96 (bs, 1H, Ac6c-NH);
6.87 (bd, 1H, J ) 7.8 Hz, NHiPr). 13C NMR (CDCl3, 75 MHz) δ 21.3,
21.4, 22.2, 22.5, 25.1, 26.2, 27.3, 27.7, 30.5, 33.1, 38.9, 41.2, 48.7,
60.4, 63.2, 171.8, 173.1, 177.9.
absorption at 3400-3450 cm-1 and a C′O band at 1650-1700 cm-1
.
When involved in a hydrogen bond these absorptions are shifted to
lower frequencies. The tertiary nature of the Pro-preceding amide group,
along with the presence of a quaternary carbon adjacent to the carbonyl,
results in a very low frequency for the free Piv-C′O (about 1620 cm-1
both in CH2Cl2 and DMSO). Thus, the pivaloyl group has a 2-fold
advantage: it excludes the cis conformation of the Piv-Pro bond for
steric reasons22 and shifts the Piv-C′O frequency out of the usual region
for peptide carbonyls, allowing easier interpretation of IR data. The
NH and C′O absorption bands for dipeptides 1, 2, and 3 were assigned
on the basis of previous studies on similar peptides,16a,17 and of the
perturbation induced by Boc/Piv and OMe/NHiPr exchange. Second-
derivative and curve-decomposition of spectra were carried out to obtain
the absorption maxima of overlapping bands.
1H NMR spectra were acquired using a Bruker ARX-300 spectrom-
eter with the solvent signal as the internal reference. Solutions of
peptides (0.01 M) in CDCl3 and DMSO-d6 were used. Proton resonances
were assigned by COSY and HOHAHA experiments. Nuclear Over-
hauser enhancement due to the proximity of Pro-CRH/Xaa-NH was
estimated through 1D-NOESY difference experiments by irradiation
at the Xaa-NH frequency. The solvent accessibility of the amide protons
was investigated by considering their resonance shift in CDCl3/DMSO-
X-ray Diffraction. Colorless single crystals of 1 (monohydrate) and
2 were obtained by slow evaporation from cyclohexane/diisopropyl
ether solutions. The X-ray diffraction data were collected at room
temperature on a Nonius CAD-4 four circle diffractometer, using
graphite-monochromated Cu KR radiation (λ ) 1.54180 Å). The
independent reflections were measured in the ω/2θ-scan mode and in
the θ range 1-70°. Unit cell parameters were determined by least-
squares refinement of the setting angles of 25 high-angle reflections
(20° < θ < 30°). The crystal structures were solved by direct methods
using SIR92.23 The E-maps revealed the whole molecules, except the
hydrogen atoms, and the existence of two independent peptide and water
molecules per asymmetric unit for 1 (molecules A and B). Structure
refinement was performed using SHELXL-93.24 Heavy atoms were
affected by anisotropic thermal factors while hydrogen atoms were
(22) (a) Liang, G. B.; Rito, C. J.; Gellman, S. H. Biopolymers 1992, 32,
293-301. (b) Nishihara, H.; Nishihara, K.; Uefuji, T.; Sakota, N. Bull.
Chem. Soc. Jpn. 1975, 48, 553-555.
(23) Altomare, A.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A. J. Appl.
Crystallogr. 1993, 26, 343-350.
(24) Sheldrick, G. M. SHELXL 93, Program for the Refinement of Crystal
Structures; University of Go¨ttingen: Germany, 1993.
(25) Taylor, R.; Kennard, O. Acta Crystallogr., Sect. B 1983, 39, 133-
138.

Phenylalanine Cyclohexane Analogues
J. Am. Chem. Soc., Vol. 122, No. 24, 2000 5815
Figure 3. Stereoviews of the âI- and âII-folded crystal molecular
structures of 1 (molecule A) and 2, indicating the intramolecular
hydrogen bond.
d6 mixtures with increasing DMSO-d6 content. The signal of a solvent-
exposed NH is rapidly downfield shifted, whereas that of a solvent-
protected proton (most probably by hydrogen bonding) is only weakly
affected by DMSO-d6 NH-solvation.16a,17,26
Figure 4. Bond distances (Å) and bond angles (deg) for the
cyclohexane ring in the crystal molecular structures of 1 (molecules A
and B) and 2. CâL and CâD denote the carbon atom in the position
corresponding to an L- and D-residue, respectively.
Theoretical Analysis. Molecular mechanics calculations on the
c6Phe residue, as well as on the Ac-Pro-Xaa-NHMe dipeptides, were
performed using the AMBER 4.1 program27 with the standard Cornell
(parm94) force field set of parameters.28 Computations were carried
out in Vacuo, using a dielectric constant of 1. Low-energy minima were
characterized using the conjugate gradient algorithm of the SANDER
module with a convergence criterium of 0.001 kcal/mol Å-1 on the
energy gradient. Ab initio calculations on the c6Phe residue were
performed using the Gaussian94 suite of programs.29 Hartree-Fock as
well as second-order Møller-Plesset (MP2) calculations were carried
out using the standard 6-31(d) basis set.30 Energy decomposition
analysis of the formamide-benzene interaction was performed using
the constrained space orbital variation method31 implemented in the
HONDO8.5 program.32 This procedure permits the partition of the
different contributions to the SCF interaction energy by computing the
frozen core-core contribution, a polarization term, and a charge-transfer
term, which is corrected for the basis set superposition error (BSSE)
with the counterpoise method.33 In addition, the dispersion contribution
Table 2. Main Torsion Anglesa (deg) with Standard Deviations for
Peptides 1b and 2
frag-
ment angle
1A
-175.3(2)
-56.2(3)
-38.1(3)
-175.8(2)
-36.7(3)
41.4(3)
-30.6(3)
7.6(3)
18.3(3)
-62.2(3)
-17.4(3)
-178.6(3)
60.3(3)
1B
-163.4(3)
-68.7(3)
-28.3(3)
-179.3(2)
22.5(4)
-33.8(4)
31.3(4)
-16.8(3)
-3.1(3)
-81.4(3)
-5.2(3)
2
Piv
Pro
ω
-178.2(3)
φc
ψc
ω
-60.5(4)
144.2(2)
169.3(2)
-18.8(4)
33.5(4)
ø1
ø2
ø3
ø4
θ
-35.0(4)
24.0(4)
-3.3(4)
65.8(3)
c6Phe φc
ψc
ω
19.1(4)
-178.1(2)
62.3(2)
84.4(3)/-96.0(3)
174.3(3)
-72.2(3)
77.9(4)/-104.1(3)
ø1 d
ø2 d
86.7(3)/-97.1(3)
a See ref 34 for definition. b Two independent molecules per asym-
metric unit (molecules A and B). c Values for ideal â-turns are (ref
42) the following: φi+1 ) -60 (âI, âII); ψi+1 ) -30 (âI), 120 (âII);
φi+2 ) -90 (âI), 80 (âII); ψi+2 ) 0 (âI, âII). d This angle refers to the
orientation of the phenyl ring.
(26) Pitner, T. P.; Urry, D. W. J. Am. Chem. Soc. 1972, 94, 1399-1400.
(27) Pearlman, D. A.; Case, D. A.; Caldwell, J. W.; Ross, W. S.;
Cheatham, T. E., III; Ferguson, D. M.; Seibel, G. L.; Singh, U. C.; Weiner,
P. K.; Kollman, P. A. AMBER 4.1; University of California: San Francisco,
1995.
(28) Cornell, W. D.; Cieplak, P.; Bayly, C. L.; Gould, I. R.; Merz, K.
M., Jr.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Cadwell, J. W.;
Kollman, P. A. J. Am. Chem. Soc. 1995, 117, 5179-5197.
(29) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Gill, P. M. W.;
Johnson, B. G.; Robb, M. A.; Cheeseman, J. R.; Keith, T.; Petersson, G.
A.; Montgomery, J. A.; Raghavachari, K.; Al-Laham, M. A.; Zakrzewski,
V. G.; Ortiz, J. V.; Foresman, J. B.; Peng, C. Y.; Ayala, P. Y.; Chen, W.;
Wong, M. W.; Andres, J. L.; Replogle, E. S.; Gomperts, R.; Martin, R. L.;
Fox, J.; Binkley, J. S.; Defrees, D. J.; Baker, J.; Stewart, J. J. P.; Head-
Gordon, M.; Gonzales, C.; Pople, J. A. Gaussian94, ReVision B.2; Gaussian
Inc.: Pittsburgh, PA, 1995.
to the interaction energy was computed as the difference between the
Hartree-Fock and MP2 energies, once the BSSE had been corrected.
Results and Discussion
Crystal Structures. Figure 3 shows the X-ray diffraction
structures of 1 (molecule A) and 2. The dimensions of the
cyclohexane ring in the c6Phe residues are indicated in Figure
4. Table 2 summarizes the most relevant torsion angles.34 The
geometry of the intra- and intermolecular hydrogen bonds is
(30) Hariharan, P. C.; Pople, J. A. Theor. Chim. Acta 1973, 28, 213-
222.
(31) Bagus, T. S.; Hermann, K.; Bauschlicher, C. W., Jr. J. Chem. Phys.
1984, 81, 1966-1974.
(32) Dupuis, M.; Johnson, F.; Marquez, J. A. HONDO8.5 from Chem-
Station, 1994; IBM Corporation: Kingston, NY 12401. Implementation of
the constrained space orbital Variation method to HONDO8.5 by Marquez,
J. A.; Rubio, J.; Illas, F. 1994.
t
given in Table 3. The Bu methyl groups in both 1 and 2, as
well as the iPr methyl groups and the water molecules in 1, are
thermally agitated. All amide bonds assume the usual trans
(34) IUPAC-IUB Commission on Biochemical Nomenclature: Biochem-
istry 1970, 9, 3471-3479.
(33) Boys, S. B.; Bernardi, F. Mol. Phys. 1970, 19, 553-566.

5816 J. Am. Chem. Soc., Vol. 122, No. 24, 2000
Jime´nez et al.
Table 3. Dimensions (Å, deg) with Standard Deviations of the Hydrogen Bondsa for Peptides 1b (Monohydrate) and 2
peptide
donor-H
acceptor
symmetry code
donor‚‚‚acceptor
H‚‚‚acceptor
donor-H‚‚‚acceptor
1b
NH(iPr)A
NH(iPr)B
W-HA
W-H′A
W-HB
Piv-C′OA
Piv-C′OB
Piv-C′OA
c6Phe-C′OB
W-OA
c6Phe-C′OA
Piv-C′O
x; y; z
x; y; z
x; y; z
x; y; z
x; 1 + y; z
x; y; z
x; y; z
x; y; -1 + z
3.039(3)
3.098(4)
2.966(4)
2.833(5)
2.875(6)
2.828(6)
3.354(4)
3.058(3)
2.05(2)
2.10(4)
2.11(4)
1.93(4)
1.99(4)
2.30(5)
2.43(3)
2.05(2)
163(3)
165(3)
144(3)
151(4)
146(3)
113(3)
151(2)
168(3)
W-H′B
2
NH(iPr)
c6Phe-NH
c6Phe-C′O
a The NH hydrogens have been replaced at 1.03 Å from N in the direction found by refinement.25 b Two independent molecules per asymmetric
unit. The subscript A or B refers to molecule A or B, respectively.
conformation,35 with only Pro-ω in 2 (169°) and Piv-ω in 1B
(-163°) deviating significantly from planarity. The Piv-Pro
amide link is forced to accommodate the trans disposition for
steric reasons.22 The proline ring adopts the twisted Câ-endo/
Cγ-exo conformation in 1A, the envelope Cγ-endo conformation
in 1B, and the envelope Cγ-exo conformation in 2.36
respective Phe analogues41 (Figure 1). As for ø2, which is not
restricted by covalent constraints, the values observed in all three
cases lie close to (90°. This ø2-conformation, which is generally
preferred by aromatic residues in peptides and proteins,41
minimizes steric interactions between the phenyl ring and the
backbone atoms. Another notable feature is the proximity
between the c6Phe amino and phenyl groups, with the distance
The dimensions of the cyclohexane ring in the c6Phe moieties
are quite similar to those reported for the Ac6c residue.37 It
should be noted, however, that the endocyclic CâL-CR-CDâ
bond angle is smaller (107-109°), and the distance between
CR and the Câ bearing the phenyl group increases with respect
to the unsubstituted Ac6c residue. These variations, together with
the large exocyclic bond angles corresponding to phenyl
branching (112-116°), must be ascribed to the steric interactions
arising from â-substitution. In all three molecules the six-
membered ring accommodates a chair conformation, with a
mean absolute value for the endocyclic torsion angles of about
57°, which is slightly higher than that theoretically predicted
for cyclohexane (54.7°).38 As expected, the phenyl ring and the
carbonyl group assume the equatorial disposition, while the
amino substituent is oriented axially. It is noteworthy that in
all crystal structures containing Ac6c so far described, with the
exception of the free amino acid,39,40d the amino group occupies
the axial position.37,40 Clearly, in the case of the c6Phe deriv-
atives, the presence of the aromatic substituent in a trans relative
stereochemistry with respect to the carbonyl group will further
stabilize this conformation. As a consequence, and according
to our expectations, the rigidly held phenyl ring adopts a gauche
(+) disposition in 1 (ø1 ) 60° in 1A and 62° in 1B) and a slightly
distorted gauche (-) orientation in 2 (ø1 ) -72°), both
corresponding to the least favored staggered rotamer in their
between the N atom and the closest aromatic ring C atom (Cipso
being 3.02 Å in 1A, 2.98 Å in 1B, and 3.19 Å in 2.
)
All three molecules are stabilized by an intramolecular
NH(iPr) to Piv-C′O hydrogen bond that closes a 10-membered
pseudocycle typical of a â-turn.42 The N‚‚‚O distance of 3.35
Å in 2 is at the upper limit for hydrogen bonding43 and is larger
than the corresponding distance in 1 (3.04 and 3.10 Å). The
orientation of the middle amide group, with the proline C′dO
and CR-H bonds in the syn disposition for 1 and the anti
disposition for 2, corresponds to the type I and II â-turn,42
respectively (Figure 3). It should be noted that, as far as the
residue in position i + 2 is concerned, the fundamental
difference between the âI- and âII-turns lies in the sign of the
φ angle. Comparison of the torsion angles gathered in Table 2
shows that the âI-folded structures adopted by molecules A and
B of dipeptide 1 differ mainly in the conformation of c6Phe.
Thus, 1B exhibits c6Phe-φ,ψ values close to those expected for
an ideal type I â-turn, while the âI structure of molecule 1A is
distorted at the c6Phe residue.
The molecular packing is completely different in the two
crystals. The âII-folded molecules of 2 are intermolecularly
hydrogen bonded by a c6Phe-NH to c6Phe-C′O interaction with
a N‚‚‚O distance of 3.06 Å. In 1 monohydrate, the molecules
interact by W-H to C′O hydrogen bonds with two connected
water molecules so that the water dyad is in contact with two
molecules A and one molecule B. Contrary to the (R,R)c6Phe-
NH in 2, the (S,S)c6Phe-NH in 1 is not involved in any
intermolecular interaction. This finding is in line with the known
lower accessibility of the Xaa-NH site in âI-folded Pro-Xaa
sequences.17
(35) Benedetti, E. In Chemistry and Biochemistry of Amino Acids,
Peptides and Proteins; Weinstein, B., Ed.; Marcel Dekker: New York, 1982;
Vol. 6, pp 105-184.
(36) (a) Nair, C. M. K.; Vijayan, M. J. Indian Inst. Sci. C 1981, 63,
81-103. (b) DeTar, D. F.; Luthra, N. P. J. Am. Chem. Soc. 1977, 99, 1232-
1244. (c) Ashida, T.; Kakudo, M. Bull. Chem. Soc. Jpn. 1974, 47, 1129-
1133.
(37) (a) Pavone, V.; Benedetti, E.; Barone, V.; Di Blasio, B.; Lelj, F.;
Pedone, C.; Santini, A.; Crisma, M.; Bonora, G. M.; Toniolo, C.
Macromolecules 1988, 21, 2064-2070. (b) Paul, P. K. C.; Sukumar, M.;
Bardi, R.; Piazzesi, A. M.; Valle, G.; Toniolo, C.; Balaram, P. J. Am. Chem.
Soc. 1986, 108, 6363-6370.
(38) Bixon, M.; Lifson, S. Tetrahedron 1967, 23, 769-784.
(39) Varughese, K. I.; Chacko, K. K.; Zand, R. Acta Crystallogr., Sect.
B 1975, 31, 866-868.
It is pertinent to mention that both Piv-L-Pro-L-Phe-NHMe17
and Piv-L-Pro-D-Phe-NHMe44 have been shown to accommodate
a type II â-turn in the solid state, the aromatic side chain lying
close to the gauche (-) (ø1 ) -42°) and gauche (+) (ø1 )
74°) dispositions, respectively. With regard to Ac6c, the two
crystal structures containing the Pro-Ac6c sequence described
(40) (a) Stra¨ssler, C.; Linden, A.; Heimgartner, H. HelV. Chim. Acta 1997,
80, 1528-1554. (b) Fabiano, N.; Valle, G.; Crisma, M.; Toniolo, C.;
Saviano, M.; Lombardi, A.; Isernia, C.; Pavone, V.; Di Blasio, B.; Pedone,
C.; Benedetti, E. Int. J. Pept. Protein Res. 1993, 42, 459-465. (c) Benedetti,
E.; Di Blasio, B.; Pavone, V.; Pedone, C.; Santini, A.; Crisma, M.; Toniolo,
C. Acta Crystallogr., Sect. C 1989, 45, 634-638. (d) Valle, G.; Crisma,
M.; Toniolo, C.; Sen, N.; Sukumar, M.; Balaram, P. J. Chem. Soc., Perkin
Trans. 2 1988, 393-398. (e) Bardi, R.; Piazzesi, A. M.; Toniolo, C.;
Sukumar, M.; Raj, P. A.; Balaram, P. Int. J. Pept. Protein Res. 1985, 25,
628-639. (f) Chacko, K. K.; Srinivasan, R.; Zand, R. J. Cryst. Mol. Struct.
1971, 1, 261-269.
(41) (a) Gould, R. O.; Gray, A. M.; Taylor, P.; Walkinshaw, M. D. J.
Am. Chem. Soc. 1985, 107, 5921-5927. (b) Benedetti, E.; Morelli, G.;
Ne´methy, G.; Scheraga, H. A. Int. J. Pept. Protein Res. 1983, 22, 1-15.
(42) Rose, G. D.; Gierasch, L. M.; Smith, J. A. AdV. Protein Chem. 1985,
37, 1-109.
(43) (a) Go¨rbitz, C. H. Acta Crystallogr., Sect. B 1989, 45, 390-395.
(b) Taylor, R.; Kennard, O.; Versichel, W. Acta Crystallogr., Sect. B 1984,
40, 280-288. (c) Ramakrishnan, C.; Prasad, N. Int. J. Pept. Protein Res.
1971, 3, 209-231.
(44) Bardi, R.; Piazzesi, A. M.; Toniolo, C.; Sen, N.; Balaram, H.;
Balaram, P. Acta Crystallogr., Sect. C 1988, 44, 1972-1976.

Phenylalanine Cyclohexane Analogues
J. Am. Chem. Soc., Vol. 122, No. 24, 2000 5817
Table 4. Amide NH and C′O Stretching Frequenciesa (cm-1) for Piv-L-Pro-Xaa-NHiPr 1-3 in CH2Cl2 and DMSO Solution (c ) 0.005 M)
and Comparison with the Analogous Dipeptides Incorporating L- and D-Pheb
Xaa-NH
bonded free
(S,S)c6Phe 3310 vw 3381 m 3435 vw 3359 s
NH(iPr)
free bonded
Piv-C′O
Pro-C′O
Xaa-C′O
solvent peptide
Xaa
free
âI-bonded âII-bonded γ-bonded âI-free âII-free
free
CH2Cl2
1
2
3
1614 s
1613 w
1614 m
1596 vw 1689 s
1686 w 1701 s
1648 s
1646 s
1655 s
1666 s
1666 s
1647 s
1648 s
1650 s
(R,R)c6Phe
Ac6c
L-Phe
3404 m
3428 m
3360 s
3362 s
1603 s
1603 m
1693 s
3413 m 3450 wc 3355 sc 1620 w 1612 s
1685 s
1687 s
D-Phe
3419 m 3450 vwc 3345 sc
1614 w
1610 s
1601 s
1692 s
DMSO
1
2
3
(S,S)c6Phe
(R,R)c6Phe
Ac6c
L-Phe
D-Phe
3380 m
3400 m
3350 s
3365 s
1606 s
1605 s
1604 m
1603 m
1689 s
1687 s
1685 m 1667 s
1684 s 1666 s
3271d
3357 s
3273c,d
3260c,d
3337 mc 1619 m
3328 sc 1619 w
a Strong (s), medium (m), weak (w), and very weak (vw) absorption band. b See ref 16a. c The NH(iPr) group is replaced by NH(Me). d Broad
absorption denoting a DMSO-solvated free NH.
Table 5. Chemical Shift and Solvent Sensitivity (ppm) of the Amide NH Proton Resonances for Piv-L-Pro-Xaa-NHiPr 1-3 (c ) 0.01 M),
Nuclear Overhauser Enhancement of the Pro-CRH Proton Resonance by Irradiation at the Xaa-NH Resonance, and Comparison with the
Analogous Dipeptides Incorporating L- and D-Phea
Xaa-NH
NH(iPr)
nOe (%)
DMSO-d6
peptide
Xaa
CDCl3
DMSO-d6
∆δb
CDCl3
DMSO-d6
∆δb
CDCl3
1
2
3
(S,S)c6Phe
(R,R)c6Phe
Ac6c
L-Phe
D-Phe
6.18
5.57
5.96
6.11
6.74
7.82
-0.07
1.17
1.86
1.81
2.37
6.20
6.76
6.87
6.55
6.44
7.13
0.35
-0.32
0.26
4
21
26
8
16
47
55
d
0.97c
0.56c
27
d
a See ref 16a. b Shift of the NH proton resonance on going from CDCl3 to DMSO-d6. c The NH(iPr) group is replaced by NH(Me). d Not determined
because of Pro-CRH and Phe-CRH resonance overlapping.
thus far37b,40b are also âII-folded. Not surprisingly, in all four
cases the Xaa-NH sites (Xaa ) L-Phe, D-Phe, Ac6c) are
intermolecularly hydrogen bonded to carbonyl groups of sym-
metry related molecules.
Structures in Solution. The conformational tendencies of
1, 2, and 3 were investigated by FTIR absorption and 1H NMR
in chlorinated solvents of low polarity (CH2Cl2, CDCl3) and in
the NH-solvating medium DMSO. Table 4 lists the amide
stretching frequencies and Table 5 shows the NMR data. Results
previously reported16a for Piv-L-Pro-L-Phe-NHMe and Piv-L-
Pro-D-Phe-NHMe are included for comparison.
NHMe for the âI structure,16a,17 and in both cases the presence
of âII-folded molecules can be excluded since no absorption is
discernible at about 1603 cm-1. These two systems do, however,
show important differences. A nonnegligible percentage of open
conformation is present for the latter, as evidenced by the IR
bands denoting free Piv-C′O and NH(Me) vibrators.16a The NH-
(iPr) in 1 also exhibits a minor contribution at high frequency,
but the absence of any absorption near 1620 cm-1 excludes the
existence of free Piv-C′O sites. The only possibility compatible
with these observations is the occurrence of an (S,S)c6Phe-NH
to Piv-C′O γ-folded45 conformer. The involvement of these
vibrators in a hydrogen bond is in fact confirmed by the weak
contributions at 3310 and 1596 cm-1, which are also found in
Piv-Pro-NHMe, where the only folded form can be γ-like. The
γ-turn structure is known to be accommodated by some Pro-
Xaa sequences in CCl4 solution, and to disappear in the presence
of more polar chlorinated solvents.17,46
As stated above, the âI and âII conformations predominantly
adopted in CH2Cl2 by 1 and 2, respectively, were attributed on
the basis of the Piv-C′O stretching frequencies. Two other
criteria allowed us to corroborate this assignment. We have
proposed16a,17 that the free Pro-C′O stretching frequency should
be higher for the type II than for the type I â-turn, respectively
translating the syn or anti disposition of the (i + 1) proline
N-CR and C′dO bonds (Figure 3). Dipeptide 1, which
exclusively adopts the type I â-turn, gives rise to a single Pro-
C′O absorption at 1689 cm-1, while 2, which is preferentially
âII-folded, presents a major contribution at 1701 cm-1 and a
weak shoulder at 1686 cm-1. Compound 3, which exhibits high
percentages of both âI- and âII-turns, presents two partially
overlapping bands centered at 1693 cm-1. The type I and II
â-turns also differ in the Pro-CRH/Xaa-NH interproton distance,
which is shorter for the latter (Figure 3) and should therefore
In CH2Cl2 solution, the NH(iPr) in 3 displays the typical
behavior of a hydrogen-bonded site. It exhibits an intense and
broad absorption near 3360 cm-1 and its proton resonance
experiences a very small shift on going from CDCl3 to DMSO-
d6. In comparison, the free Ac6c-NH gives rise to a sharp band
at 3428 cm-1 and its resonance shows a much higher sensitivity
to solvent polarity. In the CdO stretching region, the Piv-C′O
absorption is shifted to lower frequencies in comparison to that
encountered at 1619 cm-1 for Piv-Pro-OMe, where it is free
from any intramolecular interaction. It ensues that 3 is folded
in a â-turn conformation stabilized by an NH(iPr) to Piv-C′O
intramolecular hydrogen bond. The composite band observed
for Piv-C′O, with contributions at 1614 and 1603 cm-1 of
approximate relative intensity 45/55, denotes the occurrence of
two different hydrogen-bonded states for this carbonyl and we
have identified these as corresponding to the type I and II â-turn,
respectively.16a
The compound incorporating (R,R)c6Phe (2) also adopts a
â-turn structure in CH2Cl2 solution, with a clear preference for
the âII-disposition. The âI/âII ratio of about 20/80, according
to the relative intensities of their respective Piv-C′O contribu-
tions, is in fact very similar to that encountered for Piv-L-Pro-
D-Phe-NHMe.16a
(45) Ne´methy, G.; Printz, M. P. Macromolecules 1972, 5, 755-758.
(46) Boussard, G.; Marraud, M.; Aubry, A. Biopolymers 1979, 18, 1297-
1331.
The behavior of the (S,S)c6Phe-containing dipeptide (1) in
this solvent also parallels the preference of Piv-L-Pro-L-Phe-

5818 J. Am. Chem. Soc., Vol. 122, No. 24, 2000
Jime´nez et al.
be reflected by a stronger nOe effect. In line with the IR
absorption results in CH2Cl2, the enhancement of the Pro-CRH
signal in CDCl3 solution upon irradiation of the c6Phe-NH
resonance proved significantly smaller for 1 than for 2.
solution and is in agreement with the moderate or extremely
low sensitivity of the c6Phe-NH proton resonance to solvation.
The fact that in 2 this resonance is downfield shifted by 1.17
ppm on going from CDCl3 to DMSO-d6, while there is no IR
indication for NH-solvation, suggests that the DMSO molecules
are not in direct contact with the (R,R)c6Phe-NH proton to form
a hydrogen bond, but sufficiently close to perturb its magnetiza-
tion state. It is worth mentioning that the proximity of the phenyl
ring to the c6Phe-NH for both 1 and 2 inferred from the
observations discussed above constitutes the main argument in
favor of the assignment of a type I â-turn structure for 1 and a
âII disposition for 2 in DMSO solution.
Theoretical Analysis. The experimental data reveal that
dipeptides 1 and 2 display a marked propensity for the âI- and
âII-turn, respectively. The relative stability of these structures
for each compound has been analyzed by theoretical calcula-
tions, and the possible influence of a c6Phe-NH to aromatic ring
attractive interaction on the observed â-turn tendencies has been
examined.
Previously, we assessed the suitability of molecular mechanics
calculations to describe the NH‚‚‚π interaction, taking form-
amide-benzene as a model system. The ability of the π electron
density of aromatic compounds to act as a Lewis base and thus
interact with proton donors has long been noted,47 and more
recently has been recognized as an important factor in peptide
and protein stability.48 At the theoretical level, complexes
between benzene and a variety of HX systems (X ) F, Cl, OH,
SH, NH2, CN, CCl3) have been characterized.48d,g,49-51 In most
cases, a symmetric structure with the HX molecule placed over
the center of the phenyl ring has been suggested as the most
favorable topology,48d,g,49 but considerably off-centered geom-
etries with H-X pointing toward the midpoint of a C-C bond
have also been reported.50 Nevertheless, in crystallized proteins
the N-H groups are found to lie parallel to aromatic rings rather
than adopt a perpendicular disposition.48d-h
The behavior displayed by the c6Phe-NH in 1 and 2 deserves
particular comment. Apart from the minor γ-folded conformer
detected for 1 and characterized by a weak absorption at 3310
cm-1, c6Phe-NH is not hydrogen bonded to any carbonyl group
in the molecule. However, and contrary to what would be
expected for a free NH group,16a,17,26 the (S,S)c6Phe-NH proton
resonance is unaffected by addition of DMSO-d6 and that of
(R,R)c6Phe-NH is actually downfield shifted, although to a lesser
extent than is usually encountered for free NHs (2-3 ppm).
This weak or extremely weak sensitivity to solvation must be
interpreted in terms of solvent inaccessibility. Protection from
solvent certainly arises from the close proximity to the phenyl
ring, which also results in a very low stretching frequency in
CH2Cl2 for the free c6Phe-NH vibrator at 3381 and 3404 cm-1
in 1 and 2, respectively. Thus, the shift by about 50 cm-1 in 1
and 25 cm-1 in 2 with reference to the absorption at 3428 cm-1
exhibited by Ac6c-NH in 3 evidences the existence of an
interaction between the free c6Phe-NH and the aromatic ring.
Such an attractive interaction involving the Phe side chain
and NH in Pro-Phe dipeptides, and being more intense for
homochiral than for heterochiral sequences, has been invoked
to explain their respective preferences for the âI- and âII-forms
in low-polarity solvents.17 This interaction decreases the NH
frequency by about 15 cm-1 for L-Phe and 10 cm-1 for D-Phe
with respect to that found near 3430 cm-1 for L-/D-Ala-NH in
analogous dipeptides. Moreover, it would also account for the
predominance of the Phe CR-Câ gauche (+) and gauche (-)
dispositions respectively observed in CDCl3 for the L-Pro-L-
Phe and L-Pro-D-Phe sequences,17 despite being sterically
disfavored orientations41 (Figure 1). In this solvent the c6Phe-
CâH benzylic proton exhibits one small and one large vicinal
coupling constant (3.0 and 12.9 Hz for 1, 3.0 and 13.2 Hz for
2), which denote the axial orientation of the Câ-H bond and
therefore the equatorial disposition of the aromatic substituent.
Consequently, and as occurs in the crystal molecular structures,
the cyclohexane moiety forces the phenyl ring into the gauche
(+) disposition for 1 and the gauche (-) orientation for 2, thus
favoring the interaction between the c6Phe-NH and the aromatic
side chain.
The interaction curve between formamide and benzene was
computed as described in the Experimental Section. Calculations
were performed with the SCF-optimized geometries of both
(47) (a) Pimentel, G. C.; McClellan, A. L. The Hydrogen Bond;
Freeman: San Francisco, 1960. (b) Kopple, K. D.; Marr, D. H. J. Am. Chem.
Soc. 1967, 89, 6193-6200. (c) Robinson D. R.; Jencks, W. P. J. Am. Chem.
Soc. 1965, 87, 2470-2479.
(48) (a) Nishio, M.; Hirota, M.; Umezawa, Y. The CH/π Interaction:
EVidence, Nature, and Consequences; John Wiley & Sons: New York,
1998. (b) Steiner, T.; Schreurs, A. M. M.; Kanters, J. A.; Kroon, J. Acta
Cryst., Sect. D 1998, 54, 25-31. (c) Crisma, M.; Formaggio, F.; Valle, G.;
Toniolo, C.; Saviano, M.; Iacovino, R.; Zaccaro, L.; Benedetti, E. Biopoly-
mers 1997, 42, 1-6. (d) Worth, G. A.; Wade, R. C. J. Phys. Chem. 1995,
99, 17473-17482. (e) Mitchell, J. B. O.; Nandi, C. L.; McDonald, I. K.;
Thornton, J. M. J. Mol. Biol. 1994, 239, 315-331. (f) Flocco, M. M.;
Mowbray, S. L. J. Mol. Biol. 1994, 235, 709-717. (g) Mitchell, J. B. O.;
Nandi, C. L.; Ali, S.; McDonald, I. K.; Thornton, J. M. Nature 1993, 366,
413. (h) Singh, J.; Thornton, J. M. J. Mol. Biol. 1990, 211, 595-615.
(49) (a) Rodham, D. A.; Suzuki, S.; Suenram, R. D.; Lovas, F. J.;
Dasgupta, S.; Goddard, W. A., III; Blake, G. A. Nature 1993, 362, 735-
737. (b) Suzuki, S.; Green, P. G.; Bumgarner, R. E.; Dasgupta, S.; Goddard,
W. A., III; Blake, G. A. Science 1992, 257, 942-945. (c) Jorgensen, W.
L.; Severance, D. L. J. Am. Chem. Soc. 1990, 112, 4768-4774. (d) Tang,
T.-H.; Hu, W.-J.; Yan, D.-Y.; Cui, Y.-P. J. Mol. Struct. (THEOCHEM)
1990, 207, 319-326. (e) Levitt, M.; Perutz, M. F. J. Mol. Biol. 1988, 201,
751-754. (f) Bre´das, J. L.; Street, G. B. J. Am. Chem. Soc. 1988, 110,
7001-7005. (g) Menapace, J. A.; Bernstein, E. R. J. Phys. Chem. 1987,
91, 2533-2544.
(50) (a) Alkorta, I.; Rozas, I.; Elguero, J. Chem. Soc. ReV. 1998, 27,
163-170. (b) Rozas, I.; Alkorta, I.; Elguero, J. J. Phys. Chem. A 1997,
101, 9457-9463.
(51) (a) Augspurger, J. D.; Dykstra, C. E.; Zwier, T. S. J. Phys. Chem.
1992, 96, 7252-7257. (b) Cheney, B. V.; Schulz, M. W. J. Phys. Chem.
1990, 94, 6268-6273. (c) Cheney, B. V.; Schulz, M. W.; Cheney, J.;
Richards, W. G. J. Am. Chem. Soc. 1988, 110, 4195-4198. (d) Karlstro¨m,
G.; Linse, P.; Wallqvist, A.; Jo¨nsson, B. J. Am. Chem. Soc. 1983, 105,
3777-3782.
In DMSO-d6 solution, the phenyl substituent is also equatorial
in both c6Phe residues. In contrast, DMSO NH-solvation results
in a modification of the side chain rotameric preferences in the
Pro-Phe sequences,17 along with the presence of high percent-
ages of open conformers.16a,17 In addition, the folded form in
Piv-L-Pro-L-Phe-NHMe becomes of the âII-type.16a
In this solvent the NH(iPr) stretching frequency in 1-3 retains
practically the same value as in CH2Cl2, meaning that this
vibrator is still intramolecularly hydrogen bonded, a situation
that is in line with the very weak solvent sensitivity of its proton
resonance. For all three compounds, Piv-C′O gives rise to a
single peak, denoting the sole existence of one-folded conformer.
This corresponds to the âI-turn in 1 and the âII-turn in 2 and
3, as evidenced by the magnitude of the Pro-CRH/Xaa-NH nOe.
The presence of open forms can be ruled out, since no Piv-C′O
contribution can be detected near 1620 cm-1
.
DMSO is known to interact with accessible NHs and, indeed,
the Ac6c-NH absorption is actually shifted from 3428 cm-1 in
CH2Cl2 to 3271 cm-1, while the c6Phe-NH in 1 and 2 exhibits
the same frequency as in CH2Cl2. This result provides evidence
for the retention of the NH to π-orbitals interaction in DMSO

Phenylalanine Cyclohexane Analogues
J. Am. Chem. Soc., Vol. 122, No. 24, 2000 5819
Figure 5. Energy profile and decomposition analysis of the form-
amide-benzene interaction (see Experimental Section for details).
Comparison with the results of AMBER calculations.
Table 6. Geometrical Features of the Minima Obtained for
Ac-(S,S)c6Phe-NHMe by Molecular Mechanics and ab Initio
Calculations (φ,ψ, deg; ∆E, kcal/mol)
AMBER
ab initio
confor-
mation
eqa
φ
ψ
∆E
0.0 -74
47 -10 3.0 51 -1
-59 167 5.9 -64 170
-179 -179 8.1 180 180
-63 25 2.6 -76 70
61 -10 3.1 74 -72
φ
ψ
∆E(SCF) ∆E(MP2)
A
B
C
D
A
B
-56
8
30
0.0
6.2
3.5
5.9
2.9
4.1
0.0
5.4
4.0
5.9
3.0
3.2
axa
a Equatorial (eq) or axial (ax) disposition of the phenyl ring.
molecules. On keeping benzene fixed, two approaching paths
for the formamide molecule were investigated: on the center
and on a C-C bond of the benzene ring. Both pathways exhibit
a low-energy conformation at a distance of about 2.7 Å with a
well depth between 2.5 and 2.8 kcal/mol. When the system is
completely relaxed the only minimum found at the SCF level
shows the formamide molecule approximately on the center of
the aromatic ring, with a distance from the closer formamide
hydrogen to the plane of the aromatic ring of 2.68 Å. The
interaction energy computed with this geometry at the MP2 level
and corrected for the basis set superposition error is 2.94 kcal/
mol. In an attempt to better characterize the nature of this
interaction, the different contributions to the energy were
assessed following the constrained space orbital variation
method. These contributions are shown in Figure 5, and their
analysis reveals the electrostatic term to be the larger. These
results suggest that this interaction can be well accounted for
in force field calculations. For the sake of comparison, the
interaction energy of formamide with benzene computed with
the Cornell force field of AMBER is also represented in Figure
5. Although the minimum differs by about 1 kcal/mol from that
of the ab initio results, the plot clearly shows the ability of the
force field calculations to account for this interaction.
Prior to the analysis of the â-turn preferences of the Pro-
c6Phe sequences, we investigated the conformational space
available to the c6Phe residue to confirm that the theoretical
predictions agree with the experimental evidence. The Rama-
chandran maps of Ac-(S,S)c6Phe-NHMe with the cyclohexane
ring in a chair conformation and the phenyl substituent either
in an equatorial or an axial disposition were computed within
the molecular mechanics framework. Contour energy maps are
shown in Figure 6. Minima, which were characterized by energy
minimization, are depicted by letters, and their geometrical
features are described in Table 6. With the aim of assessing the
Figure 6. (φ,ψ) energy maps obtained by molecular mechanics
calculations for Ac-(S,S)c6Phe-NHMe with the cyclohexane ring in a
chair conformation and the phenyl substituent in an equatorial (up) or
an axial (down) disposition. Energy contours are drawn at intervals of
1.0 kcal/mol and minima are depicted by letters. Minima obtained from
ab initio calculations are indicated with an asterisk.
Table 7. â-Turn Low-Energy Conformations of
Ac-L-Pro-c6Phe-NHMe (φ,ψ, deg; ∆E, kcal/mol)
c6Phe
configuration
Pro-ψ
c6Phe-φ
c6Phe-ψ
∆E
structure
(S,S)
-27
115
111
-63
47
59
-10
17
16
-4.1
2.4
-2.2
âI
âII
âII
(R,R)a
a The âI-turn structure is located in a flat region at about 2.0 kcal/
mol but it does not exhibit a stationary point (the lowest energy
corresponds to Pro-ψ ) 7, c6Phe-φ ) -47, c6Phe-ψ ) 4).
performance of the force field parameters in the description of
the c6Phe residue, minima thus obtained were contrasted with
results of ab initio calculations. The molecular mechanics low-
energy structures were used as starting conformations for
geometry optimization at the Hartree-Fock level. The results

5820 J. Am. Chem. Soc., Vol. 122, No. 24, 2000
Jime´nez et al.
Table 8. Geometrical Features of the Low-Energy Conformations of Ac-L-Pro-c6Phe-NHMe Listed in Table 7a (d ) distance, Å)
c6Phe
structure
d[Ac-C′O‚‚‚NH(Me)]b
d[c6Phe-NH‚‚‚Ph]c
d[Pro-C′O‚‚‚Ph]d
d[c6Phe-C′O‚‚‚Ph]d
(S,S)
âI
âII
âII
1.99
2.08
1.97
2.60
3.98
2.64
5.11
3.05
5.06
3.45
3.20
3.56
(R,R)
a Short contact distances are indicated in bold. b Length of the hydrogen bond closing the C10 cycle. c Distance from H to the center of the
closest phenyl CsC bond. d Distance from O to the closest phenyl C atom.
of these calculations are also listed in Table 6 and minima
obtained are shown in Figure 6 with an asterisk. Comparison
of results indicates that molecular mechanics and ab initio
geometries are within a 15° tolerance, with the exception of
the ψ dihedral angle in the case of the axial conformer, due to
the shallowness of the map in this region. Furthermore, the
relative energies of conformations B and C of the equatorial
conformer are interchanged in the molecular mechanics and ab
initio calculations. Although MP2 calculations correct this
difference to a certain extent, ab initio and molecular mechanics
do not provide the same relative ordering of these two minima.
Despite these differences, both methods clearly support that the
conformation with the benzene ring in the equatorial position
is more favorable, as experimentally established both in solution
and in the solid state, and suggest an acceptable performance
of force field calculations in describing the geometries of the
conformations accessible to this residue. Obviously, the maps
of Ac-(R,R)c6Phe-NHMe exhibit the same features as those of
Ac-(S,S)c6Phe-NHMe since they are related by the correspon-
dence (φ,ψ) ) (-φ,-ψ).
We then studied the conformational propensities of Ac-L-
Pro-(S,S)c6Phe-NHMe and Ac-L-Pro-(R,R)c6Phe-NHMe using
the set of molecular mechanics parameters assessed above and
considering the phenyl substituent to be oriented equatorially.
The terminal pivaloyl and isopropylamide groups in 1 and 2
were replaced by acetyl and methylamide for the sake of
simplicity. Table 7 lists the low-energy conformations obtained.
For the compound incorporating (S,S)c6Phe both type I and II
â-turns appear as minima, the former being more stable by 6.5
kcal/mol, whereas for the (R,R)c6Phe-containing dipeptide only
a âII-turn structure can be located as a low-energy minimum.
Indeed, the âI-turn is situated on a flat section of the confor-
mational energy map about 4.2 kcal/mol above the âII confor-
mation, but it does not exhibit a stationary point. As a typical
feature of â-turns, minima are characterized by an NH(Me) to
Ac-C′O hydrogen bond. Inspection of the structural parameters
of the different low-energy conformations (Table 8) suggests
that the stability of the structures is modulated by the distance
between the c6Phe-NH and the phenyl moiety. Indeed, the two
lowest energy conformations exhibit a c6Phe-NH proton to
aromatic ring distance of about 2.6 Å, which may result in an
attractive NH‚‚‚π interaction.
Table 9. â-Turn Low-Energy Conformations of
Ac-L-Pro-Xaa-NHMe, with Xaa ) 1-Amino-2-cyclopentyl-
cyclohexanecarboxylic Acid (φ,ψ, deg; ∆E, kcal/mol)
Xaa configuration
Pro-ψ
Xaa-φ
Xaa-ψ
∆E
structure
(S,S)
-28
115
-25
108
-63
50
-49
57
2
12
1.0
5.4
3.5
3.2
âI
âII
âI
(R,R)
-14
7
âII
Table 10. Comparison of the Conformational Preferences of
RCO-L-Pro-Xaa-NHR′ Dipeptide Sequences
solution
Xaa
CH2Cl2
DMSO
solid state
(S,S)c6Phea
(R,R)c6Phea
Ac6ca,b
âI . γ
âI
âI
âII . âI
âII g âI
âI . open
âII . âI
âΙ g âII
âII . âI
âII
âII
âII
âII
âII
âII
âII
âII
L-Phec
open > âII
âII > open
âII
D-Phec,d
(S,S)c3Phee
(R,R)c3Phee
âII
a This work. b See refs 37b and 40b. c See refs 16a and 17. d See ref
44. e See ref 16.
tylcyclohexanecarboxylic acid, the âI structure is more stable
than the âII-turn by 4.4 kcal/mol, while in the case of the (R,R)
residue both structures are isoenergetic. Thus, elimination of
the NH‚‚‚π interaction leads to a decrease in the energy
difference between type I and II â-turns for both (S,S) and (R,R)
configurations, suggesting that the attractive interaction between
the c6Phe-NH and the benzene ring stabilizes the âI-turn in the
L-Pro-(S,S)c6Phe sequence and the âII-turn in L-Pro-(R,R)c6Phe.
In the case of the former, steric factors also seem to favor the
âI disposition since this folded form remains preferred in the
absence of the NH‚‚‚π interaction.
Conclusion
The conformational behavior of the Piv-L-Pro-c6Phe-NHiPr
dipeptides investigated in this work is summarized in Table 10,
where it is compared to that of the analogous compounds
incorporating Ac6c, Phe, and c3Phe. Experimental data provide
evidence that the Pro-c6Phe sequences exhibit a high tendency
to â-folding in solution, even in DMSO. The high stability of
the folded conformation in the presence of such a strong
solvating medium is also typical of the Ac6c- and c3Phe-
containing dipeptides and seems to be related to the CR-
tetrasubstituted character of these residues. In contrast, the
unmodified Pro-Phe sequences exhibit high percentages of open
conformers under the same conditions.
The â-folded modes adopted by the Pro-c6Phe dipeptides are
dictated by c6Phe chirality and are little dependent on the solvent
nature and on the solute or crystalline state. The (S,S)c6Phe
residue, analogous to L-Phe with ø1 fixed at about 60°, mainly
or exclusively occupies the i + 2 position of a type I â-turn,
while (R,R)c6Phe, analogous to D-Phe with ø1 constrained near
-60°, shows a marked preference for the âII structure. We have
presented experimental and theoretical arguments showing that
the influence of the side chain disposition on the â-turn type is
In accordance with the experimental results, theoretical
calculations reveal the preference of the L-Pro-(S,S)c6Phe and
L-Pro-(R,R)c6Phe sequences for the âI- and âII-turn, respec-
tively. In an attempt to evaluate the contribution of the aromatic
π cloud to c6Phe-NH attraction to the stability of the lowest
energy conformations obtained, we performed calculations on
similar dipeptides where the possibility for this interaction had
been eliminated. To this end, we replaced the phenyl ring of
the c6Phe residue by a cyclopentyl group. The cyclopentyl
substituent was preferred to the cyclohexyl one because the
former deviates from planarity to a lesser extent and was
therefore considered to be more appropriate to reproduce the
steric effects of the phenyl substituent. The low-energy con-
formations obtained are listed in Table 9. For the Ac-L-Pro-
Xaa-NHMe dipeptide where Xaa is (S,S)-1-amino-2-cyclopen-

Phenylalanine Cyclohexane Analogues
J. Am. Chem. Soc., Vol. 122, No. 24, 2000 5821
related to the fact that the aromatic π-orbitals are involved in
an attractive interaction with the c6Phe-NH.
of specific secondary structures. To gain a deeper knowledge
of the â-folding modulation by side chain orientation, further
investigations on other ø1-restricted surrogates of phenylalanine
are in progress.
In comparison, for the Pro-Ac6c sequence, which does not
have an aromatic substituent, both â-turn types I and II have
practically the same occurrence in low-polarity solvents, and
the latter becomes strongly favored in DMSO. This âI-to-âII
transition characterizes as well the L-Pro-L-Phe dipeptide, which
adopts a âI-turn in CH2Cl2 and a âII structure in DMSO and in
the solid state, and results from the better accessibility of the
middle Xaa-NH in the âII disposition. Solvation and molecular
aggregation involving c3Phe-NH are also able to compensate
for the energy difference between type I and II â-turns in the
case of the cyclopropane derivatives.
Acknowledgment. The authors are grateful to Drs C.
Didierjean and A. Vicherat for technical assistance. Financial
support from the Ministerio de Educacio´n y Cultura (PB97-
0998 and accio´n integrada HF97-46) and Ministe`re des Affaires
Etrange`res (action inte´gre´e 97039) is gratefully acknowledged.
Supporting Information Available: Crystal data, fractional
coordinates for the hydrogen and non-hydrogen atoms, equiva-
lent thermal parameters and anisotropic temperature parameters
for the non-hydrogen atoms, interatomic bond lengths and bond
angles, and torsion angles (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
The retention of the âI-folded conformation of Piv-L-Pro-
(S,S)c6Phe-NHiPr in DMSO and in the crystalline state is
therefore particularly noteworthy and evidences the value of
the cyclohexane analogues of phenylalanine in the induction
JA993568K