Enhanced β-turn conformational stability of tripeptides containing Δphe in cis over trans configuration

Article abstract of DOI:10.1007/s00726-013-1534-9

Conformations of three pairs of dehydropeptides with the opposite configuration of the ΔPhe residue, Boc-Gly-Δ^Z/EPhe-Phe-p- NA (Z- p -NA and E- p -NA), Boc-Gly-Δ^Z/EPhe-Phe-OMe (Z-OMe and E-OMe), and Boc-Gly-Δ^Z/EPhe-Phe-OH (Z-OH and E-OH) were compared on the basis of CD and NMR studies in MeOH, TFE, and DMSO. The CD results were used as the additional input data for the NMR-based calculations of the detailed solution conformations of the peptides. It was found that Z- p -NA, E- p -NA, Z-OMe, and Z-OH adopt the β-turn conformations and E-OMe and E-OH are unordered. There are two overlapping type III β-turns in Z- p -NA, type II' β-turn in E- p -NA, and type II β-turn in Z-OMe and Z-OH. The results obtained indicate that in the case of methyl esters and peptides with a free carboxyl group, Δ^ZPhe is a much stronger inducer of ordered conformations than Δ^EPhe. It was also found that temperature coefficients of the amide protons are not reliable indicators of intramolecular hydrogen bonds donors in small peptides.

Full text of DOI:10.1007/s00726-013-1534-9

Amino Acids
DOI 10.1007/s00726-013-1534-9
ORIGINAL ARTICLE
Enhanced b-turn conformational stability of tripeptides
containing DPhe in cis over trans configuration
•
Mariusz Jaremko Łukasz Jaremko
•
•
Adam Mazur Maciej Makowski Marek Lisowski
•
Received: 17 April 2013 / Accepted: 7 June 2013
Ó Springer-Verlag Wien 2013
Abstract Conformations of three pairs of dehydropep-
tides with the opposite configuration of the DPhe residue,
Boc-Gly-DZ/EPhe-Phe-p-NA (Z-p-NA and E-p-NA), Boc-
Gly-DZ/EPhe-Phe-OMe (Z-OMe and E-OMe), and Boc-
Gly-DZ/EPhe-Phe-OH (Z-OH and E-OH) were compared
on the basis of CD and NMR studies in MeOH, TFE, and
DMSO. The CD results were used as the additional input
data for the NMR-based calculations of the detailed solu-
tion conformations of the peptides. It was found that
Z-p-NA, E-p-NA, Z-OMe, and Z-OH adopt the b-turn
conformations and E-OMe and E-OH are unordered.
There are two overlapping type III b-turns in Z-p-NA, type
II’ b-turn in E-p-NA, and type IIb-turn in Z-OMe and Z-OH.
The results obtained indicate that in the case of methyl
esters and peptides with a free carboxyl group, DZPhe is a
much stronger inducer of ordered conformations than
DEPhe. It was also found that temperature coefficients of
the amide protons are not reliable indicators of intramo-
lecular hydrogen bonds donors in small peptides.
Keywords Dehydropeptides ꢀ Dehydrophenylalanine
configuration ꢀ Circular dichroism ꢀ Nuclear magnetic
resonance ꢀ Dehydropeptide conformation ꢀ Temperature
coefficients of amide protons
Introduction
a,b-Dehydroamino acid residues contain a double bond
between the Ca and Cb atoms. When the Cb atom bears dif-
ferent substituents, they can exist in two forms––as isomers
Z, with the higher ranking substituent in position cis to the
nitrogen atom, and isomers E, with this substituent trans to
nitrogen. Isomers Z are more thermodynamically stable than
their E counterparts and hence easier to obtain (Stammer
1982; Makowski et al. 1986; Latajka et al. 2006). A great
majority of papers published so far on dehydropeptides deal
with dehydroamino acid residues of the Z configuration. The
most studied dehydroamino acid residue has been dehydr-
ophenylalanine. It has been found that both in the solid state
and solution, a DPhe residue of the Z configuration induces
b-turns in short sequences and a 310-helix in longer ones or
peptides with more than one dehydro residue (Mathur et al.
2004; Gupta and Chauhan 2011). It may also introduce long-
range interactions in peptides (Ramagopal et al. 2001, 2002)
or break a b structure (Gupta et al. 2008).
M. Jaremko (&) ꢀ A. Mazur
Department of NMR-based Structural Biology, Max-Planck-
Institute for Biophysical Chemistry, Am Fassberg 11,
¨
37077 Gottingen, Germany
e-mail: antypater@gmail.com
Ł. Jaremko (&)
Max-Planck-Institut fu¨r Biophysikalische Chemie and Deutsches
Zentrum fu
¨r Neurodegenerative Erkrankungen (DZNE), Am
¨
Fassberg 11, 37077 Gottingen, Germany
e-mail: jaremko@gmail.com
The conformational preferences of a DPhe residue of the
E configuration have been much less studied. In the solid
state, this residue occupies the i ? 1 position in type II or
II’ b-turn (Makowski et al. 2005, 2007, 2010). Ordered
conformations have been also found for DEPhe-containing
peptides in solution (Latajka et al. 2006, 2008a, b).
M. Makowski
Institute of Chemistry, University of Opole, Oleska 48,
45-052 Opole, Poland
M. Lisowski
Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14,
50-383 Wrocław, Poland
123

M. Jaremko et al.
Still, in the literature there had been no direct compar-
ison of the conformational properties of dehydropeptides
containing one DPhe residue and differing only by its
configuration. In this connection some time ago we pub-
lished a paper (Lisowski et al. 2010) which presents the CD
and NMR conformational studies on two pairs of tetra-
peptides of a general formula Boc-Gly-DZ/EPhe-Gly-Phe-
X, with X = p-nitroanilide or methyl ester. It was found
that Boc-Gly-DEPhe-Gly-Phe-OMe is unordered and all the
other peptides adopt the b-turn conformation. There are
two overlapping b-turns in Boc-Gly-DZPhe-Gly-Phe-OMe
and Boc-Gly-DZPhe-Gly-Phe-p-NA, of type II at Gly1-
DZPhe2 and type III’ at DZPhe2-Gly3, and two overlapping
type III b-turns in Boc-Gly-DEPhe-Gly-Phe-p-NA, at
DEPhe2-Gly3 and Gly3-Phe4.
ordered structure-inducing properties of DEPhe depend on
their C-terminal blocking group. Another important reason
for this study was the observation that in one of the tetra-
peptides the temperature coefficients of amide protons
forming hydrogen bonds were larger than the cut-off value
of 0.0046 ppm/K (Cierpicki and Otlewski 2001). We
wanted to check how this criterion for detecting hydrogen
bonds in proteins and peptides works for such small mol-
ecules like the peptides studied.
The conformations of the tripeptides were studied by
CD in MeOH, TFE, and DMSO, and NMR in DMSO-d6.
Combination of the two methods is a very effective way of
conformational studies on small peptides. In the case of
such flexible and dynamic compounds NMR only is very
often not capable of providing sufficient solution structure
data due to the small number of ROE contacts and often
In this paper we present the results of our studies on the
3
three following pairs of dehydropeptides:
ambiguous JHH couplings constants. In such a case, it is
very advantageous to have additional information on the
peptide conformation from CD, concerning the type of
structure which dominates in its conformational equilib-
rium. We used then the CD results as the additional input
data for the NMR-based calculations of the detailed solu-
tion conformations of the peptides studied. Such an
approach has been successfully applied in our conforma-
tional studies on the decapeptide fragment of ubiquitin
(Jaremko et al. 2009) and the tetrapeptides mentioned
above (Lisowski et al. 2010).
Boc-Gly-DZPhe-Phe-p-NA
Boc-Gly-DEPhe-Phe-p-NA
Boc-Gly-DZPhe-Phe-OMe
Boc-Gly-DEPhe-Phe-OMe
Boc-Gly-DZPhe-Phe-OH
Boc-Gly-DEPhe-Phe-OH
Z-p-NA
E-p-NA
Z-OMe
E-OMe
Z-OH
E-OH
Their structures are presented in the Scheme 1. We
chose such peptides because they are shorter than the tet-
rapeptides described above so their conformations are
determined to a larger degree by the conformational
properties of a DPhe residue itself. At the same time their
sequences are very similar to the tetrapeptides which
allows the correlation of the results obtained for the two
groups of peptides. Moreover, we wanted to check how the
tripeptides conformation is affected by the nature of the
C-terminal blocking group or the lack of it since it has been
found for the tetrapeptides (Lisowski et al. 2010) that the
Conformational properties of Z-p-NA has been studied
earlier by X-ray, CD, and NMR (Lisowski et al. 2008). The
only conclusion reached from solution studies was that the
peptide adopts the type III b-turn conformation but without
specifying its position. Likewise, little has been deduced
from the CD and NMR studies on E-p-NA (Latajka et al.
2006). The authors found only that its conformation is
ordered. The NMR and CD studies and ab initio calcula-
tions have been also performed recently on Z-OMe (Jew-
´
ginski et al. 2013). In solution, it was found merely that the
Scheme 1 Structures of Z-p-NA, E-p-NA, Z-OMe, E-OMe, Z-OH, and E-OH
123

Enhanced b-turn conformational stability
peptide adopts the ordered conformation and no specific
information were given. In this paper, we present the
detailed solution conformations of Z-p-NA, E-p-NA,
Z-OMe, and three other related dehydropeptides on the
basis of our CD and NMR studies. The results presented
here may be helpful in the de novo design of peptides with
specific conformations in solution.
1H-NMR (800 MHz, DMSO-d6), d (ppm): 1.42 (9H, s, 3
9 CH3 Boc), 3.03–3.11 (2H, m, CbH2 Phe3), 3.71 (2H,
broad s, CaH2 Gly1), 4.49 (1H, m, CaH Phe3), 7.09 (1H, s,
CbH of DZPhe2), 7.12 (1H, t, NH Gly1), 7.21–7.29 (5H, m,
aromatic protons of Phe3), 7.37–7.56 (5H, m, aromatic
protons of DZPhe2), 8.07 (1H, d, NH Phe3), 9.44 (1H, broad
s, NH DZPhe2); 13C-NMR (201 MHz, DMSO-d6), d (ppm):
31.36 (CH3 Boc), 39.71 (Cb Phe3), 46.81 (Ca Gly1), 57.35
(Ca Phe3), 132.31 (Cb DZPhe2), 132.79 (Cd DZPhe2),
131.65 (Ce DZPhe2), 131.45 (Cd Phe3), 129.52 (Cf Phe3),
132.33 (Ce Phe3).
Materials and methods
Synthesis of peptides
Boc-Gly-DEPhe-Phe-OH (E-OH)
The syntheses of Z-p-NA, E-p-NA, and Z-OMe have been
described by Makowski et al. (2001); Latajka et al. (2006);
´
and Jewginski et al. (2013), respectively.
E-OH was obtained from E-OMe(0.964 g, 2 mmol) by a
standard basic hydrolysis with a 10 %-fold excess of 1 M
NaOH in MeOH, for 2 h at room temperature. It was
crystallized from ethyl acetate–MeOH (2:1, v/v)/hexane.
Yield 96 %; mp 145–147.5 °C; elemental analysis calcd
(%) for C25H29N4O6 (467.52): C 64.23, H 6.25, N 8.99;
found: C 64.33, H 6.12, N 9.18.
1H-NMR (800 MHz, DMSO-d6), d (ppm): 1.40 (9H, s, 3
9 CH3 Boc), 2.91–3.06 (2H, m, CbH2 Phe3), 3.69 (2H, d–d,
CaH2 Gly1), 4.44 (1H, m, CaH Phe3), 6.84 (1H, s, CbH
DEPhe2), 6.99 (1H, t, NH Gly1), 7.14–7.27 (5H, m, aro-
matic protons of Phe3), 7.16–7.24 (5H, m, aromatic protons
of DEPhe2), 8.26 (1H, d, NH Phe3), 9.51 (1H, broad s, NH
DEPhe2); 13C-NMR (201 MHz, DMSO-d6), d (ppm): 30.62
(CH3 Boc), 39.07 (Cb Phe3), 46.03 (Ca Gly1), 56.40 (Ca
Phe3), 119.80 (Cb DEPhe2), 130.64 (Cd DEPhe2), 128.63 (Ce
DEPhe2), 129.28 (Cf DEPhe2), 131.55 (Cd Phe3), 130.65 (Cf
Phe3), 130.53 (Ce Phe3).
Boc-Gly-DEPhe-Phe-OMe (E-OMe)
E-OMe was synthesized according to the method of Ma-
kowski et al. (1986). Boc-Gly-DEPhe-OH (1.602 g,
5 mmol) was coupled with H-Phe-OMe (0.896 g, 5 mmol)
in DMF (7.5 ml) by mixed anhydride method with
i-BuOCOCl (0.65 ml, 5 mmol). During the reaction exten-
sive configurational inversion took place and both E-OMe
and Z-OMe were obtained with a ratio of 1:1.3. The iso-
mers were separated on a silica gel H60 column with ethyl
acetate–benzene (2–60 % v/v of ethyl acetate) as an eluent.
The product was crystallized from chloroform–benzene
(2:1, v/v)/hexane. Yield 24 %; mp 67.5–70 °C; elemental
analysis calcd (%) for C26H31N4O6 (481.55): C 64.85, H
1
6.49, N 8.73; found: C 64.80, H 6.53, N 8.80. H-NMR
(800 MHz, DMSO-d6), d (ppm): 1.41 (9H, s, 3 9 CH3
Boc), 2.88–2.92 (2H, m, CbH2 Phe3), 3.53 (3H, s, CH3
Me), 3.68 (2H, broad s, CaH2 Gly1), 4.52 (1H, m, CaH
Phe3), 6.83 (1H, s, CbH DEPhe2), 7.01 (1H, t, NH Gly1),
7.14–7.27 (5H, m, aromatic protons of Phe3), 7.19–7.22 (5H,
m, aromatic protons of DEPhe2), 8.61 (1H, d, NH Phe3), 9.53
(1H, broad s, NH DEPhe2); 13C-NMR (201 MHz, DMSO-
d6), d (ppm): 31.36 (CH3 Boc), 39.80 (Cb Phe3), 46.64 (Ca
Gly1), 54.85 (CH3 Me), 57.13 (Ca Phe3), 120.26 (Cb
DEPhe2), 129.68 (Cd DEPhe2), 129.98 (Ce DEPhe2), 132.13
(Cd Phe3), 131.38 (Cf Phe3), 131.49 (Ce Phe3).
Circular dichroism spectroscopy
CD spectra were recorded on a Jasco J-600 spectropolar-
imeter, at room temperature. Spectra were measured in
MeOH, TFE, and DMSO. Pathlength of 1 mm was used.
Concentrations of the solutions were in the range of
0.17–0.21 mg/ml (2.6–3.9 9 10-4 M). Each spectrum
represents the average of at least four scans. The data are
presented as molar ellipticity [h].
NMR spectroscopy
Boc-Gly-DZPhe-Phe-OH (Z-OH)
NMR spectra were recorded at 11.7 T on a Varian Unity ?
500 spectrometer at 25 °C, in DMSO-d6. In all cases
5–10 mM peptide solutions were used. The 1D H-NMR
Z-OH was obtained from Z-OMe (0.964 g, 2 mmol) by a
standard basic hydrolysis with a 10 %-fold excess of 1 M
NaOH in MeOH, for 2 h at room temperature. It was
crystallized from ethyl acetate–MeOH (5:1, v/v)/hexane.
Yield 96 %; mp 181–183 °C; elemental analysis calcd (%)
for C25H29N4O6 (467.52): C 64.23, H 6.25, N 8.99; found:
C 64.12, H 6.30, N 9.13.
1
spectra were processed and analyzed with a VnmrJ soft-
ware (Varian Inc. Palo Alto, USA). The acquired two
dimensional homonuclear NMR spectra were processed by
NMRPipe (Delaglio et al. 1995) and analyzed with Sparky
(Goddard and Kneller 2003) programs. Complete
123

M. Jaremko et al.
1
assignments of the H and 13C resonances for all peptides
4.0
2.0
Z-p-NA
Z-OMe
Z-OH
were done by application of a standard procedure (Wu¨th-
rich 1986) based on inspection of the 2D homonuclear
TOCSY (with mixing times 10 and 90 ms) and ROESY
(with mixing times 200 and 500 ms) experiments. The
temperature coefficients of amide protons were measured
in DMSO-d6 at the temperature range from 291 to 318 K,
with a temperature interval of 3 K. Temperature depen-
dencies of all investigated amide protons were found to be
linear with R2 [ 0.995. Through space distances between
protons were determined by analysis of 2D 1H-1H ROESY
spectra inter proton cross peaks. Only non-trivial inter-
proton distance constraints were used in the calculations
0.0
-2.0
-4.0
E-p-NA
E-OMe
E-OH
220
240
260
280
300
320
340
360
380
Wavelength [nm]
˚
and all were kept in the range of 1.8 A (sum of Van der
˚
Waals radii) up to 5.5 A (ROE upper limit). Calculations of
Fig. 1 CD spectra of Z-p-NA, E-p-NA, Z-OMe, E-OMe, Z-OH,
and E-OH in MeOH
lowest-energy structures, carried out for peptides which
exhibited ordered conformations in solutions according to
CD, were performed with an X-PLOR NIH 2.21 program
package (Schwieters et al. 2003). Additional dihedral
angles restraints defining the b-turn type (Lewis et al.
1973) indicated by CD with the deviation of 90° were
applied. The values of u and w angles typical for the b-turn
type indicated by CD were used in the calculations for
certain amino acids along the sequence only if their pres-
ence was justified by the inter-proton ROE contacts. Fig-
ures presenting ensembles of 50 lowest-energy structures
from the 500 calculated peptide conformers were prepared
with a MolMol software (Koradi et al. 1996). Supple-
mentary data are available from the authors upon request.
4.0
Z-p -NA
Z-OMe
Z-OH
2.0
0.0
-2.0
E-p-NA
E-OMe
E-OH
-4.0
220
240
260
280
300
320
340
360
380
Wavelength [nm]
Results
Fig. 2 CD spectra of Z-p-NA, E-p-NA, Z-OMe, E-OMe, Z-OH,
and E-OH in TFE
CD studies
308 nm, and two positive at 274 and 227 nm, are observed
for E-p-NA. The bands at 308 and 274 nm are distinctly
stronger than the corresponding ones of Z-p-NA. The CD
spectra of E-OMe and E-OH differ very much from those
of other peptides. There are practically no bands in the
near-UV region, only positive ellipticities at shorter
wavelengths.
The CD spectra of Z-p-NA in MeOH, TFE, and MeCN has
been measured before (Lisowski et al. 2008). We measured
a new spectra of that peptide in MeOH and TFE, and for
the first time in DMSO, to compare them with the spectra
of its configurational counterpart, E-p-NA.
The CD spectra of all the peptides studied in MeOH are
presented in Fig. 1. The spectrum of Z-p-NA consists of a
large positive band of p-NA at 304 nm, a very distinct
negative shoulder at about 264 nm of nearly equal inten-
sity, due to the charge-transfer transition in the DPhe res-
idue (usually this band appears at 280 nm), and a strong
negative band at 236 nm, corresponding to the p–p* tran-
sition of the dehydro chromophore (Pieroni et al. 1996). In
the case of Z-OMe, there is a negative band at 279 nm and
positive ellipticities below 230 nm. The spectrum of Z-OH
shows a large negative band at 280 nm, twice bigger than
that of Z-OMe, a positive shoulder at 237 nm, and positive
ellipticities below 230 nm. Very large bands, negative at
In TFE (Fig. 2), the CD spectrum of Z-p-NA shows a
large positive band at 299 nm, a negative shoulder at
264 nm, and a very large negative band at 229 nm. The
spectra of Z-OMe and Z-OH are very similar to those in
MeOH. They show a negative band at 277 nm, twice larger
for Z-OH, a positive shoulder at about 237 nm, and posi-
tive ellipticities at shorter wavelengths. The spectrum of
E-p-NA differs very much in its shape and decreased inten-
sities from that in MeOH. It consists of a negative band at
331 nm, a positive band at 291 nm, a positive shoulder at
253 nm, and a very large, negative band below 230 nm. In
the CD spectrum of E-OMe there are only residual
123

Enhanced b-turn conformational stability
2.0
Table 1 Temperature coefficients for amide protons of Z-p-NA, E-
p-NA, Z-OMe, E-OMe, Z-OH, and E-OH in DMSO-d6
Z-p-NA
Z-OMe
Z-OH
Residue dd/dT (ppm K-1 9 1,000)
1.0
0.0
Z-p-NA E-p-NA Z-OMe E-OMe Z-OH E-OH
Gly1
DPhe2
Phe3
4.4
6.5
6.0
7.3a
2.0
5.9
5.0
6.0
–
5.3
6.5
9.6
–
5.9
4.7
5.7a
–
5.1
6.3
9.3
–
5.2
4.8a
2.8a
E-p-NA
E-OMe
E-OH
p-NA
-1.0
-2.0
Temperature coefficients were measured at the temperature range
from 291 to 318 K, with a temperature interval of 3 K. For each
peptide amide, ten data points were collected and data were fitted
using standard least-squares fit procedures in SigmaPlot to the
y = ax ? b function, where a is the amide temperature coefficient
260
280
300
320
340
360
380
400
Wavelength [nm]
a
Protons forming hydrogen bonds according to calculations
Fig. 3 CD spectra of Z-p-NA, E-p-NA, Z-OMe, E-OMe, Z-OH,
and E-OH in DMSO
bigger than the cut-off value were found for Phe3 NH in
Z-p-NA and DPhe2 NH in Z-OH, which suggests that those
protons maybe also involved in hydrogen bonds. However,
the results obtained from CD and conformational calcula-
tions (see ‘‘Discussion’’) indicate that hydrogen bonds are
formed by Phe3 and p-NA of Z-p-NA, and NH’s of Phe3 in
E-p-NA, Z-OMe, and Z-OH. It shows that in the case of
small peptides the temperature coefficients of amide pro-
tons alone are not good indicators of hydrogen bonds.
Important conformational information can be obtained
from the ROESY spectra. They allow to detect interatomic
contacts in the peptides studied. It was found that trivial,
intraresidue contacts are present in each investigated
peptide, whereas non-trivial contacts were observed for
Z-p-NA, E-p-NA, Z-OMe, and Z-OH (Table 2; Fig. 5).
The number of such contacts determined for Z-p-NA (11)
is comparable with that found for its tetrapeptide counter-
part (12) (Lisowski et al. 2010). For E-p-NA, the number
of non-trivial ROE contacts is six and for its tetrapetide
counterpart it is eight, whereas for Z-OMe it is eight and
for its tetrapetide counterpart it is five. Non-trivial contacts
are absent in E-OMe and E-OH which confirms the lack of
intramolecular hydrogen bonds in the two peptides. Such
contacts were used as the input data for calculations of the
lowest-energy conformers of Z-p-NA, E-p-NA, Z-OMe,
and Z-OH. Besides interatomic contacts, the calculations
were based on the coherent results obtained at the same
time by the NMR and CD, i.e., which amide protons form
hydrogen bonds and what type of a b-turn is present in a
given peptide. In such a case it is important to have the
results from the two methods which can be directly com-
pared. We achieved that by measurement of the CD spectra
also in DMSO, which was used for the NMR experiments.
In all the cases there is a large similarity between the CD
spectra measured in DMSO and other solvents (excluding
E-p-NA in TFE). The only exception is largely diminished
intensity of the p-NA band of E-p-NA at 331 nm in
ellipticities in the near-UV region and a weak, positive
shoulder at 244 nm. In the case of E-OH small bands,
negative at 274 nm and positive at 244 nm, are observed.
The spectra in DMSO (Fig. 3) were measured for a
direct comparison of the CD and NMR results. They are
characterized by smaller band intensities than in other two
solvents. In the case of Z-p-NA a broad positive band at
313 nm and a negative one at 272 nm are observed. For
Z-OMe and Z-OH, a negative band is present at 282 and
284 nm, respectively. In DMSO, contrary to MeOH and
TFE, the intensity of that band is larger for Z-OMe than for
Z-OH. The spectrum of E-p-NA shows a very weak,
negative band at 331 nm and a large, positive band at
289 nm. Very small, negative bands are observed for
E-OMe and E-OH, at 292 and 286 nm, respectively.
NMR studies
To check for the presence of intramolecular hydrogen
bonds in the peptides investigated the temperature depen-
dence of their amide protons was measured. Amide proton
chemical shifts are sensitive to temperature changes. If a
proton forms a hydrogen bond, then its chemical shift
shows a very small temperature dependence. The presence
or absence of hydrogen bonds is indicated by
a
dd/dT (ppm K-1) coefficient. It has been established that
amide protons in protein systems, which are characterized
by the temperature coefficient value lower than
0.0046 ppm K-1, are involved in hydrogen bonds (Cier-
picki and Otlewski 2001). This value is often used for
hydrogen bond assignments in conformational studies on
peptides and proteins. The temperature coefficients of
amide protons in the peptides studied are presented in
Table 1. They show the presence of three hydrogen bonds,
formed by amide protons of Gly1 and p-NA in Z-p-NA and
the amide proton of p-NA in E-p-NA. Values slightly
123

M. Jaremko et al.
values of their dihedral angles are given in Table 3. Fig-
ure 4e–h also shows the ensembles of 50 lowest-energy
structures of those peptides, chosen from 500 calculated.
Table 2 Interatomic non-trivial contacts in Z-p-NA, E-p-NA, Z-
OMe, and Z-OH detected from the ROESY spectra in DMSO-d6
Peptide
Interatomic contacts
Z-p-NA
CH3 Boc $ CaHa1 Gly1
CH3 Boc $ CaHa2 Gly1
CH3 Boc $ aromatic H’s DZPhe2
CH3 Boc $ aromatic H’s Phe3
NH DZPhe2 $ CaHa1 Gly1
NH DZPhe2 $ CaHa2 Gly1
NH DZPhe2 $ NH Gly1
Discussion
The CD spectra were analyzed in the near-UV region only
since there are overlapping contributions of the peptide,
aromatic, and unsaturated chromophores in the far-UV
which makes their analysis in that region quite difficult.
The dehydropeptides studied contain a DPhe residue which
gives a large CD band in the near-UV region, at about
280 nm, which is very sensitive to a dehydropeptide con-
formation (Pieroni et al. 1993, 1996; Ramagopal et al.
2002). In this connection this residue is a very good con-
formational probe in the studies on dehydropeptides. In
Z-p-NA and E-p-NA, there is also the p-NA group which
has been found to be a useful tool in the conformational
studies on peptides (Sato et al. 1981; Baldwin et al. 1994;
Halab et al. 2000; Lisowski et al. 2008, 2010). This group
gives Cotton effects in the near-UV as well which can be
either positive or negative depending on the peptide con-
formation (Latajka et al. 2006; Lisowski et al. 2008, 2010).
Large intensities of the CD bands of Z-p-NA, E-p-NA,
Z-OMe, and Z-OH in the near-UV region (Figs. 1, 2, 3)
show that the peptides adopt chiral, rigid conformations in
solution. Taking into account the conformational properties
of a DPhe residue, one can expect these are folded struc-
tures of the b-turn type stabilized by intramolecular
hydrogen bonds. The analysis of the CD spectra of Z-p-NA
in MeOH, TFE, and MeCN has been presented before
(Lisowski et al. 2008). It was found that the peptide adopts
different, solvent-dependent forms of the type III b-turn
conformation, with one or two overlapping turns. The CD
spectrum of Z-p-NA in DMSO (Fig. 3) shows that its
conformation is quite rigid. DMSO is known to lower the
amount of ordered conformations of dehydropeptides
(Pieroni et al. 1993, 1996). A smaller intensity of the p-NA
band of Z-p-NA in DMSO at 313 nm as compared with
MeOH and TFE shows that the conformational equilibrium
of Z-p-NA in that solvent shifts toward unordered struc-
tures but this change is not substantial.
NH Phe3 $ NH DZPhe2
NH Phe3 $ CbH DZPhe2
NH p-NA $ NH Phe3
NH p-NA $ CaH Phe3
E-p-NA
NH DEPhe2 $ CaHa1 Gly1
NH DEPhe2 $ CaHa2 Gly1
NH DEPhe2 $ aromatic H’s p-NA
CbH DEPhe2 $ aromatic H’s p-NA
NH p-NA $ NH Phe3
NH p-NA $ CaH Phe3
Z-OMe
CaH2 Gly1 $ CH3 Boc
NH DZPhe2 $ CaH2 Gly1
NH DZPhe2 $ NH Phe3
aromatic H’s DZPhe2 $ CaH2 Gly1
CaH Phe3 $ CH3 Boc
CbHb1Phe3 $ CH3 Boc
CbHb2Phe3 $ CH3 Boc
NH Phe3 $ CbH DZPhe2
CH3 Boc $ CaH2 Gly1
Z-OH
CH3 Boc $ aromatic H’s DZPhe2
CH3 Boc $ CbHb2 Phe3
CH3 Boc $ aromatic H’s Phe3
NH Gly1 $ NH DZPhe2
CaH2 Gly1 $ NH DZPhe2
CaH2 Gly1 $ aromatic H’s DZPhe2
CaH2 Gly1 $ NH Phe3
CaH2 Gly1 $ aromatic H’s Phe3
NH DZPhe2 $ NH Phe3
CbH DZPhe2 $ NH Phe3
DMSO. The calculations were performed with the
assumption that there are two overlapping type III b-turns
in Z-p-NA, stabilized by two hydrogen bonds Phe3
NHꢀꢀꢀBoc CO and p-NA NHꢀꢀꢀDZPhe2 CO, and type II
b-turns in Z-OMe and Z-OH, at the Gly1-DZPhe2 residues,
with no hydrogen bonds. In the case of E-p-NA, the cal-
culations were performed with a type II’ b-turn at either
Gly1-DEPhe2 or DEPhe2-Phe3 as the starting conformation.
In each case very similar results were obtained. The low-
est-energy conformers calculated for Z-p-NA, E-p-NA,
Z-OMe, and Z-OH are presented in Fig. 4a–d and the
The conclusions reached for Z-p-NA from CD are
supported by the NMR studies. The temperature coeffi-
cients of the amide protons of Phe3 and p-NA in Z-p-NA
(Table 1) indicate that they may be involved in intramo-
lecular hydrogen bonds stabilizing two overlapping b-turns
predicted by the CD. It is consistent with the results of
calculations which show the presence of two intramolec-
ular hydrogen bonds of the 4 ? 1 type, i.e., Phe3 NHꢀꢀꢀBoc
CO and p-NA NHꢀꢀꢀGly1 CO. On the other hand, the cal-
culations do not show the presence of a hydrogen bond
123

Enhanced b-turn conformational stability
a
b
c
d
e
f
g
h
Fig. 4 The lowest-energy conformers of Z-p-NA. (a), E-p-NA (b), Z-OMe (c), and Z-OH (d), and ensembles of the 50 lowest-energy
structures of Z-p-NA. (e), E-p-NA (f), Z-OMe (g), and Z-OH (h)
Table 3 Torsion angle values of Z-p-NA, E-p-NA, Z-OMe, and Z-OH calculated on the basis of NMR and CD parameters with an X-PLOR
program. Standard deviations for 50 lowest-energy structures are given in brackets
Peptide
Residue
Gly1
u
DPhe2
Phe3
w
u
w
u
w
Z-p-NA
E-p-NA
Z-OMe
Z-OH
-20° ( 89°)
48° ( 7°)
-46° ( 35°)
-48° ( 4°)
-47° ( 28°)
-110° ( 16°)
74° ( 6°)
-18° ( 34°)
-115° ( 14°)
93° ( 16°)
-60° ( 33°)
44° ( 10°)
33° ( 44°)
-17° ( 24°)
-75° ( 25°)
53° ( 6°)
-38° ( 37°)
-20° ( 63°)
-8° ( 33°)
13° ( 7°)
–
137° ( 38°)
57° ( 10°)
–
Standard torsion angles ui?1, wi?1, ui?2, wi?2 for type I, II, and III b-turns (Lewis et al. 1973) are as follows, respectively: type I -60, -30, -90,
0; type II -60, 120, 80, 0; type III -60, -30, -60, -30. Types I’, II’, and III’ have the same angles as types I, II, and III, respectively, but with
the opposite sign
123

M. Jaremko et al.
formed by the amide proton of Gly1 indicated by its tem-
perature coefficient. The dihedral angles values obtained
for Z-p-NA (Table 3) show that the 4 ? 1 hydrogen bonds
stabilize two overlapping type III b-turns, at the Gly1–
DPhe2 and DPhe2–Phe3 residues. The values presented for
Z-p-NA differ quite distinctly, however, from the standard
values of -60° and -308 for u and w angles, respectively
(Lewis et al. 1973), in a type III b-turn. Another factor
very doubtful on the basis of the peptide CD spectrum. The
type II’ b-turn is located either at the Gly1-DEPhe2 or
DEPhe2–Phe3 residues. A very large intensity of the band at
308 nm shows that the conformational freedom of the p-
NA group is distinctly restricted which can result from its
participation in the p-NA NHꢀꢀꢀGly1 CO hydrogen bond. It
suggests that E-p-NA adopts the type II’ b-turn confor-
mation at the DEPhe2–Phe3 residues. This is, however, in
some contradiction with the results obtained in DMSO
(Fig. 3). On transition from MeOH to DMSO the intensity
of the DEPhe band is decreased to a small extent while the
intensity of the p-NA band is drastically reduced. These
changes suggest that the DEPhe2 residue is a part of the
ordered, rigid structure of the same type in both solvents
whereas the p-NA group is much more conformationally
labile and becomes unordered in DMSO. This in turn
would indicate that a b-turn in E-p-NA is located at the
Gly1–DEPhe2 residues and stabilized by the Phe3 NHꢀꢀꢀBoc
CO hydrogen bond.
indicating the presence of a b-turn is the distance between
the Ca and Ca
atoms, which should be less than 7 A
˚
i
i?3
(Lewis et al. 1973). The interatomic distances derived from
the ROESY spectra are very helpful in detection of b-turns.
In the case of the first turn in Z-p-NA the Boc O–Phe3 Ca
distance (the oxygen atom of the Boc group corresponds to
the Ca atom) is 7.38 0.65 A so it is close to the limit
˚
i
value. For the second turn, the Gly1 Ca-p-NA Ca distance
(the Ca atom of the aromatic ring of p-NA corresponds to
the Cai?3 atom) is 5.23 0.44 A (the presented values are
˚
the mean values for 50 lowest-energy structures). These
results are consistent with the frequency of occurrence of
the two 4 ? 1 hydrogen bonds in the lowest-energy
structures ensemble, which is very distinctly lower for the
Phe3 NHꢀꢀꢀBoc CO hydrogen bond. Taken together, the
dihedral angles values and the values corresponding to the
The position of a b-turn in E-p-NA was determined by
NMR studies. The temperature coefficients of the amide
protons of the peptide (Table 1) indicate very distinctly
that only that of p-NA forms a hydrogen bond. Its tem-
perature coefficient (2.0) is the smallest found for the
peptides studied. It shows that a b-turn is located at
DEPhe2–Phe3. However, the calculations gave a different
result. The results presented in Table 1 were obtained with
the type II’ b-turn at Gly1–DEPhe2. They show that the only
4 ? 1 hydrogen bond present in E-p-NA is that between
Phe3 NH and Boc CO. It is interesting that the temperature
coefficient of Phe3 NH (7.3) is the largest among amide
protons of E-p-NA. The Phe3 NHꢀꢀꢀBoc CO hydrogen bond
stabilizes the b-turn at the Gly1–DEPhe2 residues. It can be
seen from the values of dihedral angles (Table 3) that this
is a b-turn of type II’, in accordance with the CD results. In
Ca –Ca
distances show that the type III b-turns present
i
i?3
in Z-p-NA are quite distorted as compared with the stan-
dard and the first one at Gly1-DZPhe2 is less stable than the
second at DZPhe2–Phe3. The solution conformation of Z-p-
NA differs from its crystal structure (Lisowski et al. 2008)
where only one type II b-turn at DPhe2–Phe3, with the p-
NA NHꢀꢀꢀGly1 CO hydrogen bond, is present. The lowest-
energy conformation of the peptide is presented in Fig. 4a.
The ensemble of 50 lowest-energy conformers (Fig. 4e)
shows that Z-p-NA retains a marked conformational free-
dom in solution.
The CD spectrum of E-p-NA in MeOH in the near-UV
(Fig. 1) consists of large DEPhe and p-NA bands which
indicates the ordered conformation of the b-turn type. It
has been found (Pieroni et al. 1996) that a negative band at
280 nm of the DPhe residue is typical of dehydropeptides
in the type II b-turn conformation, with the dehydro residue
at the i ? 2 position. In the case of dehydropeptides
adopting a type III b-turn conformation, a positive DPhe
band at 280 nm is observed, accompanied by a negative
band at 235 nm which may be diagnostic for distinguishing
between type II and type III b-turns. The positive sign of
the DEPhe residue and the absence of the negative band at
235 nm in the spectrum of E-p-NA indicates that the
peptide adopts the type II’ b-turn conformation stabilized
by the 4 ? 1 hydrogen bond. This is a single b-turn
because due to the u and w angles values typical of various
b-turn types, the only two-b-turn combination possible is
II’–III and the presence of a type III b-turn in E-p-NA is
this case the distance Boc O–Phe Ca is 5.71 0.34 A. The
˚
lowest-energy structure of E-p-NA is presented in Fig. 3b
and the ensemble of its 50 lowest-energy conformations in
Fig. 3f. The latter figure shows that E-p-NA is comparably
flexible in solution like Z-p-NA.
A surprising result obtained for E-p-NA is its CD
spectrum in TFE (Fig. 2). It is markedly different from that
in MeOH. Largely decreased intensities of the DEPhe and
p-NA bands and their shift to the longer wavelengths
suggest a substantial increase of the conformational free-
dom of the peptide and/or a change in its conformational
equilibrium consisting in appearance of larger amounts of
unordered or other structures. It is unexpected since TFE is
well known as an ordered structures-inducing solvent
(Cammers-Goodwin et al. 1996; Rajan and Balaram 1996;
Luo and Baldwin 1997). This observation indicates that the
conformation of E-p-NA is considerably solvent-
dependent.
123

Enhanced b-turn conformational stability
Fig. 5 Detailed chemical
H C
a
C H
formulas of Z-p-NA. (a), E-p-
5
6
6
5
CH
C
H2C
CH
O
C
NA (b), Z-OMe (c), and Z-OH
(d) with indicated observed
non-trivial ROESY contacts
C
O
NH
NH
C
O
NO
2
NH C H
NH CH
C
O
NH
(H C) C
O
6
4
2
3
3
C H
C H
b
6
5
6
5
HC
H2C
CH
O
C
C
O
C
O
NO
C
NH C H
NH CH
C
O
NH
(H C) C
O
2
6
4
2
3
3
H C
C H
c
5
6
6
5
H2C
CH
CH
O
C
NH
C
C
O
OCH
C
NH CH
C
NH
(H C) C
O
3
2
3
3
O
O
C H
6
5
H C
d
5
6
2
1
Hβ
CH
C
Hβ
NH
C
O
C
C
CH
C
O
OH
NH CH
C
NH
(H C) C
O
2
3
3
O
O
The CD spectra of Z-OMe and Z-OH in MeOH and
TFE (Figs. 1, 2) are very similar which shows that the
conformational equilibria of the peptides are dominated by
the same type of structures. The negative DPhe bands at
280 nm indicate that the dominant form of both peptides is
a type II b-turn with the DZPhe2 residue at the i ? 2
position (Pieroni et al. 1996). The intensities of those bands
show that type II b-turn population is about twice larger in
Z-OH than in Z-OMe. The CD spectra of Z-OMe and Z-
OH in DMSO (Fig. 3) show that their b-turn populations
are retained in that solvent which shows that they are quite
stable. The b-turns in both peptides are located at the Gly1–
DZPhe2 residues and they are stabilized by a 4 ? 1
hydrogen bond between Phe3 NH and Boc CO. The pres-
ence of such a conformation in Z-OMe and Z-OH is also
confirmed by the distances Boc O–Phe3 Ca derived from
OMe confirmed the absence of the Phe3 NHꢀꢀꢀBoc CO
hydrogen bond. At the same time they indicated its pres-
ence in Z-OH. Besides CD spectra, interatomic distances,
and a 4 ? 1 hydrogen bond in Z-OH, the presence of a
type II b-turn in Z-OMe and Z-OH was confirmed by their
calculated dihedral angles values. These values indicate
that the both peptides adopt the type II b-turn conformation
at the Gly1–DZPhe2 residues. The lowest-energy structures
of Z-OMe and Z-OH are presented in Fig. 4c, d and the
ensembles of their 50 lowest-energy conformers in Fig. 4g,
h. The presented ensembles show that the conformation of
Z-OMe is the most rigid from the peptides studied and that
the flexibility of Z-OH is comparable with that of Z-p-NA
and E-p-NA.
A very significant result obtained from the presented CD
and NMR studies concerns the temperature coefficients of
the amide protons. This parameter is used for detection of
intramolecular hydrogen bonds in peptides and proteins
(Cierpicki and Otlewski 2001). It follows from our studies
that it should be used with caution when applied to small
and flexible linear molecules like the tripeptides studied.
The temperature coefficients presented in Table 1 show
˚
the ROESY spectra. They are 5.37 0.07 A and
˚
5.49 0.42 A for Z-OMe and Z-OH, respectively, i.e.,
they are typical of b-turns. Surprisingly, the presence of
hydrogen bonds in the both peptides is decidedly con-
tradicted by the temperature coefficients values of their
Phe3 NH’s (Table 1). The calculations performed for Z-
123

M. Jaremko et al.
that there are three amide protons with the dd/dT values
smaller than 0.0046 (of Gly1 and p-NA in Z-p-NA and p-
NA in E-p-NA) and two with slightly greater values (of
Phe3 in Z-p-NA and DZPhe2 in Z-OH). Then it may be
expected that these five amide protons are involved in
hydrogen bonds. Meanwhile, the calculations based on
interatomic contacts and information from the CD spectra
indicate that this expectation is correct only in the case of
Phe3 and p-NA in Z-p-NA and three other hydrogen bonds
are wrongly predicted. Other hydrogen bonds present in the
peptides studied are formed by the amide protons of Phe3
in E-p-NA and Z-OH. CD data, interatomic distances, and
dihedral angles strongly suggest that there is also another
hydrogen bond formed by the Phe3 amide proton in Z-
OMe. In the case of analogous tetrapeptides (Lisowski
et al. 2010) a similar situation with temperature coefficients
of the amide protons occurred but it was not as drastic like
in the present case. It shows that amide protons tempera-
ture coefficients alone are not good indicators of hydrogen
bonds in small molecules and may lead to erroneous con-
clusions. Our results indicate that more reliable in detection
of hydrogen bonds in such compounds is interatomic dis-
tances and calculations based on interatomic contacts from
the ROESY spectra (Table 2; Fig. 5) in combination with
the results obtained from CD or another method like IR or
Raman.
Z-OH are both ordered and they adopt the same type II b-
turn conformation whereas their configurational isomers,
E-OMe and E-OH, are by far unordered. These results
show that a DPhe residue of the Z configuration is a much
stronger inducer of ordered conformations than its E iso-
mer. The results obtained for Z-OMe and E-OMe are
consistent with the conclusion reached for analogous tet-
rapeptide methyl esters (Lisowski et al. 2010). The dif-
ferences in the ordered structures-inducing potential of the
DPhe residue depending on its configuration result proba-
bly from the different steric interactions of the DPhe side
chain with other parts of a peptide. The results presented
show also that the nature of the C-terminal end of a peptide
is important for the DEPhe-containing peptides whereas it
is of no meaning for peptides with a DZPhe residue. Of E-
p-NA, E-OMe, and E-OH, only E-p-NA is ordered which
shows that a DEPhe residue alone is not sufficient to induce
ordered conformations in a peptide unless it is present in
combination with the p-NA group. The same result was
obtained for the analogous tetrapeptides (Lisowski et al.
2010).
It was also found that the temperature coefficients values
of the amide protons in small molecules like the peptides
studied are not a good criterion of presence of hydrogen
bond so a caution should be observed in their applications.
When used alone, they may be the source of wrong con-
clusions. Much better way of determination of hydrogen
bonds is taking into account the interatomic distances and
contacts derived from the ROESY spectra, based on their
calculations, and additional information obtained from
other methods like CD or IR.
Very small ellipticities observed in the CD spectra of E-
OMe and E-OH in the near-UV region (Figs. 1, 2, 3) in all
the solvents used indicate that the conformational equi-
libria of those peptides are distinctly dominated by unor-
dered structures. The same results were obtained from the
NMR studies. The ROESY spectra did not show any sign
of non-trivial interatomic contacts that could suggest the
presence of ordered conformations.
Acknowledgments We would like to thank prof. Andrzej Ejchart
(Institute of Biochemistry and Biophysics, Polish Academy of Sci-
ences) for making the 500 MHz Varian spectrometer available to us.
The syntheses of peptides, performed by M. Makowski, were sup-
ported by the Wrocław Research Center EIT ? under the project
‘‘Biotechnologies and advanced medical technologies––BioMed’’
(POIG 01.01.02-02-003/08-00 financed from the European Regional
Development Fund (Operational Programme Innovative Economy,
1.1.2).
Conclusions
The detailed solution conformations of three pairs of de-
hydropeptides (p-nitroanilides, methyl esters, and peptides
with a free C-terminal carboxyl group), differing by the
configuration of a dehydroamino acid residue, were
determined on the basis of CD and NMR studies, as a
function of the DPhe residue configuration and the nature
of their C-terminal ends. It was found for all the peptides
studied that the DPhe residue configuration plays an
essential role in their conformational properties. In the case
of p-nitroanilides, both Z-p-NA and E-p-NA are ordered
but there are two b-turns in the former and only one b-turn
in the latter which shows a higher order in Z-p-NA. It
distinguishes them from the analogous tetrapeptide p-ni-
troanilides (Lisowski et al. 2010) studied also by CD and
NMR which are ordered to a similar extent. Z-OMe and
Conflict of interest The authors declare that they have no conflict
of interest.
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