Preferred conformations of peptides containing tert-leucine, a sterically demanding, lipophilic α-amino acid with a quaternary side-chain C β atom

Article abstract of DOI:10.1002/chem.200400892

Terminally protected homopeptides of tert-leucine, from the dimer to the bexamer, co-oligopeptides of tert-leucine in combination with α-aminoisobutyric acid or glycine residues up to the hexamer level, and simple dipeptides representing known scaffolds for catalysts in asymmetric organic reactions were prepared by solution methods and fully characterized. The results of conformation analysis, performed by use of FT-IR absorption, NMR, CD, and X-ray diffraction techniques, indicate that this Hydrophobic α-amino acid with tetrasubstitution at the C^β atom is structurally versatile. We show that it prefers extended or semiextended conformations, but can also be accommodated in folded structures, pro vided that these are biased by the presence of helicogenic residues. The current large-scale production of Tle, combined with its conformational preferences unravelled in this work, should make this bulky, hydrophobic, C ^α-trisubstituted α-amino acid a regular building block of any strategy seeking to tailor peptides with improved catalytic and pharmacological properties.

Full text of DOI:10.1002/chem.200400892

FULL PAPER
Preferred Conformations of Peptides Containing tert-Leucine,
a Sterically Demanding, Lipophilic a-Amino Acid with a
Quaternary Side-Chain Cb Atom
Fernando Formaggio,*[a] Chiara Baldini,[a] Vittorio Moretto,[a] Marco Crisma,[a]
Bernard Kaptein,[b] Quirinus B. Broxterman,[b] and Claudio Toniolo[a]
Dedicated to the memory of Professor Murray Goodman, an outstanding peptide chemist and a great mentor
Abstract: Terminally protected homo-
peptides of tert-leucine, from the dimer
to the hexamer, co-oligopeptides of
tert-leucine in combination with a-ami-
noisobutyric acid or glycine residues up
to the hexamer level, and simple dipep-
tides representing known scaffolds for
catalysts in asymmetric organic reac-
tions were prepared by solution meth-
ods and fully characterized. The results
of conformation analysis, performed by
use of FT-IR absorption, NMR, CD,
and X-ray diffraction techniques, indi-
cate that this hydrophobic a-amino
acid with tetrasubstitution at the Cb
atom is structurally versatile. We show
that it prefers extended or semiextend-
ed conformations, but can also be ac-
commodated in folded structures, pro-
vided that these are biased by the pres-
ence of helicogenic residues. The cur-
rent large-scale production of Tle, com-
bined
with
its
conformational
preferences unravelled in this work,
should make this bulky, hydrophobic,
Ca-trisubstituted a-amino acid a regu-
lar building block of any strategy seek-
ing to tailor peptides with improved
catalytic and pharmacological proper-
ties.
Keywords: conformation analysis ·
IR spectroscopy · NMR spectros-
copy · peptides · X-ray diffraction
Introduction
a-amino acids (for a review article see reference [2]). Resi-
dues with either linear or b- or g-branched side chains were
all considered. It was shown that the position of side chain
branching, steric requirements, electronic properties, and
solvophobic character are all important factors governing
peptide conformation. However, among b-branched amino
acids only those with Cb-trisubstitution (e.g., Val, Ile) were
examined.
Fourty-five years ago, Goodman and Schmitt published the
first detailed study of the preferred conformations of well
characterized, monodisperse, low-molecular-weight pepti-
des.[1] The unordered conformation and regular secondary
structures (a helix and b sheet conformations) were all dis-
cussed. Subsequently, Goodman and his co-workers (includ-
ing one of the co-authors of this paper, C. T.) carried out
systematic analyses of a large variety of model linear pepti-
des based on protein residues and related Ca-tri-substituted
[a] Prof. F. Formaggio, C. Baldini, V. Moretto, Dr. M. Crisma,
Prof. C. Toniolo
Institute of Biomolecular Chemistry, CNR, and
Department of Chemistry
University of Padova, via Marzolo 1, 35131 Padova (Italy)
Fax : (+39)049-827-5239
We recently set out to expand this research field into in-
vestigation of the conformational preferences of peptides
heavily based on the Cb-tetrasubstituted, Ca-trisubstituted a-
amino acid Tle (tert-leucine, 3-methylvaline, or tert-butylgly-
cine) and Pen (penicillamine). Here we describe the solution
synthesis, chemical characterization and crystal-state (X-ray
diffraction) and solution (FT-IR absorption, NMR and CD
techniques) 3D structural analyses of a large set of l-Tle
E-mail: fernando.formaggio@unipd.it
[b] Dr. B. Kaptein, Dr. Q. B. Broxterman
DSM Research Life Sciences, Advanced Synthesis and Catalysis
P.O. Box 18, 6160 MD Geleen (The Netherlands)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2005, 11, 2395 – 2404
DOI: 10.1002/chem.200400892
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2395

homo-oligomers and co-oligopeptides in combination vari-
ously with Aib (a-aminoisobutyric acid), Gly, Pro, or Phe
residues.
whereas in molecule B it is cis (w = 9.7(6)8). Gly, Aib, and
ꢀ
Tle tert-butyl esters ( COOtBu) were obtained by esterifica-
tion of the corresponding Z-protected amino acid with iso-
butene in the presence of a catalytic amount of sulfuric
acid.[45] Peptide and amide bond formation was achieved in
solution by the EDC (1-(3-dimethylamino)propyl-3-ethylcar-
bodiimide)/HOAt (7-aza-1-hydroxy-1,2,3-benzotriazole)[46]
method in CH2Cl2 in the presence of N-methylmorpholine.
Despite the use of this strong C-activation approach the
yields of the coupling reactions involving the sterically hin-
dered Tle a-amino nucleophilic functionality were only
moderately good (in the 52–70% range).[47] Removal of the
Z N-protecting group was performed by catalytic hydroge-
nation.
Although Tle is a non-coded a-amino acid, it does never-
theless occur in nature. Both Tle enantiomers have been iso-
lated as components of antimicrobial and antineoplastic
agents from marine sponges (hemiasterlins, polytheona-
mides, discodermins, milnamides).[3–8] In some cases even -
(Tle)2- and -(Tle)3- sequences have been found. Synthetic
methods for Tle and applications of its derivatives and pep-
tides in catalysis and medicinal chemistry have been exten-
sively reviewed (up to 1995) by Drauz and co-workers.[9]
The number of conformation investigations of Tle deriva-
tives and peptides is limited. Sklenar and Jecny[10] reported
the crystal structure of the cyclic dipeptide composed of l-
Pro and d-Tle, whreas Hamann and co-workers[11] recently
described that of a Tle dialkylamide. According to Cordopa-
tis and co-workers,[12] it appears that the oxytocin tripeptide
tail containing a Tle residue either in the N-terminal or in
the central position folds in a b turn structure. However,
conformational energy calculations on Ac-l-Tle-NH2 pre-
dicted global minima in the pleated b sheet and semiextend-
ed (collagen-like) regions of the f, y space.[13] Interestingly,
the 5 kcalmolꢀ1 isoenergy contour encloses only 34% of the
total conformational space. More recent publications have
described exciting applications of Tle derivatives and pepti-
des as enzyme inhibitors and peptide hormone receptor an-
tagonists,[14–26] catalysts in enantioselective organic reac-
tions[27–37] and analogues of bioactive peptides.[12,38] The
steric clashes between closely neighbouring, bulky Tle ho-
mochiral residues may hamper the formation of self-assem-
bled peptide nanotubes.[39] Tle has been successfully site-spe-
cifically incorporated into proteins by biosynthetic method-
ologies.[40]
The physical properties and analytical data for the Tle de-
rivatives and peptides are listed in Table 1. The synthesis
and characterization of Boc-l-Tle-OH, of Z-l-Tle-OH and
its dicyclohexylammonium salt, and of Z-l-Tle-OtBu have
already been reported.[47,48] All newly synthesized com-
pounds were also characterized by 1H NMR spectroscopy
and ESI-TOF mass spectrometry.
l-Tle homopeptides: Figure 1a shows FT-IR absorption
spectra of the Z/OtBu-protected l-Tle homopeptide series
in CDCl3 solution (1 mm concentration) in the conforma-
ꢀ
tionally informative N H stretching region. The curves of
the oligomers are each characterized by two bands: one at
about 3430 cmꢀ1, attributable to free (solvated) NH groups,
and one at about 3340 cmꢀ1, attributable to hydrogen-
bonded NH groups.[49–52] The intensity of the low-frequency
Results and Discussion
Synthesis and characterization: For the large-scale produc-
tion of enantiomerically pure l-Tle, an economically attrac-
tive and generally applicable chemoenzymatic synthesis de-
veloped by DSM Research a few years ago[41–43] was exploit-
ed. It involves the Strecker synthesis of the a-amino nitrile
and subsequent hydrolysis to the racemic a-amino amide,
followed by optical separation by the Ochrobactrum anthro-
pi resolution technology.
Boc (tert-butyloxycarbonyl), Z (benzyloxycarbonyl), Ac,
Piv (pivaloyl), and Bz (benzoyl) N-protected or blocked de-
rivatives and peptides of l-Tle were prepared by use of tert-
butyldicarbonate, N-(benzyloxycarbonyl)succinimide, acetic
anhydride, pivaloyl chloride and benzoyl chloride, respec-
tively. The crystal structure of Boc-l-Tle-OH was solved by
X-ray diffraction (see Supporting Information). Interesting-
ꢀ
ꢀ
ly, the CONH group of the secondary Boc urethane func-
tion[44] in the crystallographically independent molecule A
of the asymmetric unit is in the more common trans confor-
Figure 1. FT-IR absorption spectra in the 3500–3250 cmꢀ1 region of:
a) the Z-(l-Tle)n-OtBu (n = 2–6) homopeptide series in CDCl3 solution
(peptide conc. 1 mm), and b) Z-(l-Tle)6-OtBu in CDCl3 solution at pep-
tide concentrations of 10 mm (A), 1.0 mm (B), and 0.1 mm (C).
mation, although somewhat distorted (w
= 163.9(3)8),
2396
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www.chemeurj.org
Chem. Eur. J. 2005, 11, 2395 – 2404

Conformations of Tle Peptides
FULL PAPER
Table 1. Physical properties and analytical data for the Tle derivatives and peptides.
Compound
m.p. [8C][a]
Recryst.
solvent[b]
[a]2D0[c]
TLC[d]
IR[e] n˜ [cmꢀ1
]
Rf(I)
Rf(II)
Rf(III)
Boc-l-Tle-OH
Z-l-Tle-OH
Z-l-Tle-OtBu
Z-(l-Tle)2-OtBu
Z-(l-Tle)3-OtBu
Z-(l-Tle)4-OtBu
Z-(l-Tle)5-OtBu
Z-(l-Tle)6-OtBu
116–117
oil
oil
EtOAc
EtOAc/PE
EtOAc/PE
DCM
EtOAc/PE
EtOAc/PE
EtOAc/PE
EtOAc/PE
MeCN
EtOAc/PE
EtOAc/PE
EtOAc/PE
EtOAc
ꢀ4.4
ꢀ4.6
0.90
0.55
0.90
0.90
0.90
0.90
0.90
0.90
0.50
0.90
0.80
0.60
0.60
0.55
0.30
0.85
0.60
0.60
0.45
0.45
0.80
0.95
0.95
0.90
0.90
0.90
0.85
0.85
0.95
0.90
0.90
0.90
0.90
0.90
0.90
0.85
0.95
0.90
0.90
0.90
0.90
0.90
0.95
0.90
0.90
0.90
0.90
0.80
0.90
0.90
0.80
0.85
0.85
0.95
0.40
0.35
0.80
0.80
0.80
0.80
0.80
0.75
0.55
0.65
0.45
0.30
0.30
0.20
0.10
0.55
0.25
0.20
0.10
0.10
0.60
0.45
0.45
0.65
0.40
0.40
0.60
3436, 1740, 1709, 1660
3321, 1715, 1520[h]
ꢀ15.4
ꢀ25.5
ꢀ38.1
ꢀ50.9
ꢀ53.0[f]
ꢀ70.4[f]
ꢀ73.9
ꢀ10.5
ꢀ3.2
3357, 1718, 1507[h]
50–52
3340, 1729, 1659, 1529
155–157
108–110
153–155
138–140
250–252
105–106
160–162
184–185
216–217
207–208
250–252
108–110
157–158
241–243
240–242
243–245
124–125
56–58
3336, 1728, 1712, 1655, 1520
3432, 3347, 1724, 1653, 1507
3436, 3352, 1717, 1651, 1507
3427, 3350, 1719, 1651, 1507
3349, 3320, 1716, 1647, 1506
3417, 3295, 1730, 1716, 1654, 1530
3416, 3338, 1726, 1652, 1532
3386, 3367, 3357, 1714, 1674, 1533
3423, 3320, 1732, 1668, 1526
3445, 3352, 1730, 1659, 1525
3369, 1736, 1657, 1523
3375, 3291, 1716, 1644, 1515
3298, 1749, 1734, 1705, 1649, 1523
3369, 3279, 1714, 1633, 1522
3290, 1740, 1632, 1534
3378, 3281, 1716, 1628, 1527
3295, 1718, 1687, 1642, 1531
3316, 1710, 1644, 1542
Ac-(l-Tle)6-OtBu
Z-l-Tle-Aib-OtBu
Z-Aib-l-Tle-Aib-OtBu
Z-(Aib)2-l-Tle-Aib-OtBu
Z-l-Tle-(Aib)2-l-Tle-Aib-OtBu
Z-Aib-l-Tle-(Aib)2-l-Tle-Aib-OtBu
Ac-Aib-l-Tle-(Aib)2-l-Tle-Aib-OtBu
Z-l-Tle-Gly-OtBu
Z-Gly-l-Tle-Gly-OtBu
Z-(l-Tle-Gly)2-OtBu
Z-Gly-(l-Tle-Gly)2-OtBu
Z-(-l-Tle-Gly)3-OtBu
Z-l-Tle-NHiPr
Z-l-Pro-l-Tle-NHiPr
Z-l-Pro-l-Tle-NHiPr
Z-l-Tle-l-Phe-NHtBu
Piv-d-Pro-l-Tle-NHiPr
Piv-l-Pro-l-Tle-NHiPr
Bz-l-Tle-l-Phe-NHtBu
ꢀ2.3
ꢀ11.7
ꢀ9.3
EtOAc
MeCN
ꢀ11.0
ꢀ19.0
ꢀ25.4
9.6[g]
EtOAc/PE
EtOAc/PE
EtOAc
DMF/H2O
DMF/H2O
EtOAc
EtOAc/PE
EtOAc/PE
DCM
EtOAc/PE
EtOAc/PE
EtOAc/PE
8.7[g]
9.2[g]
3.2
34.2
ꢀ71.3
ꢀ48.2
18.7
60–62
84–85
188–189
153–154
220–221
3312, 1710, 1644, 1545
3322, 1709, 1647, 1535
3363, 1691, 1641, 1615, 1535
3329, 1678, 1648, 1535
3326, 1637, 1548, 1522
ꢀ80.7
ꢀ43.2
[a] Determined on a Leitz model Laborlux 12 apparatus (Wetzlar, Germany). [b] EtOAc = ethyl acetate, PE = petroleum ether, DCM = dichlorome-
thane, MeCN = acetonitrile, DMF = N,N-dimethylformamide. [c] Determined on a Perkin–Elmer model 241 polarimeter (Norwalk, CT) fitted with a
Haake model L thermostat (Karlsruhe, Germany); c = 0.5 (methanol). [d] Silica gel plates (60F-254 Merck), solvent systems: I) chloroform/ethanol 9:1;
II) butan-1-ol/acetic acid/water 3:1:1; III) toluene/ethanol 7:1; the plates were viewed under a UV lamp or by use of the hypochlorite/starch/iodide chro-
matic reaction as appropriate; a single spot was observed in each case. [e] Determined in KBr pellets on a Perkin–Elmer model 580 B spectrophotometer
fitted with a Perkin–Elmer model 3600 IR data station. [f] c = 0.1 (methanol). [g ] c = 0.25 (DMF/DCM 1:1). [h] Determined in a film.
band increases relative to that of the high-frequency band as
the main chain length grows (particularly from pentamer to
hexamer). On variation of the concentration (from 10 to
0.1 mm) all peptides display a remarkable decrease in the in-
tensity of the low-frequency band (see Figure 1b for the
hexamer). At the lowest concentration examined, only the
hexamer exhibits an absorption, albeit of limited intensity,
near 3340 cmꢀ1. These findings are significantly at variance
with the IR absorption results reported for the homopepti-
des of the related protein amino acids Val, Ile, and Leu.[49]
This FT-IR absorption analysis provided convincing evi-
dence that the Tle homopeptides do not fold significantly in
the structure-supporting halohydrocarbon CDCl3, but rather
that they tend to self-associate extensively. However, this
self-association phenomenon is much weaker than that typi-
negative shoulder near 225 nm (Figure 2). This pattern,
which does not change over the temperature range from 25
to 08C (spectra not shown), is indicative of an unordered
peptide conformation.[53] Upon addition of water to the TFE
solution, the CD spectrum of this highly hydrophobic pep-
tide changes significantly, eventually (in a TFE/H2O 20:80
ꢀ
cal of the formation of b sheets, as the H-bonded N H
stretching and C=O stretching bands for the hexamer are
found near 3340 and 1650 cmꢀ1 (the latter not shown) both
at the highest concentration examined (10 mm) (Figure 1b)
and in the solid state (Table 1). These frequencies differ
markedly from those characteristic of b sheet-forming pepti-
des (ca. 3285 and 1630 cmꢀ1, respectively).[51,52]
The far-UV CD spectrum of the Ac/OtBu-blocked l-Tle
homohexamer in TFE (2,2,2-trifluoroethanol) exhibits a
strong negative maximum below 200 nm accompanied by a
Figure 2. Far-UV CD spectra of Ac-(l-Tle)6-OtBu in different TFE/water
mixtures. The TFE content in the mixtures (%) is indicated (peptide
conc. 1 mm).
Chem. Eur. J. 2005, 11, 2395 – 2404
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F. Formaggio et al.
solvent mixture) assuming the shape typical of a b sheet-like
conformation (a negative maximum at 222 nm followed by a
positive maximum near 207 nm).[54] In other words, it seems
that the Tle-rich molecules of the hexamer pack strongly in
the presence of a large percentage of water in the solvent
mixture, because of the overwhelming role played by the hy-
drophobic interactions.
Our conformational investigation of the l-Tle homo-
oligomer series was further extended by analysis of the crys-
tal structure of Z-(l-Tle)3-OtBu. The three independent
molecules (A, B, and C) in the asymmetric unit of this ho-
motrimer are conformationally similar (Table 2 and the Sup-
porting Information). Figure 3 shows the structure of mole-
cule A. None of the molecules adopts an ordered conforma-
tion. The sets of f and y torsion angles[55] of the terminal
Tle1 and Tle3 residues are close to each other (in the extend-
ed region of the conformational map).[56] On the other hand,
the conformation of the Tle2 residue is semiextended (in the
(Pro)n region), thus generating a pronounced kink in the
central part of each molecule. Because of the globally rather
extended character of the molecules, no intramolecular hy-
drogen bonds were observed. The three molecules are
packed through a complex network of nine intermolecular
Figure 3. X-ray diffraction structure of molecule A in the asymmetric
unit of Z-(l-Tle)3-OtBu with atom numbering.
hydrogen bonds (see Supporting Information): three ure-
2
ꢀ
ꢀ
thane N H···O=C (ester), three (Tle peptide) N H···O=C
3
2
ꢀ
(urethane), and three (Tle peptide) N H···O=C (Tle pep-
tide) hydrogen bonds. The N···O separations, 2.985(12)–
3.114(12) ꢁ, are in the usual range for this type of hydrogen
bonds.[57–59] Overall, the combination of the central kink
with the symmetry of the C2 space group gives rise to a cy-
lindrical arrangement of the molecules along the c direction,
with the cavity largely filled by Tle tert-butyl side chain
methyl groups (Figure 4).
Figure 4. Mode of packing of the Z-(l-Tle)3-OtBu molecules in the crys-
tal. The intermolecular hydrogen bonds are represented by dashed lines.
Table 2. Relevant backbone torsion angles [8] for the seven Tle derivatives and peptides studied in this work.
Torsion
angle
Boc-l-Tle-OH
Tle homotripeptide
Aib/Tle
Aib/Tle
Aib/Tle
Gly/Tle
dipeptide
B
Gly/Tle
tripeptide
B
tripeptide tetrapeptide hexapeptide
A
B
A
B
C
A
A
q2
ꢀ74.7(18)
169.6(12)
9.7(6) ꢀ176.8(9)
ꢀ81.0(18)
157.1(13)
ꢀ173.1(11) ꢀ173.8(9)
ꢀ76.8(17)
171.2(7)
179.6(7)
105.9(3)
171.9(3)
177.0(2)
49.8(4)
174.4(7)
ꢀ174.9(7)
ꢀ158.2(6)
ꢀ59.4(8)
ꢀ43.2(9)
ꢀ171.8(6)
ꢀ67.7(8)
ꢀ22.7(9)
ꢀ178.0(6)
ꢀ53.8(8)
ꢀ29.0(8)
ꢀ177.3(6)
ꢀ52.3(8)
ꢀ33.3(8)
ꢀ170.8(6)
ꢀ84.0(8)
ꢀ23.4(10)
179.2(7)
ꢀ91.4(8) ꢀ142.4(6)
119.6(4)
178.7(6) ꢀ172.3(4)
107.3(6)
177.0(5)
q1
ꢀ178.3(3)
178.1(4)
169.0(10)
ꢀ172.7(6)
wo
f1
y1
w1
f2
y2
w2
f3
y3
w3
f4
y4
w4
f5
y5
w5
f6
y6
w6
163.9(3)
ꢀ169.4(5)
179.4(6) ꢀ176.6(6) ꢀ176.0(5) ꢀ177.2(5)
ꢀ110.0(4) ꢀ107.0(5) ꢀ126.6(12) ꢀ124.5(12) ꢀ118.0(12)
ꢀ73.9(7)
ꢀ128.2(6) ꢀ132.2(6) ꢀ159.3(4) ꢀ164.9(4)
124.4(4)
ꢀ27.9(5)
142.3(10)
174.6(9)
142.8(10)
175.0(8)
142.9(10)
173.0(9)
ꢀ21.4(8) ꢀ128.9(3)
136.6(6)
174.9(6)
134.2(6)
175.7(7)
160.7(4)
172.5(4)
148.7(4)
171.8(4)
ꢀ174.3(5) ꢀ168.5(2)
ꢀ90.7(12)
130.7(10)
172.6(9)
ꢀ87.8(12)
132.9(11)
173.5(10)
ꢀ88.8(12) ꢀ137.2(6)
ꢀ61.1(4)
ꢀ28.2(4)
ꢀ148.7(6) ꢀ147.9(7) ꢀ118.6(4) ꢀ130.1(4)
130.1(10)
170.4(9)
150.4(5)
177.1(6) ꢀ178.7(7)
134.2(4)
138.9(4)
179.5(4)
172.1(5) ꢀ176.2(2)
ꢀ172.9(6) ꢀ170.3(6) ꢀ171.6(4)
ꢀ126.5(11) ꢀ126.9(12) ꢀ125.5(10)
49.9(7)
48.7(7)
ꢀ90.4(3)
ꢀ18.1(3)
166.7(2)
52.1(3)
39.4(3)
179.5(2)
ꢀ85.4(6) ꢀ166.4(4)
ꢀ166.4(4) ꢀ170.8(4)
ꢀ171.8(4) ꢀ179.7(5)
152.2(9)
151.2(9)
150.1(9)
ꢀ176.8(9)
ꢀ177.8(10) ꢀ174.1(9)
175.2(6)
54.5(10)
40.5(9)
171.2(6)
2398
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Chem. Eur. J. 2005, 11, 2395 – 2404

Conformations of Tle Peptides
FULL PAPER
Aib/l-Tle peptides: Aib is a strongly helicogenic residue,
due to its gem-dimethylation at the a-carbon.[60,61] The FT-
IR absorption spectra of the Z/OtBu-protected Aib/l-Tle
peptides from dimer to hexamer in CDCl3 solution
(Figure 5) in part parallel those reported for the 310-heli-
Figure 6. Plot of the bandwidths of the NH proton signals in the 1H
NMR spectra of Z-Aib-l-Tle-(Aib)2-l-Tle-Aib-OtBu as a function of in-
creasing percentages of TEMPO (w/v) added to the CDCl3 solution (pep-
tide conc. 1 mm).
Figure 5. FT-IR absorption spectra (3500–3250 cmꢀ1 region) of: Z-l-Tle-
Aib-OtBu (2), Z-Aib-l-Tle-Aib-OtBu (3), Z-(Aib)2-l-Tle-Aib-OtBu (4),
Z-l-Tle-(Aib)2-l-Tle-Aib-OtBu (5) and Z-Aib-l-Tle-(Aib)2-l-Tle-Aib-
OtBu (6) in CDCl3 solution (peptide conc. 1 mm).
d
aN(i,i+n) connectivities. The connectivity between the Tle2
CaH and Aib4 NH protons, typical of the 310-helical struc-
ture,[67] is clearly visible, whereas the connectivity between
the Tle2 CaH and Aib6 NH protons, assignable to the a-heli-
cal structure, is not observed.
cal[62,63] Aib homopeptides.[64] Here, too, the relative intensi-
ty of the low-frequency band increases significantly from the
n = 2 to the n = 6 oligomer. However, an abrupt shift is
observed from the trimer to the tetramer (3394 cmꢀ1 versus
3358 cmꢀ1). While the spectral position of the latter is typi-
cal of a helical conformation,[64] that of the former has been
associated with the fully extended, very weakly hydrogen-
bonded conformation.[65] In these peptides, unlike in the l-
Tle homopeptides discussed above, the concentration effect
is of minor significance.
The far-UV CD spectra of the Ac/OtBu-blocked Aib/l-
Tle hexapeptide in MeOH (methanol) and in TFE solutions
are similar, each exhibiting a strong negative maximum cen-
tred well above 200 nm (204 nm) and a negative shoulder at
about 222 nm (Figure 8). The intensity of the curve in TFE
is 10–15% higher than that in MeOH. The ellipticity R
ratio, [q]204/[q]222, is in the 0.15–0.30 range. These CD prop-
erties are those expected for a right-handed, 310-helical con-
formation.[68]
We initially examined the 1H NMR behavior of the Z/
OtBu-protected Aib/l-Tle hexapeptide in CDCl3 solution
through the addition of increasing amounts of the free radi-
cal TEMPO (2,2,6,6-tetramethylpiperidin-1-oxyl), which in-
duces line-broadening of the resonances of exposed NH pro-
tons[66] (Figure 6). Two classes of NH protons were observed
in the titration experiments: class 1 includes the N(1)H and
N(2)H protons, the resonances of which broaden remarka-
bly upon addition of TEMPO, whilst class 2 includes the
N(3)H–N(6)H protons, which display behavior typical of
shielded protons (relative insensitivity of linewidth to the
presence of TEMPO). This general trend closely resembles
those published for 310-helical (Aib)n peptides of comparable
main chain length.[64]
We also solved the X-ray diffraction structures of three Z/
OtBu-protected Aib/l-Tle peptides: namely, the trimer, tet-
ramer and hexamer (Figure 9, Figure 10 and Figure 11, re-
spectively). Surprisingly, the tripeptide is not folded into a b
turn conformation, as is usual for -CO-Aib-Xxx-NH- se-
quences.[61,69] Indeed, the conformation adopted by the cen-
tral Tle residue is extended, while those of the terminal Aib
residues are helical, but of opposite screw sense (Table 2).
ꢀ
Therefore, no C=O···H N intramolecular hydrogen bond
stabilizes this crystal-state 3D structure. Conversely, the hex-
amer folds into a regular, right-handed 310 helix (Table 2)
!
with four 1 4 intramolecular hydrogen bonds (see Support-
ing Information). Also the tetramer is folded, but is not heli-
cal, in that the -Aib1-Aib2- sequence is in the type-IIꢂ b turn
conformation,[70–72] which is forced to be followed by a type-
III b turn in the -Aib2-l-Tle3- sequence. This rather unusual
conformation is related to the “d-configured” semiextended
structure adopted by the achiral Aib1 residue. In the crystals
the helices of both tetramer and hexamer pack in the
common head-to-tail arrangement.[60]
We expanded the NMR analysis by recording the NOESY
spectrum of the Ac/OtBu-blocked Aib/l-Tle hexapeptide in
DMSO solution. The ROESY spectrum in the 7.1–8.3 ppm
(amide NH protons) region exhibits five intense cross-peaks
of the NH(i)!NH(i+1) type (Figure 7A), indicative of the
onset of a helical structure.[67] The type of helical structure
formed can be extracted from inspection of Figure 7B,
which shows a section of the ROESY spectrum with the
Gly/l-Tle peptides: Only the dimer and trimer of the Z/
OtBu-protected Gly/l-Tle peptides are soluble in CDCl3 so-
Chem. Eur. J. 2005, 11, 2395 – 2404
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F. Formaggio et al.
Figure 8. Far-UV CD spectra of Ac-Aib-l-Tle-(Aib)2-l-Tle-Aib-OtBu in
MeOH and TFE solutions (peptide conc. 1 mm).
Figure 9. X-ray diffraction structure of Z-Aib-l-Tle-Aib-OtBu with
atomic numbering.
Figure 7. A) Section of the ROESY spectrum of Ac-Aib-l-Tle-(Aib)2-l-
Tle-Aib-OtBu in [D6]DMSO solution, showing the NH(i)!NH(i+1)
connectivities. B) Section of the ROESY spectrum of the same peptide,
in particular showing the daN(i,i+2) connectivity (encircled). The region
of the spectrum where the daN(i,i+4) connectivity was expected is indi-
cated by an arrow. Peptide conc. 5 mm.
lution. The corresponding FT-IR absorption spectra (see
Figure 12 for the trimer) do not suggest formation of any in-
tramolecular hydrogen bond. Rather, at the highest concen-
tration examined (10 mm), a modest amount of weak inter-
molecular hydrogen bonding is apparent.[49–52] The highest
oligomers of this series are soluble in CDCl3/DMSO (di-
methyl sulfoxide)[73] mixtures. In the FT-IR absorption spec-
tra (C=O stretching region) of the tetramer (not shown),
pentamer (Figure 13a) and hexamer (Figure 13b), a strong
band is seen near 1630 cmꢀ1 at low DMSO percentages,
tending to decrease more or less rapidly (depending on pep-
Figure 10. X-ray diffraction structure of Z-(Aib)2-l-Tle-Aib-OtBu with
ꢀ
atomic numbering. The two intramolecular C=O···H N hydrogen bonds
are indicated by dashed lines.
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Conformations of Tle Peptides
FULL PAPER
Single crystals of Z-l-Tle-Gly-OtBu and Z-Gly-l-Tle-Gly-
OtBu were grown and subjected to X-ray diffraction analy-
ses (Supporting Information). Each peptide crystallizes with
two independent molecules (A and B) in the asymmetric
unit. Figure 14 and Figure 15 show the structures of mole-
Figure 11. X-ray diffraction structure of Z-Aib-l-Tle-(Aib)2-l-Tle-Aib-
ꢀ
OtBu with atomic numbering. The four intramolecular C=O···H N hy-
drogen bonds are indicated by dashed lines.
Figure 14. X-ray diffraction structure of molecule A in the asymmetric
unit of Z-l-Tle-Gly-OtBu with atom numbering.
Figure 15. X-ray diffraction structure of molecule A in the asymmetric
unit of Z-Gly-l-Tle-Gly-OtBu with atom numbering.
Figure 12. FT-IR absorption spectra (3500–3250 cmꢀ1 region) of Z-Gly-l-
Tle-Gly-OtBu in CDCl3 solution at: 10 mm (A), 1.0 mm (B), and 0.1 mm
(C) peptide concentrations.
cules A of the di- and tripeptide, respectively. The two inde-
pendent molecules of the dipeptide are conformationally
similar (except for the q2 torsion angle of the Z group) and
extended (Table 2). They are arranged in an antiparallel
fashion and alternate along the a direction, forming rows
stabilized by intermolecular hydrogen bonds (see Support-
ing Information). Globally, each molecule is involved in
four intermolecular H-bonds with the two nearest-neighbor
molecules, the NH groups in two of them playing the role of
donors while the other two act as acceptors.
The crystal structure of the trimer is remarkably similar
to that of the dimer. The sets of f, y torsion angles of both
independent molecules are in the region of the b pleated
sheet conformation (f = ꢀ1408, y = 1358),[56] with the
single exception of the f3 value of molecule A, which is
more contracted (ꢀ858) (Table 2). The two independent
molecules also alternate along the a direction in this crystal
structure, forming rows of intermolecular hydrogen bonds
(Supporting Information). Each molecule donates three hy-
drogen bonds to the two nearest-neighbour molecules and
accepts an additional three H-bonds.
Figure 13. FT-IR absorption spectra (1750–1570 cmꢀ1 region) of Z-Gly-
(l-Tle-Gly)2-OtBu (a) and Z-(l-Tle-Gly)3-OtBu (b) in DMSO/CDCl3 sol-
vent mixtures. The DMSO (v/v) percentages are indicated.
tide main chain length) as the DMSO percentage is in-
creased. This absorption, characteristic of the b sheet con-
formation,[49,51,52] completely disappears at 15% DMSO for
the tetramer and is drastically reduced in pure DMSO for
the pentamer, while still remaining remarkably intense for
the hexamer. Interestingly, this same absorption also domi-
nates the amide I spectral region of the three oligomers in
the solid state (Table 1).
Tle-based catalyst peptides: In two recent papers, Miller
and co-workers[28,29] reported that short peptides based on
the heterochiral R-CO-Pro-Tle-NH-R’ sequence—forming
an intramolecularly hydrogen-bonded b turn scaffold—are
excellent catalysts in enantioselective organic reactions and
more selective templates than those containing the diaster-
Chem. Eur. J. 2005, 11, 2395 – 2404
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F. Formaggio et al.
eomeric homochiral sequence. In addition, Hoveyda and co-
workers[30] described the use of (ortho-substituted)Bz-l-Tle-
l-aAA-NHtBu (NHtBu = tert-butylamine, aAA = aromat-
ic a-amino acid) Na-acylated dipeptide amides as catalysts
for appreciable enantioselectivity in organic reactions. We
therefore decided to investigate the preferred conformations
of these Tle-based, short peptide catalysts. To this end, the
Conclusion
The results of our conformation investigation, carried out
both in solution and in the crystal state by the use of a com-
bination of physicochemical techniques, into a large body of
Tle-rich peptides strongly point to the conclusion that this
amino acid residue may adopt a wide set of conformations:
extended or b sheet-like, semiextended or (Pro)n-like, and
folded or helical. Indeed, in the six X-ray diffraction struc-
tures of Tle peptides solved, of the seventeen Tle residues
(including those of all independent molecules in the various
asymmetric units) eleven were found to adopt the extended,
three the semiextended and three the folded conformations
(the last of these, however, clearly biased by the strongly
helicogenic Aib residue).[60,61] In structure-supporting sol-
vents the Tle homopeptides are much less folded than their
Leu, Val and Ile counterparts.[49] Furthermore, not unexpect-
edly, hydrophobic interactions play a major role in packing
the aliphatic Tle homopeptides into b sheet-like structures
in organic/aqueous solvent mixtures. The low tendency of a
Tle residue to fold is further highlighted by the nonhelical
structure of the terminally protected -Aib-Tle-Aib- tripep-
tide, as clearly extracted from our IR absorption and X-ray
diffraction analyses, despite the heavy presence of two Aib
residues. However, even two bulky Tle residues can be ac-
commodated in a longer 310-helical peptide, one on top of
the other after a complete turn of the helix. The conforma-
tional plasticity of the Tle residues is additionally under-
scored by the extended intermolecularly hydrogen-bonded
structures adopted in solution and in the crystal state, as
well by the peptides containing Gly, a residue with a strong
propensity to adopt the b sheet conformation.[2,51] On these
bases it is not surprising that the few published theoretical
and experimental works on Tle derivatives and peptides[12,13]
would not agree on Tleꢂs (extended versus folded) confor-
mational properties.
three peptides Piv-l-Pro-d-Tle-NHiPr (Piv
= pivaloyl,
NHiPr = isopropylamino), Piv-l-Pro-l-Tle-NHiPr, and Bz-
l-Tle-l-Phe-NHtBu were synthesized and studied. The Piv
Na-blocking was introduced to freeze the Pro tertiary amide
bond in the overwhelmingly more common trans conforma-
tion.[74]
ꢀ
Figure 16 shows the FT-IR absorption spectra (N H
stretching region) of the three peptides in CDCl3 solution. It
is clear that a band assignable to intramolecularly hydrogen-
Figure 16. FT-IR absorption spectra (3500–3250 cmꢀ1 region) of: Piv-d-
Pro-l-Tle-NHiPr (a), Piv-l-Pro-l-Tle-NHiPr (b), and Bz-l-Tle-l-Phe-
NHtBu (c) in CDCl3 solution (peptide conc. 1 mm).
Tle is currently extensively exploited as a fundamental
building block in the design of enzyme inhibitors, peptide
hormone receptor antagonists, analogues of bioactive pepti-
des and catalysts in asymmetric synthesis. We believe that
these 3D structural findings, combined with the availability
of Tle on large scales, should further enhance the impor-
tance of derivatives and peptides of this sterically hindered,
highly lipophilic amino acid.
bonded folded forms, at about 3360 cmꢀ1 although of moder-
ate intensity, is present only in the spectra of the two dia-
stereomeric -Pro-Tle- peptides. Apparently, a change in the
sequence chirality in these peptides does not induce any sig-
nificant variation in the IR absorption pattern. An ancillary
NMR investigation (results not shown) unambiguously indi-
cated that a type-II (II’) b turn conformation[70–72] does
indeed only populate the equilibrium mixtures of these two
l-l and l-d diastereomers, the corresponding daN(i,i+1)
cross-peaks standing out clearly in the ROESY spectra.[67] It
may be concluded that, in contrast with the -l-Tle-l-Phe-
peptide, which does not exhibit any tendency to fold, both
diastereomeric -Pro-Tle- sequences form, at least in part,
turn structures. Interestingly, if the -l-Pro-l-Tle- sequence is
folded in the type-II b turn conformation, then l-Tle is
forced to adopt f, y torsion angles typical of a (distorted)
left-handed helical structure.
Experimental Section
FT-IR absorption spectroscopy: FT-IR absorption spectra were recorded
with a Perkin–Elmer 1720 X FT-IR spectrophotometer, nitrogen flushed,
with a sample-shuttle device and at 2 cmꢀ1 nominal resolution, averaging
100 scans. Solvent (baseline) spectra were recorded under the same con-
ditions. Cells with CaF2 windows and pathlengths of 0.1, 1.0 and 10 mm
were used. Spectrograde deuterated chloroform (99.8% 2H) and dimeth-
2
yl sulfoxide (99.8% H6) were obtained from Fluka.
CD spectroscopy: The CD spectra were obtained on a Jasco J-710 dichro-
graph. Cylindrical fused quartz cells of 10, 1, 0.2 and 0.1 mm pathlengths
(Hellma) were used. The values are expressed in terms of [q]T, the total
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Chem. Eur. J. 2005, 11, 2395 – 2404

Conformations of Tle Peptides
FULL PAPER
molar ellipticity (deg cm2 dmolꢀ1). Spectrograde MeOH and TFE (Acros
Organics) were used as solvents.
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ide (99.96% 2H6 from Acros Organics). The free radical TEMPO was
purchased from Sigma.
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X-ray diffraction: Crystals of Boc-l-Tle-OH, Z-(l-Tle)3-OtBu, Z-(Aib)2-
l-Tle-Aib-OtBu, Z-Aib-l-Tle-(Aib)2-l-Tle-Aib-OtBu, and Z-Gly-l-Tle-
Gly-OtBu were grown by slow evaporation from ethyl acetate, methanol,
2,2,2-trifluoroethanol, acetone/water and methanol, respectively. Crystals
of Z-Aib-l-Tle-Aib-OtBu and Z-l-Tle-Gly-OtBu were obtained from
ethyl acetate/petroleum ether solutions by vapor diffusion. Diffraction
data were collected with a Philips PW 1100 diffractometer. Crystallo-
graphic data are given in the Supporting Information. All structures were
solved by direct methods by use of the SHELXS 97[75] or the SIR 2002[76]
programs. Refinements were carried out on F2 by full-matrix block least-
squares, with use of all data, by application of the SHELXL 97[77] pro-
gram with all non-hydrogen atoms anisotropic and their positional pa-
rameters and the anisotropic displacement parameters being allowed to
refine at alternate cycles. Hydrogen atoms were calculated at idealized
positions. During the refinement they were allowed to ride on their carry-
ing atoms with Uiso set equal to 1.2 (or 1.5 for methyl groups) times the
a
b
ꢀ
Ueq of the parent atom. Restraints were applied to the C
C bond dis-
tance of the two independent molecules (A and B) in the asymmetric
ꢀ
unit of Boc-l-Tle-OH. The location of the H atom of the COOH group
was based upon hydrogen-bonding considerations. The phenyl rings of
the Z moieties of all Tle-containing crystalline peptides were constrained
to the idealized geometry. Restraints were also applied to some of the
bonds distances involving side-chain atoms and the C-terminal tert-butyl
groups.
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Received: August 30, 2004
Published online: January 24, 2005
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