The 2.05-helix in hetero-oligopeptides entirely composed of Cα,α-disubstituted glycines with both side chains longer than methyls

Article abstract of DOI:10.1002/bip.22450

The existence of the very uncommon, but potentially quite interesting, multiple, consecutive fully-extended conformation (2.0₅-helix) has been already clearly demonstrated in homo-oligopeptides based on quaternary α-amino acids with both side chains longer than methyls, but not cyclized on the α-carbon atom. To extend the scope of this research, in this work we investigated the occurrence of this flat 3D-structure in hetero-oligopeptides, each composed of two or three different residues of that class. The synthesis of a terminally protected peptide series to the tetrapeptide level was carried out by solution methods. The resulting oligomers were chemically and conformationally characterized. The data obtained point to an overwhelming population of the fully-extended conformation in CDCl₃. However, a solvent-driven switch to a predominant 3₁₀-helical structure was seen in CD₃CN. A delicate, local balance between these two conformations is confirmed to occur in the crystalline state. Molecular dynamics simulations in CHCl₃ on a hetero-tetrapeptide converged to the fully-extended conformation even starting from the 3₁₀-helical structure.

Full text of DOI:10.1002/bip.22450

The 2.05-Helix in Hetero-Oligopeptides Entirely Composed of Ca,a-
Disubstituted Glycines With Both Side Chains Longer Than Methyls
Marco Crisma,1 Cristina Peggion,1 Alessandro Moretto,1 Raja Banerjee,2 Subhrangshu Supakar,2
Fernando Formaggio,1 Claudio Toniolo1
1Department of Chemistry, ICB, Padova Unit, CNR, University of Padova, 35131 Padova, Italy
2Department of Bioinformatics, West Bengal University of Technology, 700064 Kolkata, West Bengal, India
Received 16 September 2013; revised 15 November 2013; accepted 2 December 2013
Published online 5 December 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22450
converged to the fully-extended conformation even start-
ABSTRACT:
C
V
ing from the 310-helical structure. 2013 Wiley
The existence of the very uncommon, but potentially
quite interesting, multiple, consecutive fully-extended
conformation (2.05-helix) has been already clearly dem-
onstrated in homo-oligopeptides based on quaternary a-
amino acids with both side chains longer than methyls,
but not cyclized on the a-carbon atom. To extend the
scope of this research, in this work we investigated the
occurrence of this flat 3D-structure in hetero-oligopepti-
des, each composed of two or three different residues of
that class. The synthesis of a terminally protected peptide
series to the tetrapeptide level was carried out by solution
methods. The resulting oligomers were chemically and
conformationally characterized. The data obtained point
to an overwhelming population of the fully-extended con-
formation in CDCl3. However, a solvent-driven switch to
a predominant 310-helical structure was seen in CD3CN.
A delicate, local balance between these two conformations
is confirmed to occur in the crystalline state. Molecular
dynamics simulations in CHCl3 on a hetero-tetrapeptide
Periodicals, Inc. Biopolymers (Pept Sci) 102: 145–158,
2014.
Keywords: IR absorption; molecular dynamics simula-
tions; NMR; peptide conformation; X-ray diffraction;
Ca-tetrasubstituted a-amino acids
This article was originally published online as an accepted
preprint. The “Published Online” date corresponds to the
preprint version. You can request a copy of the preprint by email-
ing the Biopolymers editorial office at biopolymers@wiley.com
INTRODUCTION
he past 2 decades have witnessed a great progress in
our understanding of the 3D-structures induced in
peptides by non-coded a-amino acids, such as resi-
dues with a quaternary Ca-atom. More specifically,
T
two research groups1–25 have unambiguously demon-
strated that, in addition to the classical a-helix and the rela-
tively well known 310-helix,26–30 another conformation (the
2.05-helix,8,29 quite unusual in any kind of peptide molecules
and in proteins as well) is experimentally found quite often
in the presence of these residues. The overwhelmingly pre-
ferred building blocks for the construction of the latter, over-
all fully-extended structure (where the /, w, x torsion angles
have values of 180ꢀ or very close to them) are Ca,a-disubsti-
tuted Gly residues with both side chains longer than methyls
and not cyclized on the Ca-atom. In particular, this structure
has been repeatedly authenticated by FT-IR absorption and
NMR spectroscopies in solvents of low polarity, e.g. CDCl3,
and by X-ray diffraction in the crystalline state.1–25
Correspondence to: Dr. Marco Crisma, ICB, Padova Unit, CNR, Department of Chemis-
try, University of Padova, via Marzolo 1, 35131 Padova (Italy); e-mail: marco.crisma@
unipd.it
Contract grant sponsor: Government of India
Contract grant number: Sanction No. BT/BI/25/001/2006-Vol. II
Contract grant sponsor: University of Padova
Contract grant number: PRAT 2009 - CPDA095787, PRAT 2011 - CPDA114475
C
V
2013 Wiley Periodicals, Inc.
PeptideScience Volume 102 / Number 2
145

146
Crisma et al.
chromatograms were visualized by UV fluorescence or developed by
chlorine—starch—potassium iodide or ninhydrin chromatic reac-
tion as appropriate. All compounds were obtained in a chromato-
graphically homogeneous state. The solid-state IR absorption
spectra were recorded with a Perkin-Elmer (Norwalk, CT) model
1720X FTIR spectrophotometer.
All examples of the 2.05-helix published to date in both
CDCl3 solution and in the crystal state refer to homo-oligopep-
tides, that is, each of them is based on a single amino acid resi-
due. Surprisingly, however, the limited examples of hetero-
oligopeptides studied, exclusively formed by different Ca,a-dis-
ubstituted Gly residues,23,24 although all adopting the 2.05-heli-
cal conformation in CDCl3, do not generate in the crystal state
this regular secondary structure. In these cases, either a 310-hel-
ical or fully-extended/helical or fully-extended/helical/fully-
extended “mixed” conformations were shown. These irregular
3D-structural arrangements are not due to the dissymmetric
(chiral) nature of some of their amino acid constituents,
because a few, completely fully-extended homo-peptides of
this class based on chiral Ca,a-disubstituted Gly residues have
been indeed reported.12,15,22 In addition, it is relevant to
Synthesis
The DSM Innovative Synthesis BV (Geleen, The Netherlands) detailed
procedure for the preparation, enzymatic resolution, chemical hydro-
lysis, and determination of the enantiomeric excess for the chiral a-
amino acid (S)-Epg was described in Ref. 15. The syntheses and chem-
ical characterizations of the amino acid derivatives Z-Deg-OH (Z,
benzyloxycarbonyl),32 H-Deg-OtBu,32 Tfa-Deg-OH,32 Tfa-Dpng-OH
[10(OH)],23 and Tfa-(S)-Epg-OH15 were already reported.
Z-Deg-OtBu (1). This amino acid derivative was obtained as an oil
emphasize that all hetero-peptides studied bear at their N- in an almost quantitative yield from Z-Deg-OH and isobutylene in
the presence of a catalytic amount of H2SO4 in anhydrous CH2Cl2.
terminus the synthetically useful trifluoroacetyl (Tfa) protec-
Rf1: 0.95; Rf2: 0.95; Rf3: 0.90. IR (KBr): 3420, 1732, 1717 cm21
.
tion,31,32 well known to afford further stabilization to the first
fully extended form in the amino acid sequence through an F
.. . H(N) .. . O@C (peptide) bifurcated H-bond.2
1H-NMR (200 MHz, CDCl3): d 5 7.34 (s, 5H, Z-phenyl CH); 5.89 (s,
1H, NH); 5.07 (s, 2H, Z CH2); 2.31 (qd, 2H, b-CH2); 1.73 (qd, 2H,
b-CH2); 1.46 (s, 9H, OtBu); 0.76 ppm (t, 6H, 2 c-CH3).
In this work, with the aim at deepening our knowledge on
the conformational preferences of hetero-peptides of this class
in general, and at discovering the first hetero-peptide sequence
which favors the overall fully-extended conformation in the
crystal state in particular, we synthesized by solution methods
and investigated experimentally (in solution by FTIR absorp-
tion and NMR techniques, and in the crystal state by X-ray dif-
fraction) and computationally [by molecular dynamics (MD)]
the preferred conformation of a series of oligomers based on
General Procedure for the Peptide Synthesis. Removal of the
N-terminal Z protection from the amino acid derivative 1 was
achieved by 10% Pd/C catalytic hydrogenation in anhydrous CH2Cl2,
while Tfa deprotection from peptides 2 and 3 was obtained by reduc-
tion with NaBH4 in ethanol.15,21,22,31,32 Peptide bond formation for
the synthesis of compounds 2–4 was carried out in anhydrous CH2Cl2
or tetrahydrofuran using the in situ preformed 5(4H)-oxazolone from
the pertinent Tfa N-protected amino acid.15,21,22,31,32 Considering the
very hindered amino acids involved in the coupling reactions, the
yields were acceptable (48–18%), although decreasing with peptide
main-chain elongation. The 5(4H)-oxazolone intermediates from Tfa-
Dpng-OH, Tfa-Deg-OH, and Tfa-(S)-Epg-OH were synthesized by
treating the corresponding N-protected amino acid with 1-(3-dime-
thylamino)propyl-3-ethylcarbodiimide in anhydrous CH3CN under
reflux. The progress of the reaction was periodically monitored by
HPLC. The electronegative Tfa moiety is considered the main respon-
sible for the significant electrophilicity of the carbonyl group of the
otherwise poorly reactive 5(4H)-oxazolones. The final products were
purified by flash chromatography using the 8:1:1 petroleum ether /
CH2Cl2 / diethyl ether solvent mixture as eluant.
C
a,a-diethylglycine (Deg), Ca,a-di-n-propylglycine (Dpng), and
the (S)-enantiomer of Ca-ethyl, Ca-n-pentylglycine (Epg).15
The Tfa / OtBu (tert-butoxy) terminal protections usual for
this type of peptides were utilized. An additional advantage
offered by hetero-peptides is that the assignment of NMR sig-
nals is facilitated as compared with homo-peptides, where the
same amino acid residue is repeated several times.
MATERIALS AND METHODS
Tfa-Dpng-Deg-OtBu (2). This terminally protected dipeptide was
prepared in 48% yield using the 5(4H)-oxazolone from Tfa-Dpng-OH
and H-Deg-OtBu. Mp: 118–120ꢀC (from ethyl acetate/petroleum
ether). Rf1: 0.95; Rf2: 0.95; Rf3: 0.90. IR (KBr): 3365, 3304, 1736, 1712,
General
Melting points were determined in capillary tubes by use of a Stuart
(Bibby Scientific, Stone, Staffordshire, UK) SMP10 apparatus and
are uncorrected. ESI-MS spectra were obtained by using a PerSeptive
Biosystem (Framingham, MA) Mariner model instrument. Flash
chromatography was carried out by use of a Merck (Darmstadt, Ger-
many) silica gel 60 (40–63 mm mesh) stationary phase. Analytical
TLC was performed on Kieselgel F 250 precoated plates (Merck).
The retention factor (Rf) values were determined using the following
1663, 1519 cm21 1H-NMR (200 MHz, CDCl3): d 5 8.01 (s, 1H,
.
NH); 6.87 (s, 1H, NH); 2.61 (qd, 2H, Dpng c-CH2); 2.43 (qd, 2H,
Dpng b-CH2); 1.79 (qd, 2H, Dpng b-CH2); 1.55 (qd, 2H, Dpng c-
CH2); 1.51 (s, 9H, OtBu); 1.21 (m, 4H, 2 Deg b-CH2); 0.89 (t, 6H,
Dpng 2 d-CH3); 0.64 ppm (t, 6H, Deg 2 c-CH3). MS (ESI): m/z
[M1H]1 calcd for C20H35F3N2O4: 425.480, found: 425.261.
solvent mixtures as eluants: Rf1 chloroform—ethanol 9:1; Rf2 1- Tfa-Deg-Dpng-Deg-OtBu (3). This terminally protected tripep-
butanol—acetic acid—water 3:1:1; Rf3 toluene—ethanol 7:1. The tide was synthesized in 21% yield using the 5(4H)-oxazolone from
Biopolymers (Peptide Science)

2.05-Helix in Hetero-Oligopeptides
147
Tfa-Deg-OH and H-Dpng-Deg-OtBu. Mp: 1602162ꢀC (from CH2Cl2 correlation spectra. HMBC experiments37 with selective excitation
/ diethyl ether / petroleum ether). Rf1: 0.95; Rf2: 0.95; Rf3: 0.85. IR in the CO region were performed using a long-range coupling con-
.
(KBr): 3396, 3352, 3302, 1730, 1664, 1493 cm21 1H-NMR (400 stant of 7.5 Hz and a spectral width in F1 of 20 ppm centered at
MHz, CDCl3): d 5 8.01 (s, 1H, Deg1 NH); 7.44 (s, 1H, Dpng NH); 170 ppm.
6.86 (s, 1H, Deg3 NH); 2.60 (m, 2H, Deg1 b-CH2); 2.42 (m, 2H, Deg3
b-CH2); 1.77 (m, 2H, Deg3 b-CH2); 1.61 (m, 2H, Dpng c-CH2); 1.60
X-Ray Diffraction
(m, 2H, Dpng b-CH2); 1.51 (s, 9H, OtBu); 1.21 (m, 2H, Dpng
c-CH2); 0.89 (t, 6H, Dpng 2 d-CH3); 0.81 (t, 6H, Deg1 2 c-CH3); 0.76
ppm (t, 6H, Deg3 2 c-CH3). MS (ESI): m/z [M1H]1 calcd for
C26H46F3N3O5: 538.612, found: 538.338.
Single crystals of Deg and 4 were grown by slow evaporation from an
acetone / water mixture and from a methanol solution, respectively,
while those of 10(OH) and 2 from a diethyl ether / petroleum ether
mixture. X-Ray diffraction data were collected with an Agilent Tech-
nologies Gemini E four-circle kappa diffractometer equipped with a
92 mm EOS CCD detector, using graphite monochromated Cu Ka
Tfa-(S)-Epg-Deg-Dpng-Deg-OtBu (4). This terminally pro-
tected tetrapeptide was prepared in 18% yield using the 5(4H)-oxazo-
lone from Tfa-(S)-Epg-OH and H-Deg-Dpng-Deg-OtBu. Mp: 234–
236ꢀC (from ethanol). Rf1: 0.95; Rf2: 0.95; Rf3: 0.80. IR (KBr): 3388,
˚
radiation (k 5 1.54178 A). Data collection and reduction were per-
formed with the CrysAlisPro software (Agilent Technologies). A semi-
empirical absorption correction based on the multi-scan technique
using spherical harmonics, implemented in the SCALE3 ABSPACK
scaling algorithm, was applied. The structures were solved by direct
methods of the SIR 2002 program,38 and refined by full-matrix least-
squares procedures on F2, using all data, by application of the
SHELXL-97 program,39 with all non-H atoms anisotropic. In general,
H-atoms were calculated at idealized positions and refined using a
riding model. Exceptions on the location of H-atoms and their han-
dling during the refinement, together with experimental details spe-
cific to each structure, are described below. Relevant crystallographic
data and structure refinement parameters are listed in Table I. CCDC
934352 - 934355 contain the supplementary crystallographic data for
Deg, 10(OH), 2, and 4, respectively. These data can be obtained from
The Cambridge Crystallographic Data Centre via www.ccdc.cam.
ac.uk/data_request/cif
1
3343, 3295, 1727, 1656, 1500 cm21. H-NMR (400 MHz, CDCl3):
d 5 8.03 (s, 1H, Epg NH); 7.44 (s, 1H, Deg2 NH); 7.40 (s, 1H, Dpng
NH); 6.86 (s, 1H, Deg4 NH); 2.63 (m, 2H, Epg b-CH2); 2.61 (m, 2H,
Deg2 b-CH2); 2.58 (m, 2H, Dpng b-CH2); 2.43 (m, 2H, Deg4 b-CH2);
1.78 (m, 2H, Deg4 b-CH2); 1.76 (m, 2H, Epg b-CH2); 1.69 (m, 2H,
Deg2 b-CH2); 1.58 (m, 2H, Dpng b-CH2); 1.51 (s, 9H, OtBu); 1.26
(m, 2H, Dpng c-CH2); 1.25 (m, 6H, Epg 3 CH2); 1.15 (m, 2H, Dpng
c-CH2); 0.90 (t, 6H, Dpng 2 d-CH3); 0.83 (t, 3H, Epg f-CH3); 0.79 (t,
6H, Deg2 2 c-CH3); 0.77 (t, 3H, Epg c-CH3); 0.76 ppm (t, 6H, Deg4 2
c-CH3). MS (ESI): m/z [M1H]1 calcd for C35H63F3N4O6: 693.751,
found: 693.461.
FTIR Absorption
FTIR absorption spectra were recorded using a Perkin-Elmer 1720 X
FTIR spectrophotometer, nitrogen-flushed, with a sample shuttle
device and at 2 cm21 nominal resolution, averaging 100 scans. Solvent
(baseline) spectra were recorded under the same conditions. Cells
with CaF2 windows and pathlengths of 0.1, 1.0, and 10 mm were
used. Spectrograde deuterochloroform (99.8% d) was obtained from
Fluka (Bu€chs, Switzerland).
Deg Monohydrate. The asymmetric unit is composed of two mole-
cules (A and B) of the zwitterionic amino acid and two cocrystallized
water molecules. The positions of the H-atoms of the water molecules
were located on a difference Fourier map and refined with restraints
on the OAH bond distances and the HAOAH angle.
10(OH). The asymmetric unit is composed of two independent mol-
ecules (A and B). The N-terminal Tfa group of each molecule, show-
ing rotational disorder, was refined with the three fluorine atoms on
two sets of positions, with refined population parameters of 0.553(11)
/ 0.447(11) for molecule A, while 0.549(18) / 0.451(18) for molecule
B. Restraints were applied to its bond distances and bond angles.
Nuclear Magnetic Resonance
The 1H-NMR spectra were recorded with Bruker (Karlsruhe, Ger-
many) AC 200 and Avance DRX-400 spectrometers. Measurements
were carried out in CDCl3 (99.96% d; Aldrich, St. Louis, MO), deuter-
ated DMSO (99.96% d6; Acros Organics, Geel, Belgium), and CD3CN
(99.90% d3; Euriso-top, Gif-Sur-Yvette, France), with tetramethylsi-
lane as the internal standard. Splitting patterns are abbreviated as fol- 4. Data collection was limited to h 5 51.31ꢀ in that the crystal,
˚
lows: (s) singlet, (d) doublet, (t) triplet, (q) quartet, (qd) quarted of owing to its small size, did not significantly diffract above 1.0 A
doublets, (m) multiplet. The 2D-NMR experiments were performed resolution. As expected from the low diffracting power exhibited
with a Bruker AVANCE DRX-400 instrument, operating at 400 MHz by the crystal, in particular in the highest resolution shell, structure
for 1H, equipped with a 5-mm probe BBI-Z grad. Processing and eval- solution was not straightforward. The structure was eventually
uation of the experimental data were carried out using the programs solved ab initio with the SIR 2002 program38 by exploiting its
TOP-SPIN 1.3 and SPARKY.33 The DQF-COSY34 spectra were RELAX procedure, aimed at repositioning well oriented but mis-
acquired in the magnitude mode, while CLEAN-TOCSY35,36 (spin placed molecular fragments. Basically, it involves: (i) expansion of
lock period, 70 ms) and NOESY spectra were recorded in the phase- the reflections in space group P1 to relax symmetry constraints, (ii)
sensitive mode. All homo-nuclear 2D-spectra were acquired by col- search for the origin shift which maximizes the figure of merit, and
lecting 400 experiments of 64 scans each, with a relaxation delay of (iii) return to the original P212121 space group with suitable trans-
1.0–1.2 s, and 2K data points. The spectral width was 11 ppm in each formation of the P1-derived phases. The trial solution with the best
dimension.
combined figure of merit allowed the location of a nearly complete
The assignments of the bCH2 groups belonging to the same peptide molecule (45 out of 48 atoms). The remaining non-H
Deg, Epg, and Dpng residues were obtained by means of 2D 1H,13C atoms were located on subsequent difference Fourier maps. The N-
Biopolymers (Peptide Science)

148
Crisma et al.
Table I Selected Crystallographic Data and Structure Refinement Parameters for Compounds Deg, 10(OH), 2, and 4
Parameter
Deg monohydrate
10(OH)
2
4
Empirical formula
Formula weight [a.m.u.]
Temperature [K]
C6H13NO2 3 H2O
149.19
293(2)
C10H16F3NO3
255.24
293(2)
C20H35F3N2O4
424.50
293(2)
C35H63F3N4O6
692.89
293(2)
˚
Wavelength [A]
1.54178
Monoclinic
P21/c
1.54178
Triclinic
P-1
1.54178
Orthorhombic
P212121
1.54178
Orthorhombic
P212121
Crystal system
Space group
˚
a [A]
10.8511(2)
13.6886(2)
11.0456(2)
90
8.3532(8)
12.3593(11)
14.7469(13)
65.531(8)
81.625(7)
74.723(8)
7503(3)
11.2979(2)
12.1915(2)
17.0784(3)
90
10.9482(3)
15.8709(6)
24.4480(9)
90
˚
b [A]
˚
c [A]
a [ꢀ]
b [ꢀ]
c [ꢀ]
93.315(1)
90
90
90
90
90
3
˚
V [A ]
1637.93(4)
8
1.210
2352.35(7)
4
1.199
4248.0(3)
4
1.083
No. of formula units per unit cell, Z
calcd [g cm 23
Crystal size [mm3]
Absorption coefficient [m, mm 21
Max. and min. transmission
h range for data collection [ꢀ]
Reflections collected
4
1.269
q
]
0.90 3 0.40 3 0.20
0.798
0.40 3 0.20 3 0.20
1.045
0.60 3 0.35 3 0.30
0.829
0.70 3 0.06 3 0.03
0.678
]
1.0000 and 0.4744
4.08–73.34
31,275
1.0000 and 0.7599
3.30–69.07
16,618
1.0000 and 0.6620
4.46–73.40
33,903
1.0000 and 0.6301
3.32–51.31
10,395
Independent reflections
Rint
3,286
0.0389
4,870
0.0277
4,698
0.0771
4,276
0.0355
Data / restraints / parameters
Goodness of fit on F 2
3206 / 6 / 196
1.051
0.0517
4870 / 24 / 363
1.085
0.0470
4698 / 0 / 262
1.060
0.0473
4276 / 222 / 461
1.006
0.0638
Final R1 value [Iꢁ2r(I)]
Final wR2 (F 2)value [Iꢁ2r(I)]
Final R1 value (all data)
Final wR2 (F 2)value (all data)
0.1573
0.0533
0.1387
0.0616
0.1323
0.0499
0.1487
0.0943
0.1599
0.605 and 20.228
934352
0.1470
0.152 and 20.156
934353
0.1352
0.285 and 20.186
934354
0.1743
0.225 and 20.157
934355
23
˚
Residual electron density [e A
CCDC number
]
terminal Tfa group exhibits rotational disorder. Its fluorine atoms the fully extended and the 310-helical conformations were gener-
were refined on two sets of positions (atoms F1, F2 and F3 for the ated using the Accelrys Discovery Studio 2.5.5 program.46 A cubic
first set, while F10, F20 and F30 for the second set), rotated by about box was placed around the peptide, with at least 1.5 nm between
60ꢀ. Their population parameters refined to the values of 0.692(19) the periphery of the peptide and the box sides, and filled with chlo-
and 0.308(19), respectively. Restraints were applied to the bond roform. An initial energy minimization, applying the steepest
distances and bond angles involving atoms of the disordered Tfa descent algorithm, was performed. Then, the system was subjected
group and the side chains, as well as to their anisotropic displace- to NVT (constant number of particles, volume, and temperature)
ment parameters (the latter to approach isotropic behavior). Over- ensemble dynamics to allow the relaxation of the chloroform mole-
all, many crystallographic parameters suffer from the far from
optimal crystal size. We are confident, however, that the basic con-
formational features of the molecule, as discussed in this work, are
firmly established.
cules, while the peptide was restrained to its initial coordinates
with a force constant of 1000 Kj mol21, followed by NPT (constant
number of particles, pressure, temperature) equilibration47 for 100
ps. Subsequently, MD simulations at constant pressure and tem-
perature (300 K) were carried out for 15 ns with a time step of 2 fs
using the Berendsen temperature coupling method.47 The tempera-
ture and pressure time coupling constants used were 0.1 and 0.5
ps, respectively. The LINCS algorithm48 was applied to constrain
all bonds during the simulations. The PME method49 with a cut-
off of 1.0 nm was used to calculate the nonbonded interactions,
and the neighbor-list was updated every 10 steps. The structures
were saved at 1 ps intervals along the MD trajectory, and analyzed
using various GROMACS utilities and a custom written PERL
program.
Molecular Dynamics Simulations
MD simulations on tetrapeptide 4 were carried out using version
5.4 of the GROMACS package40 and the OPLSAA force field41 in
explicit CHCl3, starting from two different conformations, namely
(i) the fully extended conformation (/ 5 180ꢀ, w 5 180ꢀ), and (ii)
a
regular, right-handed 310-helical structure (/ 5 257ꢀ,
w 5 230ꢀ).26 The OPLSAA topology was used for CHCl3,42–44
whereas the molecular topology of peptide 4, based on noncoded
amino acids, was developed using the SwissParam server.45 Both
Biopolymers (Peptide Science)

2.05-Helix in Hetero-Oligopeptides
149
FIGURE 1 FTIR absorption spectra (A: amide A region; B: amide
I and amide II regions) of the 2–4 peptides (for clarity, in part B
only the curve of peptide 4 is shown) in CDCl3 solution. Peptide
concentration: 1.0 mM.
SCHEME 1 Chemical structures of the compounds synthesized
and investigated in this work.
formers the NAH .. . O@C H-bond is intra-residue (i, i or
C5).52,53 Main-chain lengthening to the tri- (3) and the tetra-
peptide (4) induces a marked shift of the 3335 cm21 band to
higher wavenumbers (3343 and 3347 cm21, respectively) and a
significant increase of its relative intensity. We propose that the
internal C5 conformer III, (peptide) Ni-Hi .. . Oi@Ci (pep-
tide), would be the one giving an enhanced contribution to the
spectral modifications observed in peptides 3 and 4. If we sub-
tract the contributions of conformers I and II, both present in
2, from the spectra of 3 and 4, the resulting IR absorption
maximum (not shown), associated to the C5 conformer III,15
is exactly aligned at 3357 cm21. The FTIR absorption spectra
remain almost unchanged in the peptide concentration range
(10–0.1 mM) investigated. As a result, the experimentally
observed NAH . . . O@C H-bonds can be safely assumed to be
of the intramolecular type.
RESULTS AND DISCUSSION
Solution Conformational Analysis
The preferred conformations of the peptide series 2–4 (Scheme
1) were first determined in two solvents of different polarities
(CDCl3 and CD3CN) at various concentrations (10–0.1 mM)
using FTIR absorption and NMR.
Figure 1 shows the FTIR absorption spectra in CDCl3 solu-
tion at 1.0 mM peptide concentration. The band at 3446 6 1
cm21, associated with the free (non H-bonded) NAH stretch-
ing vibration (amide A),50–52 is very weak. For the dipeptide
(2), two much more intense bands are seen in the 3391–3335
cm21 region. We attribute them to stretching vibrations of
NH groups involved in intramolecular H-bonds of the type
F .. . H(N) .. . O@C (peptide) (conformer I in Scheme 2) and
(peptide) NAH . . . O@C (ester) (conformer II) at the N- and
C-ends of the peptide backbone.15 Remarkably, in both con-
In the amide I and amide II regions (1750–1450 cm21) of
peptide 4 (shown as an example in Figure 1), a strong absorp-
tion is seen at 1720 cm21, which we assign to an overlapping
Biopolymers (Peptide Science)

150
Crisma et al.
SCHEME 2 Intramolecularly and intra-residue H-bonded conformers occurring in the Tfa-
protected peptides discussed in this work.
of the C@O vibrations of the amide Tfa- and ester –CO–OtBu combination of two-dimensional NMR HMBC and NOESY
moieties.5,15 Amide I peaks for the peptide C@O groups, spectral experiments.
In the 88:12% CDCl3/DMSO solvent mixture (Figure 2A),
found at 1674 (weaker) and 1650 (stronger) cm21, are attrib-
uted to the coupling of consecutive carbonyls, each involved in the accessibilities of the peptide investigated show only one
an intra-residue H-bond, as computationally demonstrated by class of NH protons: all of them are almost insensitive to the
Maekawa et al.14 The position of the very intense single peak at addition of the perturbing agent. Taken together, it seems rea-
1490 cm21 is typical of the amide II band of overall fully sonable to interpret the results of the present FTIR absorption
1
extended peptides.14,54
and one-dimensional H-NMR studies in CDCl3 as arising
These FTIR absorption findings in CDCl3 agree nicely from single or multiple, consecutive C5 peptide conformations,
with those reported for other peptide series unequivocally where all NH protons are involved as donors in intra-residue
shown to adopt multiple, consecutive C5 conformers (2.05- H-bonds. In addition, these data fit nicely with those already
helices).2,5,14,15,21,22
published for the Deg, Dpng, and (S)-Epg homo-peptide
To check the role played by solvent properties on the overall series.2,5,15,21,22 Conversely, in the CD3CN/DMSO solvent mix-
fully extended peptide conformation,16 we expanded our FTIR ture (Figure 2B), the trend for the solvent accessibilities resem-
absorption analysis in the amide I and II regions of tetrapep- bles closely that expected for a peptide folded in a well
tide 4 to CD3CN (results not shown). The splitting of the developed 310-helix conformation,17 with the N(1)H and
amide I band is less marked in this solvent (1672, shoulder, N(2)H protons, not involved in the intramolecular H-bonding
and 1652 cm21), the amide II band moves to higher wave- scheme, remarkably more sensitive to DMSO addition.
numbers (1494 cm21) and is much less intense, and the
Two-dimensional NMR is the most reliable technique to
shoulder at 1518 cm21 (hardly visible in CDCl3), characteristic acquire local, detailed information on the secondary structure
of the amide II vibrational mode of the 310-helical peptide con- propensity of peptides in solution. Our results for tetrapeptide
formation, becomes stronger. From this preliminary analysis, 4 in CDCl3 are shown in Figure 3. Out of the three NH(i) !
we are inclined to conclude that the overall fully-extended NH(i11) sequential space connectivities expected, only two,
structure, although overwhelmingly populated in CDCl3 solu- Epg(1) ! Deg(2) and Dpng(3) ! Deg(4), were observed,
tion, is not robust, generating a mixture of stretched and although very weak (Figure 3A). This first finding indirectly
folded conformers in CD3CN.
supports the occurrence of an overall fully extended conforma-
To obtain additional information on the conformational tion, where the NH(i) to NH(i11) groups are spatially located
16
˚
preferences of the terminally protected peptide 4, the NH far apart (ꢁ3.50 A), in this solvent. In our peptides entirely
1
chemical shifts in its H-NMR spectra were investigated as a based on Ca-tetrasubstituted a-amino acids, where the aCH
function of the addition of dimethylsulfoxide (DMSO), known proton for all residues is missing, the observation of the bCH
to interact effectively with the exposed amide NH protons via ! NH connectivities proved to be essential for the conforma-
NAH . . . O@S H-bonds, thus inducing a shift in their tional assignments.16 For related peptides in the overall fully-
resonances.55,56 Assignments of the NH protons were in part extended conformation, we already reported that inter-residue
facilitated by the known observations in this type of peptides bCH(i21) ! NH(i) correlations are much more intense than
that: (i) the N-terminal Tfa-NH proton is unambiguously the intra-residue bCH(i) ! NH(i) correlations. This is pre-
identified by virtue of its low-field position (near 8.0 ppm).2,5 cisely what we did observe for peptide 4 in CDCl3 (Figure 3B).
(ii) The C-terminal NH signal is typically found at 6.80–6.90
Interestingly, in CD3CN solution (Figures 4 and 5) the rel-
ppm. These assignments were subsequently confirmed by a ative intensities of the cross-peaks for peptide 4 are quite
Biopolymers (Peptide Science)

2.05-Helix in Hetero-Oligopeptides
151
FIGURE 2 Histograms showing the variations of the NH proton chemical shifts for each of the
residues of peptide 4 as a function of the addition of DMSO (12% v/v) to the CDCl3 solution (A)
and to the CD3CN solution (B). Peptide concentration: 1.0 mM.
different from those seen in CDCl3. More specifically: (i) All in this work and their simpler synthetic precursors. We were
able to solve the 3D-structures of the Deg amino acid, the
three NH(i) ! NH(i11) cross-peaks are visible and they are
Dpng derivative 10(OH), and the di- (2) and tetra- (4) hetero-
rather intense (Figure 4). (ii) The inter-residue and intra-
oligopeptides (Figure 7). The most relevant geometric and
residue bCH ! NH cross-peaks exhibit comparable inten-
conformational results, and intramolecular H-bond parame-
ters are listed in Tables II and III, respectively.
sities (Figure 5). Another interesting observation is that in
peptide 4 the remarkable splitting of the signals of the diaster-
eotopic bCH2 protons in CDCl3, typical of the more rigid
and highly sterically hindered, overall fully-extended confor-
mation,16 is much attenuated in CD3CN (Figure 6A). The
logical conclusion we extracted from all these data in CD3CN
is that peptide 4 is overwhelmingly folded in a helical struc-
ture in this solvent, without excluding a limited population
of the fully extended structure in the conformational equilib-
rium mixture. These results confirmed that a solvent-driven
conformational switch is an interesting and characteristic
property of this type of peptides.16 In particular, a CDCl3 /
CD3CN titration experiment on the 3D-structurally sensitive
bCH2 protons16 of peptide 4 between 2.65 and 1.58 ppm
clearly shows that the fully-extended to helical conforma-
tional transition is relatively sharp, as it is almost complete
between 75% and 60% CDCl3 (Figure 6B).
The geometric parameter s (N-Ca-C0) bond angle57 for the
four structures (Table II) can be classified in two groups: (i)
103.2(2)ꢀ 2 105.0(4)ꢀ, markedly narrower than that of a regu-
lar tetrahedral sp3 atom (109.5ꢀ) and typical of the fully-
extended conformation,1,4,8,17,21,22 shown by compounds
10(OH) (molecule B), 2 (residue 1), and 4 (residues 1, 2, and
4); and (ii) 108.2(1)2110.5(1)ꢀ, characteristic of a helical con-
formation, exhibited by Deg (molecules A and B), 10(OH)
(molecule A), 2 (residue 2), and 4 (residue 3).
Five out of the total ten amino acid residues investigated
exhibit values for the /,w backbone torsion angles close to the
all-trans conformation (180ꢀ,180ꢀ), the largest deviation being
6.5ꢀ (Table II). This finding unambiguously indicates that the
pertinent Deg, Dpng, and (S)-Epg residues adopt the fully-
extended conformation. Two C5 forms are consecutively
observed in the tetrapeptide (4), which therefore generates a
short 2.05-helix (residues 1 and 2). On the average, the intra-
˚
Crystal-State Conformational Analysis
We performed a number of attempts to grow single crystals
molecular N . . . O separation is 2.56 A (Table III). Also, all
peptide and ester moieties (x torsion angles) are trans. The
suitable for X-ray diffraction analysis on the peptides discussed largest deviation (12.8ꢀ) is seen in the peptide bond between
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152
Crisma et al.
FIGURE 4 NH ! NH region of the NOESY spectrum of peptide
4 in CD3CN solution. Peptide concentration: 1.0 mM.
III). The same observation applies to peptides 2 and 4, where
˚
˚
the N . . . F separations are 2.647(2) A and 2.652(14) A, respec-
tively. These results, combined with the set of /,w values of
molecule A of 10(OH) discussed above, are strongly indicative
of the occurrence of an intramolecular, bifurcated H-bond of
the type F . . . H(N) . . . O@C (peptide) (conformer I in
Scheme 2) for molecule B of the amino acid derivative 10(OH)
and peptides 2 and 4. All n-propyl, and n-pentyl side chains
(v2, v3, and v4 torsion angles) of the amino acids forming the
compounds studied in this work are found in a fully-extended
conformation, pointing orthogonally out of the peptide main
chain. This spatial disposition is energetically favorable in that
too close intramolecular non-bonded interactions are avoided.
Interestingly, in the overall L-shaped and chiral tetrapeptide 4,
the achiral helical residue 3, generating the molecular kink, is
left-handed. From this 3D-analysis, we conclude that in the
crystal state both hetero-oligopeptides examined are partially
(50%; compound 2) or even predominantly (75%; compound
4) fully-extended, but none of them is entirely elongated in a
2.05-helical structure.
In each carboxylate moiety of molecules A and B of the
FIGURE 3 (A): NH ! NH and (B): side-chain CH ! NH
regions of the NOESY spectrum of peptide 4 in CDCl3 solution.
Peptide concentration: 1.0 mM.
amino acid Deg, one of the two CAO bond distances is
˚
slightly longer that the other [C1-O1 1.2613(14) A vs. C1-O1T
˚
1.2326(14) A in molecule A, and C2-O2 1.2563(13) A vs. C2-
˚
˚
O2T 1.2397(14) A in molecule B]. These values compare well
the fully-extended residue 2 and the helical residue 3 of tetra-
peptide 4. The Tfa-NH groups of compounds 10(OH), 2 and 4
are close to planarity, the related x torsion angles being
179.1(2)ꢀ [for molecule A of 10(OH)], 177.4(2)ꢀ [for molecule
B of 10(OH)], 173.6(2)ꢀ (for 2), and 2178.1(7)ꢀ (for 4). The
N-terminal NH group is within a H-bonding distance from
o0ne of the fluorine atoms of the CF3 moiety in molecule A of
with those in the literature for the carboxylate anion of ali-
phatic acids [CAO 1.254(10) A].58 For a ACOOH moiety, on
˚
the other hand, the average values for the C@O and the
˚
CAO(H) bond distances would be 1.214(19) A and 1.308(19)
˚
A, respectively. The zwitterionic nature of both Deg molecules
is also supported by the location of the H-atoms and the
H-bonding scheme (see below). The Deg packing mode is
characterized by a complex network of H-bonds. The
˚
1 (OH), where the N . . . F separation is 2.715(5) A, and in
˚
molecule B of the same derivative, N .. . F5 2.556(6) A (Table
Biopolymers (Peptide Science)

2.05-Helix in Hetero-Oligopeptides
153
FIGURE 5 Overlapping of the NOESY spectra in the side-chain CH ! NH region for peptide 4
in CDCl3 and CD3CN solutions. Peptide concentration: 1.0 mM.
protonated amino group of molecule A acts as the donor of cule B, and a (2x, y11/2, 2z11/2) symmetry equivalent of
three H-bonds, the acceptors of which are the O2 atom of a the O2W water molecule.
(x11, y, z) symmetry equivalent of molecule B, the O1 atom
In the packing mode of the derivative 10(OH) (for atom
of a (2x11, 2y11, 2z) symmetry equivalent of molecule A, numbering see Figure 7), the carboxylic acid hydroxyl OTB-H
and a (2x11, y21/2, 2z11/2) symmetry equivalent of the group of molecule B is H-bonded to the O0A atom of mole-
O1W water molecule. The acceptors of the three H-bonds cule A within the same asymmetric unit, and the N1-H group
formed by the protonated amino group of molecule B are: the of molecule A is H-bonded to a (x, y21, z) symmetry equiva-
O1 atom of molecule A within the same asymmetric unit, the lent of the O0B atom of molecule B. These two intermolecular
O2 atom of a (2x, 2y11, 2z) symmetry equivalent of mole- H-bonds give rise to chains of molecules A and B, alternating
FIGURE 6 (A): Histogram showing the differences in chemical shifts between the split Hb1 and
Hb2 resonances for each of the residues of peptide 4 in CDCl3 (blue) and CD3CN (green) solutions.
(B): Variations in the partial (3.8–1.0 ppm) 1 H-NMR spectrum of peptide 4 in the CDCl3/CD3CN
titration experiment. Peptide concentration: 1.0 mM.
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154
Crisma et al.
FIGURE 7 X-ray diffraction structures of Deg monohydrate, and compounds 10(OH), 2, and 4
with atom numberings. There are two independent molecules (A and B) in the asymmetric unit of
both Deg and 10(OH). The cocrystallized water molecules are also reported in the structure of Deg
monohydrate. Only the major occupancy conformer of the disordered Tfa group is shown in the
structures of 10(OH) and 4. The intramolecular H-bonds are represented by dashed lines.
Biopolymers (Peptide Science)

2.05-Helix in Hetero-Oligopeptides
155
Table II Relevant Geometric and Conformational Parameters
(ꢀ) Observed for Compounds Deg, 10(OH), 2, and 4 in the Crystal
State
Compound
Residue
s
/
w
H-Deg-OH (Deg)
mol. A 108.98(8)
mol. B 108.90(8)
—
—
175.6(1)a
176.4(1)b
Tfa-Dpng-OH
[10(OH)]
Tfa-Dpng-Deg-OtBu (2)
mol. A 108.2(1) 245.1(2) 247.6(2)c
mol. B 103.2(2) 177.9(2) 177.5(2)d
1
2
1
2
3
4
103.7(1) 2174.6(2) 178.1(1)
110.5(1) 254.7(2) 246.6(2)e
103.3(5) 178.9(7) 2173.5(5)
105.0(4) 2179.0(5) 179.1(4)
109.8(4)
104.4(5) 178.0(5) 179.0(5)f
Tfa-(S)-Epg-Deg-
Dpng-Deg-OtBu (4)
58.0(6)
41.6(5)
a N1-C1A-C1-O1T.
b N2-C2A-C2-O2T.
c N1-C1A-C1-OTA.
d N2-C2A-C2-OTB.
e N2-C2A-C2-OT.
f N4-C4A-C4-OT.
along the b direction. A third type of intermolecular H-bond is
observed between the carboxylic acid hydroxyl OTA-H group
and the (2x, 2y, 2z11) symmetry equivalent of O1, thus
generating centrosymmetric, doubly H-bonded carboxylic acid
dimers of molecule A. Interestingly, the N2-H group of the
fully-extended molecule B does not participate in the intermo-
lecular H-bonding scheme.
The packing mode of peptide 2 is characterized by the
occurrence of a single type of intermolecular H-bond, which
involves the N2-H group as the donor, and a (2x11, y11/2,
z21/2) symmetry equivalent of the O0 atom as the acceptor.
As a result, rows of molecules, related by a twofold screw axis,
are generated along the b direction. Again, the NAH group of
FIGURE 8 Time evolution of the /,w backbone torsion angles
for tetrapeptide 4 in the MD simulations in CHCl3 starting from
(A): the fully extended conformation (/ 5 180ꢀ, w 5 180ꢀ), and
(B): a regular, right-handed 310-helical structure (/ 5 257ꢀ,
w 5 230ꢀ). 26
the fully extended residue 1 is not involved in any intermolecu- a (x21/2, 2y21/2, 2z) symmetry equivalent of the O3 car-
lar H-bond.
bonyl oxygen, generating rows of molecules related by a two-
Also in the packing mode of 4 a single type of intermolecu- fold screw axis along the a direction. In this structure too, the
lar H-bond is observed, namely between the N3-H3 group and NAH and C@O groups of residues 1, 2, and 4,
Table III Intramolecular H-bond Parameters Observed for Compounds 10(OH), 2, and 4 in the Crystal State
Compound
Donor D-H Acceptor A Distance (A) D. . .A Distance (A) H. . .A Angle (ꢀ) D-H. . .A
˚ ˚
Tfa-Dpng-OH [10(OH)] (mol. A and B) N1-H1
F2
F4
2.715(5)
2.556(6)
2.575(2)
2.647(2)
2.546(2)
2.652(14)
2.553(7)
2.552(6)
2.593(6)
2.35
2.13
2.13
2.22
2.09
2.24
2.09
2.09
2.15
106
110
112
110
113
109
113
113
112
N2-H2
N2-H2
O2
F1
Tfa-Dpng-Deg-OtBu (2)
N1-H1
N1-H1
N1-H1
N1-H1
N2-H2
N4-H4
O1
F3
Tfa-(S)-Epg-Deg-Dpng-Deg-OtBu (4)
O1
O2
O4
Biopolymers (Peptide Science)

156
Crisma et al.
intramolecularly involved in the C5 structures, do not partici- acterized by the longest possible separation between two adja-
cent Ca-atoms in an a-amino acid sequence.
pate in any intermolecular H-bond.
In this article, for hetero-oligopeptides entirely based on
appropriate, but different, Ca,a-disubstituted Gly residues, we
showed that: (i) In CDCl3 the 2.05-helix structure is stable. (ii)
However, in CD3CN this is not a robust conformation, as it
tends to switch to the much shorter 310-helix structure to a
large extent.16,62 We propose that this result would be related
to the different H-bonding properties of the two solvents. In
particular, the 310-helix, with its two (N-terminal) free amide
NH groups26–30 interacts more effectively with the good H-
bonding acceptor solvent CD3CN63 than the 2.05-helix, where
all amide NH groups are involved in (intramolecular) H-bond
formation. (iii) In the crystal state, examples of only partially
developed 2.05-helices were achieved. This last finding might
be, at least in part, related to the observation that an efficient
packing of extended side chains of unequal length protruding
orthogonally from a fully-extended backbone is problematic.
(iv) Molecular dynamics simulations in CHCl3 support the
experimental results, in that a hetero-tetrapeptide converged to
the 2.05-helix conformation even starting from the right-
handed 310-helical structure.
Molecular Dynamics Simulations
Additional information about the conformational preferences
of tetrapeptide 4 was obtained by MD simulations. MD runs
of 15 ns were performed in explicit CHCl3 starting from two
different conformations, namely (i) the fully-extended confor-
mation (/ 5 180ꢀ, w 5 180ꢀ); and (ii) a regular, right-handed
310-helical structure (/ 5 257ꢀ, w 5 230ꢀ).26 The evolution
of the /,w backbone torsion angles along the MD trajectories
are reported in Figure 8.
When the MD simulation starts with all residues in the
fully-extended conformation (Figure 8A), only residue 4 seems
to enjoy a limited flexibility (accessing a semi-extended confor-
mation for a large part of the trajectory and returning to the
fully-extended conformation towards the end of the run),
whereas the other three residues are essentially locked into the
fully-extended conformation. It is worth recalling that the
C@O group of residue 4, being involved in an ester function,
is a poorer acceptor of the intra-residue H-bond (typical of the
fully extended conformation) if compared to the peptide C@O
groups of residues 1–3.
In conclusion, we confirm23,24 that hetero-peptides of this
class may adopt a fully-extended 2.05-helix, but its stability is
governed by the environmental conditions. In particular, the
discovery of a fully developed 2.05-helix in hetero-peptides
from Ca,a-disubstituted Gly residues in the crystal state
remains elusive.
A completely different scenario is observed when the MD
simulation starts from the right-handed 310-helical structure
(Figure 8B). Indeed, residue 1 almost immediately shifts to
the fully-extended conformation, whereas within 250 ps resi-
dues 2 and 3 undergo a transition toward a semi-extended
[poly (Pro)n II] structure which persist until 7.5 ns, when they
transform to the fully extended conformation that is retained
up to the end of the simulation. Only the torsion angles of res-
idue 4 fluctuate around the original helical values throughout
the MD run.
R.B. and S.S. acknowledge the BIF (Bioinformatics Infra-
structural Facility) -DBT (Department of Biotechnology) Centre
at the Department of Bioinformatics, West Bengal University of
Technology, Kolkata, for the computational facility. M.C. is grate-
ful to Prof. Alessandro Dolmella, Department of Pharmaceutical
Sciences, University of Padova, for granting access to the Gemini
diffractometer and for his help with X-ray diffraction data collec-
tion and processing.
Taken together, the results of the MD simulations support
the view that in CHCl3 peptide 4 has a strong inclination
towards an extensively developed fully-extended structure, irre-
spective of the starting conformation. A fully developed, right-
handed 310-helix seems to be disfavored in that solvent, at least
under our simulation conditions.
REFERENCES
1. Benedetti, E.; Toniolo, C.; Hardy, P.; Barone, V.; Bavoso, A.; Di
Blasio, B.; Grimaldi, P.; Lelj, F.; Pavone, V.; Pedone, C.; Bonora,
G. M.; Lingham, I. J Am Chem Soc 1984, 106, 8146–8152.
2. Bonora, G. M.; Toniolo, C.; Di Blasio, B.; Pavone, V.; Pedone,
C.; Benedetti, E.; Lingham, I.; Hardy, P. J Am Chem Soc 1984,
106, 8152–8156.
CONCLUSIONS
The multiple, consecutive fully-extended conformation (2.05-
helix), authenticated in both CDCl3 solution and in the crystal
state exclusively with homo-peptides,1,4,8,15,21,22,59 and con-
firmed by conformational energy computations,60,61 is a very
important peptide secondary structure for the construction of
exceptionally efficient spacers and templates because it is char-
3. Valle, G.; Bonora, G. M.; Toniolo, C.; Hardy, P. M.; Leplawy, M.
T.; Redlinski, A. J Chem Soc Perkin Trans II 1986, 885–889.
4. Benedetti, E.; Barone, V.; Bavoso, A.; Di Blasio, B.; Lelj, F.; Di
Blasio, B.; Bonora, G. M.; Toniolo, C.; M. T. Leplawy, M. T.;
Kaczmarek, K.; Redlinski, A. Biopolymers 1988, 27, 357–371.
Biopolymers (Peptide Science)

2.05-Helix in Hetero-Oligopeptides
157
5. Toniolo, C.; Bonora, G. M.; Bavoso, A.; Benedetti, E.; Di Blasio, 32. Leplawy, M. T.; Kaczmarek, K.; Redlinski, A. In Peptides: Chem-
B.; Pavone, V.; Pedone, C.; Barone, V.; Lelj, F.; Leplawy, M. T.;
Kaczmarek, K.; Redlinski, A. Biopolymers 1988, 27, 373–379.
6. Crisma, M.; Valle, G.; Bonora, G. M.; De Menego, E.; Toniolo,
C.; Lelj, F.; Barone, V.; Fraternali, F. Biopolymers 1990, 30, 1–11.
7. Valle, G.; Crisma, M.; Bonora, G. M.; Toniolo, C.; Lelj, F.;
Barone, V.; Fraternali, F.; Hardy, P. M.; Langran-Godsmith, A.;
Maia, H. L. S. J Chem Soc Perkin Trans 2 1990, 1481–1487.
8. Toniolo, C.; Benedetti, E. In Molecular Conformation and Bio-
logical Interactions; Balaram, P.; Ramaseshan, S., Eds.; Indian
Academy of Sciences: Bangalore, India, 1991; pp 511–521.
9. Crisma, M.; Valle, G.; Bonora, G. M.; Toniolo, C.; Lelj, F.;
Barone, V.; Fraternali, F.; Hardy, P. M.; Maia, H. L. S. Biopoly-
mers 1991, 31, 637–641.
istry and Biology; Marshall, G. R., Ed.; ESCOM: Leiden, The
Netherlands, 1988; pp 239–241.
33. Goddard, T. D.; Kneller, D. G. SPARKY 3, University of Califor-
nia, San Francisco, CA; http://www.cgl.ucsf.edu/home/sparky/.
34. Rance, M.; Sïrensen, O. W.; Bodenhausen, G.; Wagner, G.;
Ernst, R. R.; Wu€thrich, K. Biochem Biophys Res Commun 1983,
117, 479–485.
35. Bax, A.; Davis, D. G. J Magn Reson 1985, 65, 355–360.
36. Griesinger, C.; Otting, G.; Wu€thrich, K.; Ernst, R. R. J Am
Chem Soc 1988, 110, 7870–7872.
37. Bax, A.; Summers, M. F. J Am Chem Soc 1986, 108, 2093–2094.
38. Burla, M. C.; Camalli, M.; Carrozzini, B.; Cascarano, G. L.;
Giacovazzo, C.; Polidori, G.; Spagna, R. J Appl Crystallogr 2003,
36, 1103.
10. Di Blasio, B.; Pavone, V.; Isernia, C.; Pedone, C.; Benedetti, E.;
Toniolo, C.; Hardy, P. M.; Lingham, I. N. J Chem Soc Perkin
Trans 2 1992, 523–526.
39. Sheldrick, G. M. Acta Crystallogr A 2008, 64, 112–122.
40. van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A.
E.; Berendsen, H. J. C. J Comput Chem 2005, 26, 1701–1718.
41. Jorgensen, W. L.; Tirado-Rives, J. J Am Chem Soc 1988, 110,
1657–1666.
11. Valle, G.; Crisma, M.; Toniolo, C. Zeit Kristallogr 1992, 199,
203–210.
12. Aubry, A.; Del Duca, V.; Pantano, M.; Formaggio, F.; Crisma,
M.; Toniolo, C.; Kamphuis, J. Lett Pept Sci 1994, 1, 157–162.
13. Moretto, A.; De Zotti, M.; Crisma, M.; Formaggio, F.; Toniolo,
C. Int J Pept Res Ther 2008, 14, 307–314.
42. Marcus, Y. The Properties of Solvents; Wiley: New York, NY,
1998.
43. Caleman, C.; van Maaren, P. J.; Hong, M.; Hub, J. S.; Costa, L.
T.; van der Spoel, D. J Chem Theor Comput 2012, 8, 61–74.
44. Lide, D. R. CRC Handbook of Chemistry and Physics, 90th ed.;
CRC Press: Cleveland, OH, 2009.
14. Maekawa, H.; Ballano, G.; Toniolo, C.; Ge, N.-H. J Phys Chem B
2011, 115, 5168–5182.
15. Crisma, M.; Moretto, A.; Peggion, C.; Panella, L.; Kaptein, B.;
Broxterman, Q. B.; Formaggio, F.; Toniolo, C. Amino Acids
2011, 41, 629–641.
45. Zoete, V.; Cuendet, M. A.; Grosdidier, A.; Michielin, O. J Com-
put Chem 2011, 32, 2359–2368.
16. Peggion, C.; Crisma, M.; Toniolo, C.; Formaggio, F. Tetrahedron
2012, 68, 4429–4433.
46. Accelrys Software Inc., Discovery Studio Modeling Environ-
ment, Release 2.5.5, Accelrys Software Inc.: San Diego, CA,
2010.
17. Formaggio, F.; Crisma, M.; Peggion, C.; Moretto, A.; Venanzi,
M.; Toniolo, C. Eur J Org Chem 2012, 167–174.
47. Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; Di
Nola, A.; Haak, J. R. J Chem Phys 1984, 81, 3684–3690.
48. Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M. J
Comput Chem 1997, 18, 1463–1472.
18. Formaggio, F.; Crisma, M.; Ballano, G.; Peggion, C.; Venanzi,
M.; Toniolo, C. Org Biomol Chem 2012, 40, 2413–2421.
19. Peggion, C.; Moretto, A.; Formaggio, F.; Crisma, M.; Toniolo, C.
Biopolymers (Pept Sci) 2013, 100, 621–636.
49. Darden, T.; York, D.; Pedersen, L. J Chem Phys 1993, 98, 10089–
10092.
20. Crisma, M.; Formaggio, F.; Toniolo, C.; Yoshikawa, T.;
Wakamiya, T. J Am Chem Soc 1999, 121, 3272–3278.
21. Tanaka, M.; Imawaka, N.; Kurihara, M.; Suemune, H. Helv
Chim Acta 1999, 82, 494–509.
50. Baron, M. H.; De Lozꢀe, C.; Toniolo, C.; Fasman, G. D. Biopoly-
mers 1978, 17, 2225–2239.
51. Moretto, V.; Crisma, M.; Bonora, G. M.; Toniolo, C.; Balaram,
H.; Balaram, P. Macromolecules 1989, 22, 2939–2944.
52. Cung, M. T.; Marraud, M.; Nꢀeel, J. Ann Chim (Paris) 1972, 7,
183–209.
22. Imawaka, N.; Tanaka, M.; Suemune, H. Helv Chim Acta 2000,
83, 2823–2835.
23. Oba, M.; Tanaka, M.; Kurihara, M.; Suemune, H. Helv Chim
Acta 2002, 85, 3197–3217.
53. Toniolo, C. CRC Crit Rev Biochem 1980, 9, 1–44.
54. Kennedy, D. F.; Crisma, M.; Toniolo, C.; Chapman, D. Biochem-
istry 1991, 30, 6541–6548.
24. Tanaka, M.; Nishimura, S.; Oba, M.; Demizu, Y.; Kurihara, M.;
Suemune, H. Chem Eur J 2003, 9, 3082–3090.
25. Tanaka, M. Chem Pharm Bull 2007, 55, 349–358.
26. Toniolo, C.; Benedetti, E. Trends Biochem Sci 1991, 16, 350–
353.
55. Martin, D.; Hauthal, H. G. Dimethyl Sulphoxide; van Nostrand-
Reinhold: Wokingham, U.K., 1975.
56. Kopple, K. D.; Ohnishi, M.; Go, A. J Am Chem Soc 1969, 91,
4264–4272.
27. Karle, I. L.; Balaram, P. Biochemistry 1990, 29, 6747–6756.
28. Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C.; Toniolo, C.;
Crisma, M. Biopolymers 1992, 32, 453–456.
57. Touw, W. G.; Vriend, G. Acta Crystallogr D 2010, 66, 1341–
1350.
29. Toniolo, C.; Crisma, M.; Formaggio, F.; Peggion, C. Biopolymers
(Pept Sci) 2001, 60, 396–419.
58. Allen, F. H.; Kennard, O.; Watson, D.; Brammer, L.; Orpen, G.;
Taylor, R. J Chem Soc Perkin Trans 2 1987, S1–S19.
59. Pavone, V.; Lombardi, A.; Saviano, M.; De Simone, G.; Nastri,
F.; Maglio, O.; Omote, Y.; Yamanaka, Y.; Yamada, T. Biopoly-
mers 2000, 53, 161–168.
30. Banerjee, R.; Chattopadhyay, S.; Basu, G. Proteins Struct Funct
Bioinform 2009, 76, 184–200.
31. Hardy, P. M.; Lingham, I. N. Int J Pept Protein Res 1983, 21,
392–405.
Biopolymers (Peptide Science)

158
Crisma et al.
60. Barone, V.; Lelj, F.; Bavoso, A.; Di Blasio, B.; Grimaldi, P.; 62. Lettieri, R.; Bischetti, M.; Gatto, E.; Palleschi, A.; Ricci, E.;
Pavone, V.; Pedone, C. Biopolymers 1985, 24, 1759–1767.
61. Casanovas, J.; Revilla-Lopez, G.; Crisma, M.; Toniolo, C.;
Aleman, C. J Phys Chem B 2012, 116, 13297–13307.
Formaggio, F.; Crisma, M.; Toniolo, C.; Venanzi, M. Biopoly-
mers (Pept Sci) 2013, 100, 51–63.
63. Nain, A. K. Bull Chem Soc Jpn 2006, 79, 1688–1695.
Biopolymers (Peptide Science)