Effect of sequence on peptide geometry in 5-tert-butylprolyl type VI β-turn mimics

Article abstract of DOI:10.1021/ja012442w

The influence of sequence on turn geometry was examined by incorporating (2S,5R)-5-tert-butylproline (5-^tBuPro) into a series of dipeptides and tetrapeptides. (2S,5R)-5-tert-Butylproline and proline were respectively introduced at the C-termina

Full text of DOI:10.1021/ja012442w

Published on Web 02/20/2002
Effect of Sequence on Peptide Geometry in 5-tert-Butylprolyl
Type VI â-Turn Mimics
Liliane Halab and William D. Lubell*
Contribution from the De´partement de chimie, UniVersite´ de Montre´al, C. P. 6128,
Succursale Centre Ville, Montre´al, Que´bec, Canada H3C 3J7
Received October 26, 2001
Abstract: The influence of sequence on turn geometry was examined by incorporating (2S,5R)-5-tert-
butylproline (5-tBuPro) into a series of dipeptides and tetrapeptides. (2S,5R)-5-tert-Butylproline and proline
were respectively introduced at the C-terminal residue of N-acetyl dipeptide N′-methylamides 1 and 2. The
conformational analysis of these analogues was performed using NMR and CD spectroscopy as well as
X-ray diffraction to examine the factors that control the prolyl amide (in this text, the term “prolyl amide”
refers to the tertiary amide composed of the pyrrolidine nitrogen of the prolyl residue and the carbonyl of
the N-terminal residue) equilibrium and stabilize type VI â-turn conformation. The high cis-isomer population
with aromatic residues N-terminal to proline was shown to result from a stacking interaction between the
partial positive charged prolyl amide nitrogen and the aromatic π-system as seen in the crystal structure
of 1c. The effect of sequence on the prolyl amide equilibrium of 5-tBuPro-tetrapeptides (Ac-Xaa-Yaa-5-t-
BuPro-Zaa-XMe, 13 and 14) was studied by varying the amino acids at the Xaa, Yaa, and Zaa positions.
High (>80%) cis-isomer populations were obtained with alkyl groups at the Xaa position, an aromatic residue
at the Yaa position, and either an alanine or a lysine residue at the Zaa position of the 5-tBuPro-tetrapeptide
methyl esters in water. Tetrapeptides Ac-Ala-Phe-5-tBuPro-Zaa-OMe (Zaa ) Ala, Lys), 14d and 14f, with
high cis-isomer content adopted type VIa â-turn conformations as shown by their NMR and CD spectra.
Although a pattern of amide proton temperature coefficient values indicative of a hairpin geometry was
observed in peptides 14d and 14f, the value magnitudes did not indicate strong hydrogen bonding in water.
Introduction
protein folding by catalyzing the conversion of the cis isomer
to its more thermodynamically stable trans conformation.5,6 Type
Reverse turns play important roles in protein folding.1 Local
sequence-specific interactions can initiate the folding process
by enhancing turn structures that nucleate hairpins and thereby
stabilize â-pleated sheets.2 On the other hand, cis-trans isomer-
ization about prolyl amide (in this text, the term “prolyl amide”
refers to the tertiary amide composed of the pyrrolidine nitrogen
of the prolyl residue and the carbonyl of the N-terminal residue)
bonds in turn regions can be a rate-limiting step in the folding
mechanism.3 The factors that favor specific isomer geometry
about prolyl amides can thus contribute significantly toward
controlling peptide folding.
The type VI â-turn is a relatively rare secondary structure
that features uniquely an amide cis isomer N-terminal to a
proline residue situated at the i + 2 position of the peptide
bend.1,4 Type VI â-turns play important roles in protein
folding.5-7 They have been shown to be recognition sites for
peptidyl prolyl isomerases (PPIases) which can accelerate
VI â-turns have also been implicated in other important
recognition events of bioactive proteins. For example, a type
VI â-turn conformation has been proposed for thrombin-
catalyzed cleavage of the V3 loop of HIV gp120, a prerequisite
to viral infection.7 In addition, in the X-ray structure of the
ribonuclease S protein, a type VIa â-turn was located at the
central position of a hairpin conformation.4
Type VI â-turns are classified into two sub-types based on
the dihedral angle values of their central i + 1 and i + 2
residues.4 In the type VIa â-turn, the proline ψ-dihedral angle
is equal to 0°, and a 10-membered intramolecular hydrogen bond
exists between the carbonyl oxygen of the i residue and the
amide hydrogen of the i + 3 residue. This intramolecular
hydrogen bond is not present in the type VIb â-turn in which
the proline ψ-dihedral angle value is equal to 150°.4
As a minor isomer, the prolyl amide cis conformer is often
difficult to observe in natural peptides.8 Minor cis conformers
may, however, exhibit significant effects on the transport,
metabolism, and reactivity of biologically active peptides.9 To
(1) (a) Wilmot, C. M.; Thornton, J. M. J. Mol. Biol. 1988, 203, 221. (b) Rose,
G. D.; Gierasch, L. M.; Smith, J. A. AdV. Protein Chem. 1985, 37, 1.
(2) Reviewed in: (a) Dobson, C. M. Curr. Opin. Struct. Biol. 1993, 3, 57. (b)
Fersht, A. R.; Dill, K. A. Curr. Opin. Struct. Biol. 1994, 4, 67.
(3) (a) Grathwohl, C.; Wu¨thrich, K. Biopolymers 1981, 20, 2623 and refs 7-14
therein. (b) Brandts, J. F.; Halvorson, H. R.; Brennan, M. Biochemistry
1975, 14, 4953.
(4) Mu¨ller, G.; Gurrath, M.; Kurz, M.; Kessler, H. Proteins: Struct., Funct.
Genet. 1993, 15, 235.
(5) Reviewed in: (a) Fischer, G. Angew. Chem., Int. Ed. Engl. 1994, 33, 1415.
(b) Liu, J.; Chen, C.-M.; Walsh, C. T. Biochemistry 1991, 30, 2306.
(6) (a) Fischer, S.; Michnick, S.; Karplus, M. Biochemistry 1993, 32, 13830.
(b) Kallen, J.; Walkinshaw, M. D. FEBS Lett. 1992, 300, 286.
(7) Johnson, M. E.; Lin, Z.; Padmanabhan, K.; Tulinsky, A.; Kahn, M. FEBS
Lett. 1994, 337, 4.
(8) Reviewed in: MacArthur, M. W.; Thornton, J. M. J. Mol. Biol. 1991, 218,
397.
9
2474 VOL. 124, NO. 11, 2002 J. AM. CHEM. SOC.
10.1021/ja012442w CCC: $22.00 © 2002 American Chemical Society

Effect of Sequence on Peptide Geometry
A R T I C L E S
enhance the cis-isomer population, several approaches have been
tried to stabilize this geometry by the means of conformational
constraint using structural links and steric interactions.10-18
Alternatively, double bond isosteres have been employed to
mimic the spatial orientation presented by the cis conformer.19
These approaches have achieved effective replication of the
backbone geometry of the type VI â-turn as well as analogues
exhibiting inhibitory activity against PPIases.20 Moreover,
stabilization of a hairpin conformation has been achieved in a
model linear tetrapeptide possessing an indolizidinone amino
acid mimic of the central residues of type VIa â-turn as
demonstrated by NMR in DMSO and IR spectroscopy in
dichloromethane.14d These approaches have succeeded in rep-
licating cis conformer geometry; however, an important aspect
of prolyl amides that many such examples by design fail to
mimic has been the conformational equilibrium exhibited by
prolyl peptides.
contingent upon the stereochemistry of the N-terminal residue.22
Dipeptides possessing Ala and Leu residues adopted type VIa
and VIb â-turn conformations when the N-terminal amino acid
possessed respectively L- and D-configuration as shown by NMR
and CD spectroscopy as well as X-ray analysis.22 Furthermore,
the presence of phenylalanine at the N-terminal of 5-tBuPro
caused a remarkable increase in cis-isomer population (Ac-
Phe-5-tBuPro-NHMe exhibited >90% prolyl amide cis isomer
in water).22
As well as its power to augment the cis-isomer population,
the 5-tert-butyl substituent influences the barrier for amide
isomerization.21b In the case of the (2S,5R)-diastereomer, the
sterically bulky tert-butyl group interacts with the N-terminal
residue such as to twist the amide bond away from planarity.22
In N-acetylproline N′-methylamides, twisting of the prolyl amide
was among factors that caused a reduction in the barrier for
isomerization of 3.7 kcal/mol in the (2S,5R)-5-tBuPro analogue
relative to its proline counterpart.21b The influences of sequence
and stereochemistry on the amide equilibrium become thus more
apparent in (2S,5R)-5-tBuPro peptides, because of the combina-
tion of the reduced isomerization barrier and the enhanced cis-
isomer population.
We have synthesized and used (2S,5R)-5-tert-butylproline (5-
tBuPro) to explore both prolyl amide cis-isomer geometry as
well as the amide isomer equilibrium N-terminal to proline in
various peptides.21-23 In Ac-Xaa-5-tBuPro-NHMe, the 5-tBuPro
residue stabilized type VIa and VIb â-turn conformations
We have synthesized a diverse array of 5-tBuPro peptides
by employing N,N′-bis(2-oxo-3-oxazolidinyl)phosphonic chlo-
ride (BOP-Cl)24 as a coupling reagent to attach different amino
acid electrophiles onto the sterically hindered prolyl residue.
This synthesis achievement has allowed us to explore the
influence of sequence on the equilibrium N-terminal to the prolyl
residues. Study of the effect of sequence on isomer equilibrium
in natural prolyl peptides has previously shown that aromatic
residues adjacent to proline caused an augmentation in the cis-
isomer population.8,25-27a Although aromatic residues N-terminal
to proline have been shown to cause a 10-fold reduction in the
cis to trans isomerization rate,3a to the best of our knowledge,
little has been reported about the factors by which aromatic
residues augment the cis-isomer population and increase the
isomerization energy barrier. Amino acid residues possessing
side chains with hydrogen-bond acceptor and donor moieties
have been shown to stabilize turn conformations when adjacent
to proline.1a,27b We report now the influence of hydrogen-
bonding residues on the prolyl amide equilibrium and the cis-
isomer population.
Examining the influence of sequence on turn geometry, we
have introduced 5-tBuPro into a series of dipeptide and tetra-
peptide analogues possessing aromatic and hydrogen-donor and
acceptor residues. By studying the conformations of these ana-
logues using NMR and CD spectroscopy as well as X-ray
diffraction, we have itemized factors that control the prolyl
amide equilibrium and stabilize type VI â-turn geometry. The
high cis-isomer populations and preponderance of type VIa
â-turn conformation brought about by aromatic amino acid
(9) (a) Yaron, A.; Naider, F. CRC Biochem. Mol. Biol. 1993, 28, 31. (b)
Williams, K. A.; Deber, C. M. Biochemistry 1991, 30, 8919. (c) Brandl,
C. J.; Deber, C. M. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 917. (d) Markley,
J. L.; Hinck, A. P.; Loh, S. N.; Prehoda, K.; Truckses, D.; Walkenhorst,
W. F.; Wang, J. Pure Appl. Chem. 1994, 66, 65. (e) Richards, N. G.; Hinds,
M. G.; Brennand, D. M.; Glennie, M. J.; Welsh, J. M.; Robinson, J. A.;
Biochem. Pharmacol. 1990, 40, 119. (f) Lin, L.-N.; Brandts, J. F.
Biochemistry 1979, 18, 43.
(10) Reviewed in: Etzkorn, F. A.; Travins, J. M.; Hart, S. A. AdV. Amino Acid
Peptidomimetics 1999, 2, 125.
(11) (a) Brady, S. F.; Paleveda, W. J. Jr.; Arison, B. H.; Saperstein, R.; Brady,
E. J.; Raynor, K.; Reisine, T.; Veber, D. F.; Freidinger, R. M. Tetrahedron
1993, 49, 3449. (b) Cumberbatch, S.; North, M.; Zagotto, G. Tetrahedron
1993, 49, 9049. (c) Cumberbatch, S.; North, M.; Zagotto, G. J. Chem. Soc.,
Chem. Commun. 1993, 641. (d) Horne, A.; North, M.; Parkinson, J. A.;
Sadler, I. H. Tetrahedron 1993, 49, 5891.
(12) O¨ sapay, G.; Zhu, Q.; Shao, H.; Chadha, R. K.; Goodman, M. Int. J. Peptide
Protein Res. 1995, 46, 290.
(13) (a) Zabrocki, J.; Dunbar, J. B.; Marshall, K. W.; Toth, M. V.; Marshall, G.
R. J. Org. Chem. 1992, 57, 202. (b) Garofolo, A.; Tarnus, C.; Remy, J.-
M.; Leppik, R.; Piriou, F.; Harris, B.; Pelton, J. T. In Peptides: Chemistry,
Structure and Biology; Rivier, J. E.; Marshall, G. R.; Eds.; ESCOM Science
Publishers B. V.: Leiden, The Netherlands, 1990; pp 833-834. (c) Beusen,
D. D.; Zabrocki, J.; Slomczynska, U.; Head, R. D.; Kao, J. L.-F.; Marshall,
G. R. Biopolymers 1995, 36, 181. (d) Abell, A. D.; Foulds, G. J. J. Chem.
Soc., Perkin Trans. 1 1997, 2475.
(14) (a) Dumas, J.-P.; Germanas, J. P. Tetrahedron Lett. 1994, 35, 1493. (b)
Kim, K.; Dumas, J.-P.; Germanas, J. P. J. Org. Chem. 1996, 61, 3138. (c)
Kim, K.; Germanas, J. P. J. Org. Chem. 1997, 62, 2847. (d) Kim, K.;
Germanas, J. P. J. Org. Chem. 1997, 62, 2853. (e) Gramberg, D.; Robinson,
J. A. Tetrahedron Lett. 1994, 35, 861. (f) Gramberg, D.; Weber, C.; Beeli,
R.; Inglis, J.; Bruns, C.; Robinson, J. A. HelV. Chim. Acta 1995, 78, 1588.
(g) Hoffmann, T.; Lanig, H.; Waibel, R.; Gmeiner, P. Angew. Chem., Int.
Ed. 2001, 40, 3361.
(15) (a) Didierjean, C.; Del Duca, V.; Benedetti, E.; Aubry, A.; Zouikri, M.;
Marraud, M.; Boussard, G. J. Peptide Res. 1997, 50, 451. (b) Zouikri, M.;
Vicherat, A.; Aubry, A.; Marraud, M.; Boussard, G. J. Peptide Res. 1998,
52, 19.
(16) (a) Curran, T. P.; McEnaney, P. M. Tetrahedron Lett. 1995, 36, 191. (b)
Lenman, M. M.; Ingham, S. L.; Gani, D. Chem. Commun. 1996, 85.
(17) (a) Magaard, V. W.; Sanchez, R. M.; Bean, J. W.; Moore, M. L. Tetrahedron
Lett. 1993, 34, 381. (b) An, S. S. A.; Lester, C. C.; Peng, J.-L.; Li, Y -J.;
Rothwarf, D. M.; Welker, E.; Thannhauser, T. W.; Zhang, L. S.; Tam, J.
P.; Scheraga, H. A. J. Am. Chem. Soc. 1999, 121, 11558.
(18) Keller, M.; Sager, C.; Dumy, P.; Schutkowski, M.; Fischer, G. S.; Mutter,
M. J. Am. Chem. Soc. 1998, 120, 2714.
(19) (a) Hart, S. A.; Sabat, M.; Etzkorn, F. A. J. Org. Chem. 1998, 63, 7580.
(b) Andres, C. J.; Macdonald, T. L.; Ocain, T. D.; Longhi, D. J. Org. Chem.
1993, 58, 6609.
(20) Hart, S. A.; Etzkorn, F. A. J. Org. Chem. 1998, 64, 2998.
(21) (a) Beausoleil, E.; L’Archeve´que, B.; Be´lec, L.; Atfani, M.; Lubell, W. D.
J. Org. Chem. 1996, 61, 9447. (b) Beausoleil, E.; Lubell, W. D. J. Am.
Chem. Soc. 1996, 118, 12902. (c) Beausoleil, E.; Lubell, W. D. Biopolymers
2000, 53, 249.
(22) (a) Halab, L.; Lubell, W. D. J. Org. Chem. 1999, 64, 3312. (b) Halab, L.;
Lubell, W. D. J. Peptide Sci. 2001, 7, 92. (c) Halab, L.; Lubell, W. D.
Peptides 2000, Proc. Eur. Peptide Symp., 26th 2000, 815-816.
(23) Be´lec, L.; Slaninova, J.; Lubell, W. D. J. Med. Chem. 2000, 43, 1448.
(24) (a) Van Der Auwera, C.; Anteunis, M. J. O. Int. J. Peptide Protein Res.
1987, 29, 574. (b) Tung, R. D.; Rich, D. H. J. Am. Chem. Soc. 1985, 107,
4342.
(25) (a) Hetzel, R.; Wu¨thrich, K. Biopolymers 1979, 18, 2589. (b) Grathwohl,
C.; Wu¨thrich, K. Biopolymers 1976, 15, 2025.
(26) (a) Yao, J.; Bru¨schweiler, R.; Dyson, H. J.; Wright, P. E. J. Am. Chem.
Soc. 1994, 116, 12051. (b) Yao, J.; Dyson, H. J.; Wright, P. E. J. Mol.
Biol. 1994, 243, 754. (c) Yao, J.; Feher, V. A.; Espejo, B. F.; Reymond,
M. T.; Wright, P. E.; Dyson, H. J. J. Mol. Biol. 1994, 243, 736. (d) Dyson,
H. J.; Rance, M.; Houghten, R. A.; Lerner, R. A.; Wright, P. E. J. Mol.
Biol. 1988, 201, 161.
(27) (a) Wu, W.-J.; Raleigh, D. P. Biopolymers 1998, 45, 381. (b) Marraud,
M.; Aubry, A. Int. J. Peptide Protein Res. 1984, 23, 123.
9
J. AM. CHEM. SOC. VOL. 124, NO. 11, 2002 2475

A R T I C L E S
Halab and Lubell
Scheme 1. Synthesis of N-(Acetyl)dipeptide N′-Methylamides 1a-f and 2a-f
residues has been shown to result from an interaction between
the pyrrolidine ring and the aromatic π-system in the cis-amide
as observed in the X-ray structure of Ac-Tyr-5-tBuPro-NHMe.
Furthermore, hydrogen-bonding residues situated N-terminal to
5-tBuPro were found to destabilize the amide cis isomer.
Studying tetrapeptide analogues, we have examined the pro-
pensity for type VIa â-turns to nucleate hairpins. A combination
of aromatic residues at the N-terminal and small alkyl groups
at the C-terminal of tetrapeptide esters has led to high popula-
tions of cis confomers that exhibited hydrogen-bonding char-
acteristics of â-hairpins.
N-Boc-asparagine, the 5-tBuPro and Pro dipeptides 1f and 2f
were synthesized by converting their respective aspartic benzyl
esters, 7a and 8a, to the corresponding amides, 1f and 2f, using
liquid ammonia in methanol in 97-99% yield (Scheme 1).
Synthesis of Tetrapeptides. Longer peptide sequences have
been synthesized with 5-tBuPro using a combination of solution
and solid-phase chemistry.21c,22c,23 Tetrapeptides 13-16 were
prepared on oxime resin28a by employing a Boc-protected
dipeptide possessing 5-tBuPro at the C-terminal. The first
N-(Boc)amino acid was loaded onto oxime resin using N,N-
dicyclohexylcarbodiimide (DCC) in dichloromethane for 18 h
(Scheme 2).28 The loading of the resin (mmol/g) was then
determined by treatment of a precise amount of the resin with
n-propylamine in chloroform28b and subsequent measurement
of the weight and purity of the resulting N-(Boc)amino N′-pro-
pylamide as assessed by NMR spectroscopy. Sequential elonga-
tion involved deprotections using TFA in dichloromethane,
couplings of N-(Boc)amino acids using TBTU and DIEA in
DMF and acetylation using Ac2O and DIEA in CH2Cl2.
The sterically hindered 5-tert-butylproline was introduced into
the peptides as a dipeptide unit that was synthesized in solution
(Scheme 2). Protection of N-Boc-5-tBuPro with benzyl bromide
and DIEA in dichloromethane at reflux for 18 h gave ester 10
which was exposed to HCl (g) in dichloromethane to remove
the Boc group and then coupled to N-(Boc)amino acids using
BOP-Cl to provide the N-(Boc)dipeptide benzyl esters
11a-c. The benzyl group was removed by hydrogenation to
afford the N-(Boc)dipeptides 12a-c that were coupled to the
resin using TBTU and the solid-phase protocol described above.
The peptides were cleaved from the resin to afford the peptide
N′-methylamides and methyl esters by using respectively
Results
Synthesis of Dipeptides 1 and 2. Dipeptides Ac-Xaa-
5-tBuPro-NHMe (1a-f) were normally synthesized using Boc
and benzyl (Bn) protecting groups for the respective protection
of the amine and side-chain groups. The protected amino acids
were coupled to the N-terminal of 5-tBuPro-NHMe21a using
BOP-Cl and diisopropylethylamine (DIEA) in dichloromethane
to furnish dipeptides 6 in 67-94% yield (Scheme 1). For
comparison, dipeptides possessing natural proline, Ac-Xaa-Pro-
NHMe (2a-f), were synthesized by coupling similarly protected
amino acids to proline N′-methylamide using benzotriazol-1-
yl-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and
DIEA in acetonitrile which furnished dipeptides 5 in 59-70%
yield. Removal of the Boc group with TFA in dichloromethane
followed by acetylation of the amine with acetic anhydride
(Ac2O) and potassium carbonate in dichloromethane provided
N-acetyl dipeptide N′-methylamides. The benzyl ester and ether
groups of the N-acetyl O-benzyl dipeptide N′-methylamides
7a-c and 8a-c were then deprotected by hydrogenation using
1 atm of H2 with Pd/C in methanol and afforded the N-acetyl
dipeptide N′-methylamides 1a-c and 2a-c in 92-99% yield.
Because of the low yields obtained in coupling reactions with
(28) (a) DeGrado, W. F.; Kaiser, E. T. J. Org. Chem. 1982, 47, 3258. (b) Voyer,
N.; Lavoie, A.; Pinette, M.; Bernier, J. Tetrahedron Lett. 1994, 35, 355.
9
2476 J. AM. CHEM. SOC. VOL. 124, NO. 11, 2002

Effect of Sequence on Peptide Geometry
A R T I C L E S
Scheme 2. Solid Phase of 5-tert-Butylprolyl Tetrapeptides 13 and
14
gave lower cis-isomer populations. An increase in cis-isomer
population was observed when the aromatic residue was varied
from phenylalanine to tyrosine to tryptophan in 5-tert-butylprolyl
dipeptides, which has also been the trend seen in prolyl
peptides.26,27a This tendency may be caused by the increase in
electron density in the aromatic rings which interacts effectively
with the partial positive charge of the prolyl nitrogen thus
increasing the cis-isomer population.
In 5-tBuPro-peptides 1c-e and 1g-j, which have amino acids
of L-configuration possessing aliphatic and aromatic side chains,
the cis-isomer population was augmented on switching solvent
from DMSO to water and from DMSO to chloroform. On the
contrary, higher cis-isomer populations were observed in DMSO
relative to water when the N-terminal residue was of L-con-
figuration possessing a side-chain capable of forming hydrogen
bonds (1a, 1b, and 1f), as well as when a D-amino acid residue
was N-terminal to 5-tBuPro (1d′, 1h′, and 1i′). In the natural
prolyl peptides which possess D-amino acid residues (2d′, 2h′,
and 2i′), a large increase of cis isomer was also observed in
DMSO relative to water and chloroform. However, in prolyl
peptides 2 possessing a side-chain capable of forming hydrogen
bonds (2a, 2b, and 2f), a greater amount of the cis isomer was
found in water relative to DMSO.
methylamine in chloroform and calcium acetate in MeOH:THF
at 40 °C.29 The final peptides 13-16 were obtained in 21-
61% overall yields after purification by C18 reverse-phase
HPLC and lyophilization. The purity of the peptides 13-16 was
examined by analytical HPLC and their compositions were
verified by fast atom bombardment mass spectrometry.
The influence of solvent composition on the chemical shifts
of the amide signals was used to identify amides engaged in
intramolecular hydrogen bonds. In the major amide cis isomer
of 5-tBuPro-peptides 1, the signal for the N′-methylamide proton
appeared always downfield relative to that for the acetamide
proton in chloroform. The N′-methylamide proton signals
appeared between 7.29 and 8.52 ppm whereas the acetamide
proton signals came between 5.97 and 7.21 ppm for the cis
isomer of peptides 1. The downfield shifted amide proton signal
suggested an intramolecular hydrogen bond between the N′-
methylamide and acetamide carbonyl in a type VIa â-turn
conformation. In contrast, the signals of the N′-methylamide
and acetamide of the major trans conformer of prolyl peptides
2 showed no significant differences in their chemical shifts
which appeared between 6.03 and 7.17 ppm. Solvent changes
from chloroform to water and to DMSO caused little changes
in the chemical shift of the N′-methylamide proton signal of
peptide 1, which moved 0.04-0.60 ppm and 0.17-1.12 ppm
downfield, respectively. The signals for the acetamide proton
of peptides 1 and 2 and the N′-methylamide proton of peptide
2 shifted significantly (0.40-2.55 ppm) with changes in solvent.
In conclusion, the influence of solvent composition on the chem-
ical shift of the amide signals demonstrated the N′-methylamide
proton of the cis isomer of peptide 1 to be in an intramolecular
hydrogen bond of a type VIa â-turn conformation.
Circular Dichroism Spectroscopy. Circular dichroism (CD)
spectra of peptides 1a-f and 2a-f were measured in water and
acetonitrile to study the influence of solvent composition on
peptide conformation. The CD spectra of 5-tert-butylprolyl
peptides possessing aromatic side chains 1c-e exhibited a strong
negative band at 190 ( 5 nm, a strong positive band at 205 (
6 nm and a weak negative band at 230 ( 10 nm in water (Figure
2A). When changing the solvent from water to acetonitrile, the
shape of the CD curves of peptides 1c-e remained unchanged
(Figure 2B). This type of CD curve has been identified as a
class B CD spectrum which we have previously assigned to
type VIa â-turn conformations.22,32 Similar curve shapes were
Conformational Analysis of the Dipeptides
NMR Spectroscopy. The conformation of peptides 1 and 2
was analyzed by NMR spectroscopy in chloroform, DMSO, and
water. The relative populations of the amide cis and trans
isomers N-terminal to the prolyl residues were measured by
integration of the isomeric tert-butyl singlets and N′-methyl
1
doublets in the H NMR spectra. As previously noted,22 the
tert-butyl singlet of the amide cis isomer appeared always
upfield from that of the trans isomer in the 5-tert-butylprolyl
peptides 1. Nuclear Overhauser effect experiments were used
to confirm the assignment of the cis isomer in dipeptides 1 and
2 on the basis of observation of a cross-peak between the
N-terminal amino acid and the proline R-hydrogens in the
NOESY and ROESY spectra.
In the prolyl dipeptides 2, the amide trans isomer geometry
N-terminal to the prolyl residue was the major conformer (Figure
1) in all three solvents, as observed in linear prolyl pep-
tides.7,26,27,30,31 Aromatic residues N-terminal to proline exhibited
a 2-3-fold increase in the cis isomer population relative to their
alanine counterpart. The largest amount of cis-amide (44%) was
observed with Ac-L-Trp-Pro-NHMe among the prolyl peptides
in water. On the contrary, the 5-tert-butylprolyl peptides 1
adopted the amide cis isomer in the major conformer (Figure
1). The presence of an aromatic amino acid N-terminal to 5-tert-
butylproline caused a significant increase in the cis isomer
population. The highest cis isomer population (96%) was
observed for Ac-L-Trp-tBuPro-NHMe in water. Relative to their
aliphatic and aromatic amino acid counterparts, hydrogen-bond
donor and acceptor residues N-terminal to 5-tert-butylproline
(29) Moraes, C. M.; Bemquerer, M. P.; Miranda, M. T. M. J. Peptide Res. 2000,
55, 279.
9
J. AM. CHEM. SOC. VOL. 124, NO. 11, 2002 2477

A R T I C L E S
Halab and Lubell
Figure 1. Amide equilibrium of N-acetyl dipeptide N′-methylamide.
obtained for 5-tert-butylprolyl peptides 1a, 1b, and 1f which
possessed hydrogen-bond donor and acceptor side chains (Figure
3, parts A and B).
N-terminal to the 5-tert-butylprolyl residue with twisted dihedral
angle values of ω ) 8.5° and ω ) 13.0° (Figure 4). As
previously shown, the sterically bulky 5-tert-butyl substituent
influenced the amide bond N-terminal to the 5-tert-butylprolyl
residue and distorted it from planarity.22a-b Furthermore, an
intramolecular hydrogen bond could be inferred to exist between
the N′-methylamide proton and the acetamide carbonyl oxygen
from measurement of an interatomic distance of 2.08 and 2.18
Å in the X-ray structure of 1c. The dihedral angles of peptide
1c ressembled those of the central i + 1 and i +2 residues of
an ideal type VIa â-turn. For comparison, the dihedral angles
of peptide 1c, the ideal values of type VIa and VIb â-turn
conformations as well as those for Ac-L-Leu-tBuPro-NHMe and
Ac-D-Leu-tBuPro-NHMe are listed in Table 1.4,22 The 5-tBuPro
ψ-dihedral angle of dipeptide 1c placed the N′-methylamide
hydrogen at a 2.4 Å interatomic distance from the prolyl
nitrogen, which has also been observed in the X-ray structure
of Ac-L-Leu-tBuPro-NHMe.22a The interaction between the
prolyl amide nitrogen and the N′-methylamide hydrogen has
been suggested to stabilize the pyramidalized amide in the
transition state and thereby accelerate isomerization N-terminal
to proline.42
The CD spectra of the prolyl peptides possessing aromatic
side chains 2c-e varied significantly with changes in solvent
composition and exhibited no distinct CD curve shapes (Fig-
ure 2, parts C and D). On the other hand, the CD curves of
prolyl peptides 2a and 2f which possessed Asp and Asn residues
with hydrogen-bonding side chains were characterized by
negative maxima at around 210 ( 2 and 220 ( 2 nm in
acetonitrile that converged to a single band at around 205 nm
in water (Figure 3, parts C and D). This type of CD curve has
been assigned to a class C CD spectrum that has been reported
for peptides adopting prolyl amide trans isomers in type II′
â-turn conformations.22,32,33
X-ray Crystallographic Analysis. Crystals of N-acetyl-L-
tyrosyl-5-tert-butylproline N′-methylamide were obtained from
a mixture of MeOH, EtOAc, and hexane. The analysis of the
crystal structure by X-ray diffraction revealed an asymmetric
unit with a molecule of water and two molecules of 1c which
adopted similar turn conformations.34 The crystallographic
analysis of 1c demonstrated the presence of an amide cis isomer
(30) (a) Valdeavella, C. V.; Blatt, H. D.; Pettitt, B. M. Int. J. Peptide Protein
Res. 1995, 46, 372. (b) Wang, Y.; Scott, P. G.; Sejbal, J.; Kotovych, G.
Can. J. Chem. 1996, 74, 389.
(34) The structure of 1c was solved at l’Universite´ de Montre´al X-ray facility
using direct methods (SHELXS96) and refined with NRCVAX and
SHELXL96: C42H64N6O9; Mr ) 796.992; orthorhombic, colorless crystal;
space group P212121; unit cell dimensions (Å) a ) 9.294 (2), b ) 12.895
(31) Oka, M.; Montelione, G. T.; Scheraga, H. A. J. Am. Chem. Soc. 1984,
106, 7959.
(3), c ) 36.837 (7); volume of unit cell (Å3) 4415.12 (16); Z ) 4; R1
)
(32) Woody, R. W. In Peptides, Polypeptides and Proteins; Blout, E. R., Bovey,
F. A., Goodman, M., Lotan, N., Eds.; John Wiley: New York, 1974, pp
338-348.
(33) (a) Kawai, M.; Nagai, U. Biopolymers 1978, 17, 1549. (b) Bush, C. A.;
Sarkar, S. K.; Kopple, K. D. Biochemistry 1978, 17, 4951.
0.0614 for I > 2σ(I), wR2 ) 0.1569 for all data; GOF ) 1.054. The author
has deposited the atomic coordinates for the structure of 1c with the
Cambridge Crystallographic Data Center. The coordinates can be obtained,
on request, from the Cambridge Crystallographic Data Center, 12 Union
Road, Cambridge, CB2 1EZ, U.K.
9
2478 J. AM. CHEM. SOC. VOL. 124, NO. 11, 2002

Effect of Sequence on Peptide Geometry
A R T I C L E S
Figure 2. Circular dichroism spectra of dipeptides 1c-e (A, in water, and B, in acetonnitrile) and 2c-e (C, in water, and D, in acetonitrile) at 0.l mM.
In the structure of peptide 1c, the Tyr side-chain adopted a
ø1 dihedral angle value of 172°. This trans conformer (Figure
5) positioned the tyrosyl aromatic ring beneath the proline ring
(distance of 3.4-4.9 Å between the proline nitrogen and the
tyrosyl phenyl group, Figure 4). Prior to our work, computational
study had suggested that the amino-aromatic interactions occur
between positively charged or δ(+) amine groups in side chains
and the δ(-) π-electrons of the aromatic ring of Phe, Tyr, and
Trp when they are separated by 3.4-6.0 Å.35 Stacking interac-
tions between the aromatic and pyrrolidine rings can be found
in the crystal structures of proteins and cyclic peptides pos-
sessing aromatic residues N-terminal to proline.8,36 For example,
in the X-ray structure of the hexapeptide cyclo(L-Phe-L-Pro-D-
Ala)2, the L-Phe-L-Pro amide bonds are in the cis isomer with
the ø1 dihedral angle of the Phe residue in the trans conformer
(Figure 5).36a Interactions between the aromatic and proline rings
in the amide cis isomer of prolyl peptides containing aromatic
residues have been observed by NMR spectra in which the
chemical shift of the R-proton of proline was shifted upfield.37
(35) (a) Burley, S. K.; Petsko, G. A. FEBS 1986, 203, 139. (b) Dougherty, D.
A. Peptides 1999 Proc. Am. Pept. Symp., 16th 1999, 550-552. (c) Gallivan,
J. P.; Dougherty, D. A. J. Am. Chem. Soc. 2000, 122, 870.
(36) (a) Kartha, G.; Bhandary, K. K.; Kopple, K. D.; Go, A.; Zhu, P.-P. J. Am.
Chem. Soc. 1984, 106, 3844. (b) Chiang, C. C.; Karle, I. L. Int. J. Peptide
Protein Res. 1982, 20, 133.
(37) (a) Poznanski, J.; Ejchart, A.; Wierzchowski, K. L.; Ciurak, M. Biopolymers
1993, 33, 781. (b) Juy, M.; Lam-Thanh, H.; Lintner, K.; Fermandjian, S.
Int. J. Peptide Protein Res. 1983, 22, 437. (c) Stimson, E. R.; Montelione,
G. T.; Meinwald, Y. C.; Rudolph, R. K. E.; Scheraga, H. A. Biochemistry
1982, 21, 5252.
(41) (a) Imperiali, B.; Shannon, K. L.; Rickert, K. W. J. Am. Chem. Soc. 1992,
114, 7942. (b) Abbadi, A.; Mcharfi, M.; Aubry, A.; Pre´milat, S.; Boussard,
G.; Marraud, M. J. Am. Chem. Soc. 1991, 113, 2729.
(38) (a) Fro¨mmel, C.; Preissner, R. FEBS Lett. 1990, 277, 159. (b) Stewart, D.
E.; Sarkar, A.; Wampler, J. E. J. Mol. Biol. 1990, 214, 253.
(39) Wu¨thrich, K. In NMR of Proteins and Nucleic Acids; John Wiley & Sons:
New York, 1986.
(42) (a) Fischer, S.; Dunbrack, Jr. R. L.; Karplus, M. J. Am. Chem. Soc. 1994,
116, 11931. (b) Cox, C.; Young, Jr. V. G.; Lectka, T. J. Am. Chem. Soc.
1997, 119, 2307. (c) Beausoleil, E.; Sharma, R.; Michnick, S.; Lubell, W.
D. J. Org. Chem. 1998, 63, 6572. (d) Cox, C.; Lectka, T. J. Am. Chem.
Soc. 1998, 120, 10660.
(40) Reviewed in: Kessler, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 512.
9
J. AM. CHEM. SOC. VOL. 124, NO. 11, 2002 2479

A R T I C L E S
Halab and Lubell
Figure 3. Circular dichroism spectra of dipeptides 1a, 1b, and 1f (A, in water, and B, in acetonnitrile) and 2a, 2b, and 2f (C, in water, and D, in acetonitrile)
at 0.l mM.
Table 1. Comparison of the Dihedral Angles (deg) of Ideal Type
VIa â-Turn and X-ray Structure of
N-(Acetyl)tyrosyl-5-tert-butylproline N′-Methylamide (1c)
entry
φi+1
ψi+1
ω
φi+2
ψi+2
ideal type VIa â-turn4
-60
-58
-58
-61
-120
93
120
137
137
139
120
141
0
-90
-101
-94
-95
-60
-81
0
26
11
19
150
157
Ac-L-Tyr-5-tBuPro-NHMe (A)
Ac-L-Tyr-5-tBuPro-NHMe (B)
Ac-L-Leu-5-tBuPro-NHMe22a
ideal type VIb â-turn4
8
13
17
0
Ac-L-Leu-5-tBuPro-NHMe22b
29
spectroscopy in 9:1 H2O/D2O. The signals in the 1H NMR were
assigned using COSY, TOCSY and ROESY experiments. The
relative populations of the prolyl amide cis and trans isomers
of tetrapeptides 13-16 were measured by integration of the tert-
butyl singlets and the N′-methylamide or methyl ester signals
in the proton NMR spectra (Table 2).
Figure 4. Structure of Ac-L-Tyr-5-tBuPro-NHMe 1c from X-ray crystal-
lography.34 Hydrogens are shown on nitrogens and chiral carbons (C, gray;
N, blue; O, red; H, turquoise).
Conformational Analysis of the Tetrapeptides
The conformations of tetrapeptides Ac-Xaa-Yaa-5-tBuPro-
Zaa-XMe 13 (X ) NH) and 14 (X ) O) and Ac-Xaa-Yaa-Pro-
Zaa-XMe 15 (X ) NH) and 16 (X ) O) were studied by NMR
The cis-isomer geometry was found to be stabilized by
aliphatic residues such as alanine and valine relative to the
9
2480 J. AM. CHEM. SOC. VOL. 124, NO. 11, 2002

Effect of Sequence on Peptide Geometry
A R T I C L E S
population of tetrapeptide 13c (47%) was augmented 26% on
conversion to its methyl ester 14a (73%, Table 2). This aug-
mentation may be caused by the removal of competitive hy-
drogen-bonding conformations in the tetrapeptides N′-meth-
ylamides that favor the prolyl amide trans isomer.
The coupling constant values (3JNH-CRH) for the i + 1 and i
+ 2 residues of tetrapeptides 14d and 14f exhibiting the highest
cis-isomer populations were characteristic of turn structure.39
Figure 5. Rotamers around the CR-Câ axis in amino acids.
Table 2. Yaa-5-tBuPro Amide Isomer Equilibrium of Tetrapeptides
Ac-Xaa-Yaa-5-tBuPro-Zaa-XMe and Ac-Xaa-Yaa-Pro-Zaa-XMe in
10% D2O/H2O
3
The JNH-CRH coupling constant value for the Phe residue in
tetrapeptides 14d and 14f was 3.7 Hz and in good agreement
with a 4 Hz coupling constant corresponding to the φ dihedral
angle of -60° for the i + 1 residue in a type VIa â-turn
entry
Xaa (i)
Yaa (i+1)
(i+2)
Zaa (i+1)
X
% cis isomer
13a
13b
13c
14a
13d
13e
13f
12g
13h
14b
14c
13i
14d
13j
14e
14f
15
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ser
Ser
Val
Ala
Ala
Ala
Ala
Ala
Ala
Leu
Leu
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
5-tBuPro
5-tBuPro
5-tBuPro
5-tBuPro
5-tBuPro
5-tBuPro
5-tBuPro
5-tBuPro
5-tBuPro
5-tBuPro
5-tBuPro
5-tBuPro
5-tBuPro
5-tBuPro
5-tBuPro
5-tBuPro
Pro
Ala
Leu
Phe
Phe
Leu
Phe
Phe
Leu
Val
Val
Lys
Ala
Ala
Ala
Ala
Ala
Ala
Ala
NH
NH
NH
O
NH
NH
NH
NH
NH
O
O
NH
O
NH
O
O
49
44
47
73
50
43
62
65
52
68
79
68
84
70
73
80
22
9
3
conformation. The JNH-CRH coupling constant values for the
other residues in the tetrapeptides 14d and 14f were in the range
of 6.2-8.4 Hz. Sequential HRN(i, i +1) NOEs were observed
for all expected residues in the tetrapeptide 14d. In the ROESY
spectra of tetrapeptide 14d, an additional HRN(i, i +2) NOE
was observed between the R-hydrogen of Phe and amide proton
of the C-terminal Ala characteristic of a â-turn conformation.
The temperature coefficient values (∆δ/∆T) were measured
for the amide protons in peptide 14d, which possessed the
highest cis-isomer population, its amide counterpart 13i, and
their respective prolyl analogues 16 and 15 in 10% D2O/H2O
(Table 3). The amide protons of the alanine residues at the Xaa
and Zaa positions of 5-tBuPro-peptide 13i and 14d exhibited
lower temperature coefficient values than the phenylalanine and
N′-methylamide amide protons, which may indicate their
participation in intramolecular hydrogen bonds. This tendency
was not observed in the prolyl tetrapeptides 15 and 16 where
the chemical shift variation with temperature for the amide
protons were in the range of -6.1 to -10.4 ppb/K. The
temperature coefficient values were measured in DMSO for the
amide protons in 5-tBuPro-tetrapeptides 14d and 14f, which
possessed high cis-isomer populations. The amide proton of the
alanine residue at the Zaa position of peptide 14d exhibited
similar temperature coefficient values in DMSO (-4.7 ppb/K)
and water (-4.9 ppb/K). In DMSO, peptides 14d and 14f
exhibited similar temperature coefficient values. Although
temperature coefficient values greater than -3 ppb/K in DMSO
have been suggested to indicate amide protons engaged in
intramolecular hydrogen bonds, such values are normally
measured in cyclic and larger peptides than those reported here.40
The pattern of the temperature coefficient magnitudes in 13i,
14d, and 14f did conform to a hairpinlike structure; however,
the values themselves suggested solvent-exposed amides pre-
sumably due to the flexibility of the linear tetrapeptide.
The CD spectra of tetrapeptides 14d and 14f exhibited a
negative maximum at 230 nm, a positive maximum at 215 nm,
an intense negative maximum at 200 nm and a positive
maximum at 190 nm in water (Figure 7). This type of CD curve
has been classified as a class D CD spectrum which has been
previously assigned to a â-turn conformation.32
NH
O
16
Pro
Figure 6. Influence of sequence on the amide equilibrium (% cis isomer)
in 5-tBuPro-tetrapeptides.
hydrogen-bonding residue serine at the i position (Figure 6). In
the natural sequence Xaa-Tyr-Pro-Tyr-Asp-Val, an alanine
residue (57%) gave rise to higher cis-isomer population than a
serine residue (52%) at the i position.26d Protein X-ray analysis
in contrast has shown the highest occurrence of serine at the i
position in peptides possessing proline at the i + 2 position in
the amide cis conformer.38 As observed in the 5-tBuPro-
dipeptides, a significant augmentation of the cis-isomer popula-
tion occurred when an aromatic residue was at the i + 1 position,
N-terminal to 5-tBuPro in the N-acetyl tetrapeptide N′-methyl-
amides. When the 5-tBuPro residue was replaced by proline,
the cis-isomer population decreased from 68-84% to 9-22%
(Table 2). Thus, the steric interactions between the tert-butyl
substituent and the side-chain of the N-terminal residue disfa-
vored the prolyl amide trans isomer and increased the cis-isomer
population. At the i + 3 position of the tetrapeptides, alanine
and lysine were found to give rise to higher amide cis-isomer
populations relative to the â-branched alkyl residue valine. In
protein X-ray data, alanine C-terminal to proline gave rise to
the highest cis-conformer population.38 Among the modifications
to 5-tBuPro-tetrapeptides, a significant increase in cis-isomer
population was observed when the N′-methylamide was replaced
by its methyl ester counterpart. For example, the cis-isomer
Discussion
We have studied the influence of sequence and stereochem-
istry of the residue N-terminal to 5-tBuPro by the synthesis
and analysis of a series of dipeptide and tetrapeptide analogues
(Ac-Xaa-5-tBuPro-NHMe and Ac-Xaa-Yaa-5-tBuPro-Zaa-XMe).
All of the 5-tBuPro-dipeptide analogues adopted predominant
cis-isomer conformations about the prolyl amide bond. In the
9
J. AM. CHEM. SOC. VOL. 124, NO. 11, 2002 2481

A R T I C L E S
Halab and Lubell
Table 3. Influence of Temperature on the NH Chemical Shifts of the Major Isomer of the Tetrapeptides 13i, 14d, 14f, 15, and 16 in Water
and in DMSO
−∆δ/∆T (ppb/K)a
peptides
NHXaa
NHPhe
NHZaa
6.9
4.9 (4.7)b
10.4
NHMe
Ac-Ala-Phe-5-tBuPro-Ala-NHMe (13i)
Ac-Ala-Phe-5-tBuPro-Ala-OMe (14d)
Ac-Ala-Phe-Pro-Ala-NHMe (15)
Ac-Ala-Phe-Pro-Ala-OMe (16)
7.7
8.8
8.4
6.9 (6.0)b
7.2
10.0 (6.9)b
7.8
7.1
9.5
8.6
6.1
Ac-Val-Phe-5-tBuPro-Ala-OMe (14f)
6.0b
6.7b
4.5b
a Determined by 600 MHz NMR in 10% D2O/H2O at 5 mM concentration. b Values are in DMSO.
ring and the N-terminal residue in the cis conformer. The
downfield shift for the N′-methylamide proton signal of
5-tBuPro-dipeptides 1 was indicative of an intramolecular
hydrogen bond between the N′-methylamide proton and the
acetamide carbonyl in a type VIa â-turn conformation. The
presence of a type VIa â-turn geometry for 5-tBuPro-dipeptides
in solution, independent of solvent composition, was supported
by the circular dichroism spectra of dipeptides 1a-f in water
and acetonitrile. In chloroform, the intramolecular hydrogen
bond in the type VIa â-turn conformation may be stabilized,
resulting in an increased cis-isomer population in 5-tBuPro
dipeptides with L-configuration possessing alkyl and aromatic
residues; in DMSO, the cis-isomer population was reduced
presumably by the disruption of this intramolecular hydrogen
bond.3b In the prolyl peptides possessing hydrogen-bonding
residue (2a, 2b, and 2f), higher cis populations were observed
in water which may compete with the side-chain hydrogen donor
and perturb intramolecular hydrogen bonding that can stabilize
a trans conformer.
In the solid state, N-acetyl-L-tyrosyl-5-tert-butylproline N′-
methylamide (1c) existed in a type VIa â-turn conformation as
shown by X-ray diffraction. In addition, the aromatic and proline
rings were stacked in the crystal structure in a way that may
stabilize the cis-isomer geometry by an interaction between the
partial positive charge of the amide nitrogen and the electron-
rich aromatic π-system. The tert-butyl substituent distorted the
prolyl amide from planarity which may result in lowering of
the prolyl amide isomerization energy barrier. Stabilization of
the transition state for prolyl isomerization may also occur from
interaction between the lone pair of the pyramidalized prolyl
amide nitrogen and the N′-methylamide hydrogen, which were
inferred to be separated by 2.4 Å in the crystal structure of
1c.6a,42 A second stabilizing force in the transition state may
arise from the aromatic side chain of tyrosine because as the
prolyl amide carbonyl rotates, it may interact with the aromatic
ring protons which from the crystal structure were inferred to
be at a distance of 3.7-4.6 Å. Such a C-H‚‚‚O interaction
between the side chains of the aromatic residues in the binding
pocket of the PPIase FKPB and the tertiary amide carbonyl of
its substrate has previously been suggested to stabilize the
transition state for enzyme-catalyzed isomerization.6a,43 The in-
creased cis-isomer population and relative increase in the prolyl
amide isomerization energy barrier that have been observed in
natural prolyl peptides possessing aromatic amino acids, both
Figure 7. Circular dichroism spectra of tetrapeptides 14d and 14f in water
at 0.1 mM.
dipeptide series, the turn type was contingent on the configu-
ration of the amino acid N-terminal to 5-tBuPro and adopted,
respectively, type VIa and VIb â-turn conformations when the
N-terminal amino acid was of L- and D-configurations.22a,b
In the N-acetyl dipeptide N′-methylamides, the cis-isomer
population increased as the amino acid N-terminal to 5-tBuPro
was varied from a hydrogen-bonding residue to an alkyl residue
to an aromatic residue. This pattern was also observed in the
natural prolyl dipeptides where aromatic residues gave higher
cis-isomer populations than alkyl and hydrogen-bonding resi-
dues. The augmentation of cis isomer with aromatic residues
N-terminal to proline appears to be caused by the interaction
between the δ(+) nitrogen of 5-tBuPro and the aromatic ring
of the tyrosyl side-chain which adopted a ø1 value of 172° as
observed in the X-ray structure of 1c. This stacking interaction
cannot be achieved in the trans-isomer geometry because the
side-chain ø1 dihedral angle adopts a gauche conformer (Figure
5).37 In the dipeptides possessing hydrogen-bonding side chains
(1a, 1b, 2a, and 2b), a hydrogen-bond between the N′-
methylamide hydrogen and the carbonyl oxygen of the side-
chain in an Asx-turn may favor the trans isomer and therefore
decrease the cis-isomer population.41
The influence of solvent composition on the cis-isomer
equilibrium was contingent on the sequence of the dipeptide.
Alkyl and aromatic residues in L-Xaa-5-tBuPro dipeptides gave
higher cis-isomer population in water than DMSO presumably
due to favorable hydrophobic interactions between the proline
(43) In a preliminary investigation, the tetrapeptide succinyl-Ala-Ala-5-tBuPro-
Phe-OMe (71% cis isomer in 10% D2O/H2O; tR ) 16.1; HRMS calcd for
C
29H43N4O8 (MH+) 575.3081; HRMS found 575.3097) was synthesized,
was tested, and exhibited no inhibition against the PPIase cyclophilin at
364.7 µM. We thank Professor Felicia Etzkorn and Dr. Scott, A. Hart for
their analysis of the inhibitory activity of this tetrapeptide.
9
2482 J. AM. CHEM. SOC. VOL. 124, NO. 11, 2002

Effect of Sequence on Peptide Geometry
A R T I C L E S
however, appear to be due to ground-state stabilization through
interactions between the δ(+) nitrogen and π-aromatic sys-
tem.
dynamic process such as hydrophobic effects, solvation of polar
groups and hydrogen bonding which involve both van der Waals
and electrostatic interactions.46 Prolyl amide isomerization in
proteins can be a rate-limiting step in the folding mechanism.
Although the trans-isomer geometry has been shown to be of
lower energy, the cis isomer may be augmented by the hydro-
phobic stacking interaction of the N-terminal residue to proline
with the pyrrolidine ring as observed in the X-ray structure of
1c. The prolyl amide cis isomers in proteins may thus begin
folding processes by a series of hydrophobic interactions prior
to isomerization to the more stable trans isomer.
In conclusion, we have demonstrated that the prolyl amide
equilibrium was influenced by the sequence in the 5-tBuPro-
dipeptides and tetrapeptides. X-ray analysis of Ac-L-Tyr-tBuPro-
NHMe has illustrated the stabilization of the prolyl amide cis
isomer by a stacking interaction of the aromatic and pyrrolidine
rings. By using steric constraints of a tert-butyl substituent, we
were able to favor a type VIa â-turn conformation in N-acetyl
tetrapeptide methylesters, possessing a prolyl amide cis isomer
and a 10-membered intramolecular hydrogen bond. Incorpora-
tion of these type VI â-turn mimics into larger peptides is being
pursued to provide more understanding of the impact of prolyl
amide cis isomers and type VI â-turns on the folding of peptide
structures.
The effect of sequence on the prolyl amide equilibrium of
5-tBuPro-tetrapeptides (Ac-Xaa-Yaa-5-tBuPro-Zaa-XMe) was
studied by varying the amino acids at the Xaa, Yaa, and Zaa
positions. The highest cis-isomer populations were obtained with
alkyl groups at the Xaa position, an aromatic residue at the Yaa
position, and either an alanine or a lysine residue at the Zaa
position of the 5-tBuPro-tetrapeptide methyl esters. It has been
previously illustrated that replacement of amide bonds with
esters in peptides prevented the formation of undesired hydrogen-
bonding conformations.44 In natural prolyl peptides, high cis-
isomer populations have been obtained by placing aromatic
residues at both the N- and C-termini of proline.26 In the
5-tBuPro-tetrapeptide 13f, a combination of Phe residues at the
N- and C-termini to 5-tBuPro did not produce the highest cis-
isomer populations. On the contrary, amino acids with linear
alkyl groups, such as alanine and lysine, at the C-terminal of
5-tBuPro-tetrapeptide N′-methylamides 13g and 13i produced
higher cis-isomer populations. Alkyl-branched residues at the
C-terminal of 5-tBuPro may not favor the cis isomer because
of the steric interactions between the side-chain and tert-butyl
subsituents.
Turn formation in peptides has been illustrated by the
Experimental Section
presence of a significant HRN(i, i +2) NOE connectivity.26
A
strong HRN(i, i +2) NOE was observed between the R-hydrogen
of Phe and amide proton of the C-terminal Ala in tetrapeptide
14d. The circular dichroism spectra of peptides 14d and 14f
exhibited a similar curve shape that has been classified as a
type D CD spectrum, assigned to â-turn conformations.32 Thus,
peptide 14d was shown to adopt a type VIa â-turn conformation
in water.
General Data. Solvents and reagents were purified as previously
described. NMR and CD data were measured as described in ref 22.
Mass spectral data, HRMS (EI and FAB), were obtained by the
Universite´ de Montre´al Mass Spectroscopy facility.
General Procedure for Coupling to 5-tert-Butylproline N′-
Methylamide. A solution of (2S,5R)-5-tert-butylproline N′-methylamide
hydrochloride (0.37 mmol, prepared according to refs 21a and 22a),
N-(Boc)amino acid (0.74 mmol), and DIEA (0.26 mL, 1.5 mmol) in
CH2Cl2 (4 mL) was cooled to 0 °C, treated with BOP-Cl (188 mg,
0.74 mmol), stirred for 1 h, and allowed to warm to room temperature
with stirring for 18 h. Brine was added, and the solution was extracted
with EtOAc. The combined organic layers were washed with 0.1 M
HCl (2 × 10 mL), 5% NaHCO3 (2 × 10 mL), and brine (10 mL),
dried, and evaporated to a residue that was purified by chromatography
on silica gel using 35% EtOAc in hexane as eluant. Evaporation of the
collected fractions furnished N-(Boc)dipeptide N′-methylamides.
General Procedure for Acetamide Synthesis. A solution of
N-(Boc)dipeptide N′-methylamide (20.5 mg, 0.05 mmol) in 25% TFA
in CH2Cl2 (1 mL) was stirred for 1 h, and the solvent was evaporated.
The residue was dissolved in CH2Cl2 (1 mL), treated with K2CO3 (65.6
mg, 0.5 mmol) and acetic anhydride (45 µL, 0.5 mmol), and stirred
for 18 h. The solution was filtered, washed with CH2Cl2 (2 × 5 mL),
and evaporated to give the N-acetyl dipeptide N′-methylamide.
General Procedure for Benzyl Group Deprotection. A solution
of N-acetyl-O-benzyl-dipeptide N′-methylamide 8a-c (110.0 mg, 0.26
mmol) in MeOH (3 mL) was treated with Pd/C (26 mg, 10 wt %)
under 1 atm of H2 and stirred for 18 h at room temperature. The solution
was filtered onto Celite and washed with MeOH, and the filtrate was
evaporated to furnish N-acetyl dipeptide N′-methylamides 1a-c.
General Procedure for Asparaginyl Dipeptide Synthesis. A
solution of N-acetyl-O-benzyl-(2S)-aspartyldipeptide N′-methylamides
7a and 8a (100.0 mg, 0.23 mmol), respectively, in MeOH (2 mL) was
treated with NH3 (g) bubbles at 0 °C and stirred for 18 h at room
temperature. The solution was evaporated and triturated with Et2O to
give N-acetyldipeptide N′-methylamides 1f and 2f, respectively.
The temperature coefficient study of tetrapeptides 13i, 14d,
and 14f in water and DMSO did not provide values that
corresponded to intramolecular hydrogen bonding amide pro-
tons. However, a pattern was observed where the amide protons
of the residues at the Xaa and Zaa positions of 5-tBuPro-peptides
13i, 14d, and 14f exhibited lower temperature coefficient values
than the phenylalanine and N′-methylamide amide protons. Polar
solvents such as water and DMSO are known to perturb
intramolecular hydrogen-bonds in peptides. Steric interactions
of 5-tert-butylproline could not stabilize a hairpin conformation
in a linear tetrapeptide relative to the stabilizing structural link
in an indolizidinone amino acid type VIa â-turn mimic.14d
Although a type VIa â-turn was located at the central position
of a hairpin conformation about residues 92-94 in the X-ray
structure of the ribonuclease S protein, the type VIa â-turn
conformation has rarely been observed to stabilize a hairpin
geometry in natural peptides.4,45 Our data reflect the difficulty
of stabilizing a hydrogen-bonded network in a small peptide
possessing an amide cis isomer in polar solvents.
Protein folding implicates a set of conformational changes
and multiple transition states to adopt finally the low energy
native structure. Different interactions are involved in this
(44) (a) Haque, T. S.; Little, J. C.; Gellman, S. H. J. Am. Chem. Soc. 1996,
118, 6975. (b) Haque, T. S.; Little, J. C.; Gellman, S. H. J. Am. Chem.
Soc. 1994, 116, 4105. (c) Aubry, A.; Marraud, M. Biopolymers 1989, 28,
109. (d) Boussard, G.; Marraud, M. Ne´el, J.; Maigret, M.; Aubry, A.
Biopolymers 1977, 16, 1033.
(45) (a) Sibanda, B. L.; Thorton, J. M. Nature (London) 1985, 316, 170. (b)
Sibanda, B. L.; Thorton, J. M. J. Mol. Biol. 1993, 229, 428.
(46) Dobson, C. M.; Sali, A.; Karplus, M. Angew. Chem., Int. Ed. Engl. 1998,
37, 868.
9
J. AM. CHEM. SOC. VOL. 124, NO. 11, 2002 2483

A R T I C L E S
Halab and Lubell
General Protocol for the Synthesis of N-Acetyl Dipeptide N′-
Methylamides Possessing Natural Proline. A solution of N-(Boc)-
amino acid (1.4 mmol) in CH3CN (6 mL) was treated with DIEA (0.49
mL, 2.8 mmol), proline N′-methylamide hydrochloride (115 mg, 0.7
mmol, prepared according to ref 22a) and TBTU (0.45 g, 1.4 mmol),
stirred at room temperature for 18 h, and partitioned between brine
(10 mL) and EtOAc (10 mL). The organic phase was washed with 0.1
M HCl (2 × 8 mL), 5% NaHCO3 (2 × 8 mL), and brine (10 mL),
dried, and evaporated to a residue that was purified by chromatography
on silica gel (35% EtOAc in hexane). The N-(Boc)dipeptide N′-
methylamides 5a-e were treated with 25% TFA in CH2Cl2 (10 mL)
for 1 h and evaporated. The resulting dipeptide N′-methylamide
trifluoroacetates were dissolved in CH2Cl2 and treated under the same
acetylation and benzyl deprotection conditions as described above.
Peptide Synthesis. The synthesis of the tetrapeptides were carried
out manually by a stepwise solid-phase procedure using oxime resin.
Serine and lysine were introduced as Boc-L-Ser(Bn)-OH and Boc-L-
Lys(Cbz)-OH. Couplings were performed with Boc-protected amino
acids (200 mol %), TBTU (200 mol %), and DIEA (400 mol %) in
DMF for 1 h. The resin was agitated with N2 bubbles during the
coupling, rinsing, and deprotection sequences. Coupling reactions were
monitored by the Kaiser ninhydrin test.47 In cases of incomplete
couplings, the resin was resubmitted to the same coupling conditions.
Deprotections were performed with 25% TFA in CH2Cl2 (2 × 30 min)
and the resin was free-based with 10% DIEA in CH2Cl2 (2 × 5 min).
N-Terminal acetylation of the peptides was accomplished by treating
the resin with Ac2O (1000 mol %) and DIEA (1000 mol %) in CH2Cl2
for 1 h. The tetrapeptide N′-methylamides were obtained by cleaving
the peptides from the resin with 10% methylamine in CHCl3 upon
agitation with a mechanical shaker for 24 h. The tetrapeptide methyl-
esters were obtained by treating the resin with Ca(OAc)2 in MeOH:
THF (1:4) at 40 °C for 48 h. The crude material was purified with
semipreparative RP-HPLC (Higgins C18 column, 20 × 250 mm,
particle size 5 µm) with solvent A, H2O (0.05% TFA), and solvent B,
75% CH3CN/H2O (0.05% TFA). Analytical RP-HPLC was performed
on a Higgins C18 (4.6 × 250 mm, particle size 5 µm) using a gradient
of 0-90% eluant B (CH3CN 0.003% TFA) in A (H2O 0.03% TFA)
over 30 min with a flow rate of 1.5 mL/min and the detector centered
at 214 nm; retention times (tR) are reported in minutes.
Acknowledgment. This research was supported in part by
the Natural Sciences and Engineering Research Council of
Canada and the Ministe`re de l′EÄ ducation du Que´bec. We thank
Sylvie Bilodeau and Dr. M. T. Phan Viet of the Regional High-
Field NMR Laboratory for their assistance.
Supporting Information Available: Text listing spectro-
scopic, chromatographic and analytical data for 1a-c, 1e, 1f,
2a-f, 6a-c, 6e, 8a-c, 10, 11a-c, 12a-c, 13a-j, 14a-f, 15,
and 16, as well as details on purification of specific compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(47) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. L. Anal. Biochem.
1970, 34, 595-598.
JA012442W
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2484 J. AM. CHEM. SOC. VOL. 124, NO. 11, 2002