Effect of the Δphe residue configuration on a didehydropeptides conformation: A combined CD and NMR study

Article abstract of DOI:10.1002/bip.21522

Conformations of two pairs of dehydropeptides with the opposite configuration of the ΔPhe residue, Boc-Gly-Δ^ZPhe-Gly-Phe- OMe (Z-OMe), Boc-Gly-Δ^EPhe-Gly-Phe-OMe (E-OMe), Boc-Gly-Δ^ZPhe-Gly-Phe-p-NA (Z-p-NA), and Boc-Gly- Δ<

Full text of DOI:10.1002/bip.21522

Effect of the DPhe Residue Configuration on a Didehydropeptides
Conformation: A Combined CD and NMR Study
Effect of the DPhe Residue Configuration on a Didehydropeptides
Conformation: A Combined CD and NMR Study
Marek Lisowski,1 Łukasz Jaremko,2,3 Mariusz Jaremko,2 Adam Mazur,4 Rafał Latajka,5
Maciej Makowski6
1
Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wrocław, Poland
2
´
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland
3
4
Faculty of Chemistry, Warsaw University, Pasteura 1, 02-093 Warsaw, Poland
Department of NMR-based Structural Biology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077
¨
Gottingen, Germany
5
´
Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeze Wyspianskiego 27,
˙
50-370 Wrocław, Poland
6
Institute of Chemistry, University of Opole, Oleska 48, 45-052 Opole, Poland
Received 23 April 2010; revised 25 June 2010; accepted 8 July 2010
Published online 14 July 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.21522
the DEPhe one has no such properties. In p-nitroanilides,
ABSTRACT:
both isomers of DPhe cause the peptides to adopt ordered
Conformations of two pairs of dehydropeptides with the
opposite configuration of the DPhe residue, Boc-Gly-DZPhe-
#
structures to a similar extent. 2010 Wiley Periodicals, Inc.
Biopolymers 93: 1055–1064, 2010.
Gly-Phe-OMe (Z-OMe), Boc-Gly-DEPhe-Gly-Phe-OMe
Keywords: dehydropeptides; dehydrophenylalanine
(E-OMe), Boc-Gly-DZPhe-Gly-Phe-p-NA (Z-p-NA), and
configuration; circular dichroism; nuclear magnetic
Boc-Gly-DEPhe-Gly-Phe-p-NA (E-p-NA) were compared
resonance; dehydropeptide conformation
on the basis of CD and NMR studies in MeOH,
trifluoroethanol (TFE), MeCN, chloroform, and
dimethylsulfoxide (DMSO). The CD results were used as
This article was originally published online as an accepted
the additional input data for the NMR-based determination
of the detailed solution conformations of the peptides. It was
found that E-OMe is unordered and Z-OMe, Z-p-NA, and
E-p-NA adopt the b-turn conformation. There are two
overlapping b-turns in each of those peptides: type II and
type III0 in Z-OMe and Z-p-NA, and two type III in
E-p-NA. The ordered structure-inducing properties of
DZPhe and DEPhe in the peptides studied depend on the
C-terminal blocking group. In methyl esters, the DZPhe
residue is a strong inducer of ordered conformations whereas
preprint. The ‘‘Published Online’’date corresponds to the preprint
version. You can request a copy of the preprint by emailing the
Biopolymers editorial office at biopolymers@wiley.com
INTRODUCTION
b-Dehydroamino acid residues (a,b-un-
saturated or D-amino acid residues)
contain a double bond between the Ca
and Cb atoms. This structural feature
endows them with the ability to limit
a,a
the conformational freedom of peptides. These D-amino acids,
which have different substituents on the Cb carbon atom, can
exist in two forms — as isomers Z, with the older substituent
in position cis to the nitrogen atom, and isomers E, with this
substituent trans to nitrogen. Isomers Z are more thermody-
namically stable than their E counterparts and hence easier to
Correspondence to: Marek Lisowski; e-mail: mbl@wchuwr.pl
C
VV
2010 Wiley Periodicals, Inc.
Biopolymers Volume 93 / Number 12
1055

1056
Lisowski et al.
obtain. It has been shown recently, though, that poly-DAbu
exists in alternate DZAbu and DEAbu form rather than the all
Z-form.1 A great majority of articles published so far on dehy-
dropeptides deal with D-amino acid residues of the Z configu-
ration. The most studied dehydroamino acid residue has been
dehydrophenylalanine. It has been found that in the solid state
a DPhe residue of the Z configuration induces b-turns in short
sequences2–6 and a 310-helix in longer ones or peptides with
more than one dehydro residue.4,7–19 Similar conformational
properties of DZPhe have been observed in solution by
NMR8,11,19–34 and CD,18,19,27–44 though other, unusual struc-
tures have also been reported.45 The DZPhe residues have also a
potential to introduce long-range interactions in peptides46,47
or to be a b structure breaker.48
Much less is known about the conformational preferences
of a DPhe residue of the E configuration. Few crystal struc-
tures of DEPhe-containing peptides show that this residue
occupies the i 1 1 position in type II or II0 b-turn.17,49,50
NMR and CD studies on DEPhe-containing peptides51–54
revealed that this residue favors formation of the folded con-
formations of the b-turn type.
So far, in the literature there has been no article with a
direct comparison of the conformational properties of dehy-
dropeptides containing a single DPhe residue and differing
only by its configuration. Therefore, we wanted to check how
the dehydroamino acid residue configuration influences a
dehydropeptide conformation. In this connection, we inves-
tigated the conformations of two following peptides (Scheme 1):
SCHEME 1 Structures of Z-OMe, E-OMe, Z-p-NA, and E-p-NA.
to obtain the detailed solution conformations of the peptides
studied. We used successfully such an approach in our confor-
mational studies on the decapeptide fragment of ubiquitin.55
Basing on CD and NMR studies we present the detailed
solution conformations of Z-OMe and E-OMe. The confor-
mational properties of the methyl esters were compared with
those of two very similar peptides (Scheme 1):
Boc-Gly-DZPhe-Gly-Phe-OMe (Z-OMe)
Boc-Gly-DEPhe-Gly-Phe-OMe (E-OMe)
The absorption and CD spectra of Z-OMe in MeOH have
been presented in Ref. 17, but the peptide has been used
there as a model compound and its conformation has not
been discussed at all. We investigated the conformations of
the two methyl esters by CD in MeOH, TFE, MeCN, and
chloroform, and NMR in CDCl3 and DMSO-d6.
Boc-Gly-DZPhe-Gly-Phe-p-NA (Z-p-NA)
Boc-Gly-DEPhe-Gly-Phe-p-NA (E-p-NA)
So far, the detailed solution conformations of dehydropep- differing from the first pair only by the C-terminal blocking
tides have been derived from the NMR alone. This approach group. The X-ray1 and CD studies17 have shown that Z-p-NA
has some disadvantages because a peptide in solution is a adopts a type II b-turn conformation at the DZPhe2 and Gly3
dynamic system. In the cases of small and dynamic systems residues, in the solid state and solution. Both p-nitroanilides
when there is a small number of ROE contacts per residue and have also been studied by NMR,18,51 but these investigations
often ambiguous 3JHH couplings information, the NMR is not have not allowed to present the detailed solution conforma-
capable of sufficient solution structure determination. In such tions of the peptides. In this connection, we carried out anew
a case the CD can give additional information on the peptide the NMR studies on Z-p-NA and E-p-NA in dimethylsulfox-
conformation, for example, whether a peptide is folded or ide (DMSO). The latter peptide was also studied by CD in
unordered or what type of a b-turn dominates in its confor- MeOH, MeCN, TFE, and DMSO. The CD spectrum of Z-p-
mational equilibrium. In this article, we applied an integrated NA in DMSO was measured as well. The CD and NMR stud-
approach that consists in combining the NMR and CD results ies on Z-p-NA and E-p-NA yielded the detailed solution
Biopolymers

Effect of the DPhe Residue Configuration
1057
Table I Absorption Features of Z-OMe, E-OMe, Z-p-NA, and
E-p-NA in MeOH, in the Near-UV Region
structures of both peptides. The results presented in this arti-
cle may have implications for the de novo design of peptides
with specific conformations in solution.
Peptide
k
max (nm)
emax
Z-OMea
E-OMe
Z-p-NAa
E-p-NA
280
280
286
289
19,500
22,900
30,500
30,200
EXPERIMENTAL PROCEDURES
Synthesis of Peptides
The syntheses of Z-OMe, Z-p-NA, and E-p-NA have been
described in Refs. 18,56, and 51, respectively.
a
Ref. 17.
Boc-Gly-DEPhe-Gly-Phe-OMe
(E-OMe). Boc-Gly-DEPhe-
resents the average of at least four scans. The data are pre-
Gly-OH was obtained from Boc-Gly-DEPhe-Gly-OMe51 by a
standard basic hydrolysis with a 30% excess of 1M NaOH in
MeOH. Yield 98%; mp 127–1308C; elemental analysis calcd
(%) for C18H23N3O6 (377.39): C 57.29, H 6.14, N 11.40;
found: C 57.59, H 6.00, N 11.11.
sented as molar ellipticity [y].
NMR Spectroscopy
NMR spectra of Z-OMe and E-OMe were recorded at 11.7 T
on a Varian Unity 1500 spectrometer at 258C, in CDCl3 and
DMSO-d6. NMR spectra of Z-p-NA and E-p-NA were meas-
ured at 11.7 T with a Bruker Avance 500 spectrometer at
258C, in DMSO-d6. Two-dimensional (2D) homonuclear
NMR spectra were processed with NMRPipe57 and analyzed
TEA (0.143 mL, 1.05 mmol) was added to the solution of
Boc-Gly-DEPhe-Gly-OH (0.189 g, 0.5 mmol) and HCl ꢀ Phe-
OMe (0.108 g, 0.5 mmol) in MeCN (3 mL). After 5 min,
TBTU (0.168 g, 0.55 mmol) was added and the solution was
stirred for 10 h at room temperature. Then MeCN was
evaporated and the resulting oil was dissolved in ethyl acetate
(50 mL). The solution was washed with 1M HCl (3 3 3 mL),
saturated solution of KHCO3 (3 3 3 mL), and brine (3 mL).
Then it was dried with MgSO4 and ethyl acetate was evapo-
rated in vacuo. Crude E-OMe was crystallized from CHCl3-
ethyl acetate (9:1, v/v)/hexane. Yield 0.247 g (92%); mp 100–
1028C; elemental analysis calcd (%) for C28H34N4O7
(538.58): C 62.44, H 6.36, N 10.40; found: C 62.56, H 6.20, N
1
with SPARKY57 programs. Complete assignments of the H
and 13C resonances for all the peptides were done by applica-
tion of a standard procedure58 based on inspection of the 2D
homonuclear TOCSY (with mixing times 10 and 80 ms) and
ROESY (with mixing times 300 and 500 ms) experiments.
3
Accuracies of the estimated proton chemical shifts and JHH
coupling constants are 0.02 ppm and 0.4 Hz, respectively.
The temperature coefficients of amide protons of methyl
esters and p-nitroanilides were measured in CDCl3 and
DMSO-d6, respectively, at the temperature range from 291 to
318 K, with a temperature interval of 3 K. Chemical shifts are
given as d in relation to SiMe4 as internal standard. In all
cases 15 mM peptide solutions were used. Calculations of
lowest-energy structures, carried out for peptides that exhib-
ited ordered conformations in solution according to CD,
were performed with an X-PLOR NIH 2.21 program pack-
age.59 Figures presenting the calculated structures were pre-
pared with a MolMol software.60 Supplementary data are
available from the authors upon request.
1
10.21. H NMR (500 MHz, CDCl3) d (ppm): 1.43 (9H, s, 3
3 CH3 Boc); 3.09 (2H, d-d, CbH2 Phe4); 3.66 (3H, s,
COOCH3); 3.71 (1H, d-d, CaH1 Gly3); 3.85 (1H, d-d, CaH2
Gly3); 3.90 (2H, broad s, CaH2 Gly1); 4.75 (1H, q, CaH
Phe4); 5.37 (1H, t, NH Gly1); 6.39 (1H, broad t, NH Gly3);
7,11 (1H, d, NH Phe4); 7.19–7.31 (11H, m, aromatic of Phe4
and DEPhe2; CbH DEPhe2); 8.69 (1H, broad s, NH DEPhe2).
Absorption Spectra
Absorption spectra were measured with a Cary 500 Scan
UV–vis–NIR spectrophotometer in MeOH, at room temper-
ature. Pathlength of 1 mm was used. Concentration of the
solutions was 5 3 1024M.
RESULTS
Circular Dichroism Spectroscopy
Absorption Spectra
CD spectra were recorded on a Jasco J-600 spectropolarime- The absorption features of Z-OMe, E-OMe, Z-p-NA, and E-
ter, at room temperature. Spectra were measured in MeOH, p-NA in the near-UV region are presented in Table I. The
TFE, MeCN, chloroform, and DMSO. Pathlength of 1 mm spectra of Z-OMe and E-OMe show a large, broad band at
was used. Concentrations of the solutions were in the range 280 nm, similar to those observed for other mono-unsatu-
of 0.17–0.21 mg/mL (2.6–3.9 3 1024M). Each spectrum rep- rated DZPhe peptides.35,36,42 It is an intramolecular charge-
Biopolymers

1058
Lisowski et al.
FIGURE 1 CD spectra of Z-OMe and E-OMe.
FIGURE 2 CD spectra of Z-p-NA and E-p-NA.
transfer band originating from the cinnamic chromophore
C6H5C¼¼CꢁꢁC¼¼O of the DPhe residue, from the highest
occupied orbital of the electron-donating styryl group to the
vacant orbital of the electron-accepting carbonyl group.35,61
In the case of Z-p-NA and E-p-NA, the DPhe residue band at
280 nm is overlapped by a charge-transfer band of the p-NA
group at 315 nm.18 The spectra of Z-p-NA and E-p-NA are
very similar. They show a band at 286 and 289 nm, respec-
tively, with a shoulder at about 325 nm that is quite distinct
for Z-p-NA and poorly visible for E-p-NA. The spectra of
both pairs of peptides show that the DPhe residue configura-
tion has only a slight influence on their shape.
between the CD spectra of Z-p-NA in DMSO and other sol-
vents. In DMSO, the positive p-NA band at 318 nm and the
negative DZPhe band at 280 nm are distinctly smaller than in
other solvents, especially the latter. The spectra of E-p-NA
are less differentiated than those of Z-p-NA. In MeOH, TFE,
and MeCN, there is a large negative p-NA band at 309–314
nm and a large positive DEPhe band at 274–278 nm. The
spectrum in DMSO is similar in its shape to the spectra in
other solvents but it is shifted somewhat to longer wave-
lengths, with a negative and a positive band at 319 and
284 nm, respectively.
NMR Studies
To detect intramolecular hydrogen bonds in the peptides
CD Studies
The CD spectra of Z-OMe and E-OMe in MeOH, TFE, investigated, the temperature dependence of their amide pro-
MeCN, and chloroform are presented in Figure 1. The spec- tons was measured. It is well known that amide proton
tra of Z-OMe in MeOH and chloroform are very similar. chemical shifts are sensitive to temperature changes. If a pro-
They show a large negative band at 280 nm, a little more in- ton forms a hydrogen bond, then its chemical shift shows a
tensive in the latter solvent. In trifluoroethanol (TFE) and very small dependence on the temperature. The parameter
MeCN this band, at 278 and 273 nm, respectively, is about that indicates the presence or absence of hydrogen bonds is a
twice smaller than in chloroform and MeOH. The CD spec- dd/dT (ppm K21) coefficient. It has been established that
tra of E-OMe are much less differentiated. They show a very amide protons in protein systems, which are characterized by
small, broad, positive band at 270–279 nm, the most inten- the temperature coefficient value lower than 0.0046 ppm
sive in MeOH and the smallest in MeCN.
K21, are involved in hydrogen bonds.62 This value is often
The CD spectra of Z-p-NA and E-p-NA in MeOH, TFE, used for checking if any amide protons are involved in
MeCN, and DMSO are shown in Figure 2. They look to a hydrogen bonds. The temperature coefficients of amide pro-
some extent like mirror images. The spectra of Z-p-NA in tons in the studied peptides are presented in Table II. They
MeOH and TFE are very similar. There is a large, positive show that there are two hydrogen bonds in Z-OMe, Z-p-NA,
p-NA band at 311 and 308 nm, and a large, negative DZPhe and E-p-NA. They are formed by amide protons of Gly3
band at 275 and 273 nm, in those spectra. The spectrum of and Phe4 in Z-OMe and Z-p-NA, and of Phe4 and p-NA in
Z-p-NA in MeCN differs from those in MeOH and TFE by E-p-NA. The values of temperature coefficients of Gly3 and
the bands intensity. In MeCN, the p-NA band at 308 nm is Phe4 amide protons in Z-OMe are larger than the 0.0046
much stronger, and the DZPhe residue band at 273 nm much limit value, but other results presented below confirm that
weaker, than in MeOH and TFE. There is a large difference the two protons form hydrogen bonds. A low value of the
Biopolymers

Effect of the DPhe Residue Configuration
1059
Table II Temperature Coefficients for Amide Protons of Z-OMe,
E-OMe, Z-p-NA, and E-p-NA
gen-bonded, and the other whose type and location could
not be determined exactly by CD, then only this first b-turn
with its hydrogen bond were used in the calculations. Such
an approach was applied for Z-OMe and Z-p-NA. In the case
of E-p-NA, the CD results are not unambiguous. The CD
spectra show the presence of two b-turns whose position
could be determined only by the hydrogen bonds detected by
the NMR (Table II). These turns are located at the DEPhe2-
Gly3 and Gly3-Phe4 residues. They can be either of type II0 or
III. It gives four possibilities: II0/II0, II0/III, III/II0, and III/III.
It was found that independently of which of those combina-
tions was chosen, the calculations yielded very similar results.
The results presented for E-p-NA (Table V) were obtained
with the two hydrogen bonds, Phe4 NH ꢀ ꢀ ꢀ Gly1 CO and p-
dd/dT (ppm K21 3 1000)
Residue
Z-OMea
E-OMea
Z-p-NAb
E-p-NAb
Gly1
7.2
7.4
5.2c
5.4c
—
6.9
7.2
8.1
4.7
—
5.6
6.4
5.5
DPhe2
4.8
Gly3
Phe4
p-NA
3.0c
0.7c
5.6
6.6
3.5c
2.6c
a
In CDCl3.
In DMSO-d6.
Protons forming hydrogen bonds.
b
c
Phe4 amide proton temperature coefficient in E-OMe, lower NA NH ꢀ ꢀ ꢀ DEPhe2 CO, and the b-turns at the DEPhe2-Gly3
than for Gly3 and Phe4 amide protons in Z-OMe, suggests and Gly3-Phe4 residues, assumed to be of type II0 and III,
that this proton also forms an intramolecular hydrogen respectively, as the input data. In the case of calculations
bond. Still, other results obtained for that peptide, described based on combined NMR and CD results, it was important
below, are in contradiction with such a possibility. Another to have the results from the both methods that could be
way of checking for the presence of intramolecular hydrogen directly compared. Such a situation took place in the case of
bonds in peptides is determination of the dependence of the Z-OMe and E-OMe whose NMR spectra were measured in
amide protons chemical shifts on the solvent polarity. In the chloroform that was also one of the solvents used for
case of protons involved in hydrogen bonds that dependence CD studies. Because of a very poor solubility of Z-p-NA and
is distinctly smaller than for nonhydrogen bonded ones. The E-p-NA in chloroform, the NMR spectra of those peptides
results obtained for the methyl esters, measured in CDCl3 were measured in DMSO. Therefore, the CD spectra of
and DMSO-d6, are shown in Table III. They confirm the Z-p-NA and E-p-NA were measured in DMSO as well. For
presence of two hydrogen bonds in Z-OMe, formed by amide E-p-NA, there is a large similarity between the CD spectra
protons of Gly3 and Phe4, and indicate the absence of measured in DMSO and other solvents. However, the CD
a hydrogen bond in E-OMe. Such studies were not per- spectra of Z-p-NA in DMSO and other solvents differ very
formed for the p-nitroanilides due to their poor solubility in much from each other (see Figure 2). The CD spectrum of
chloroform.
Z-p-NA in DMSO indicates that its conformation, contrary
Important information on the conformational properties to that in MeOH, TFE, and MeCN and to the NMR results,
of Z-OMe, E-OMe, Z-p-NA, and E-p-NA were obtained from is unordered to a large extent. Still, it is rather not the case
the ROESY spectra. On their basis, interatomic contacts in here. So for that peptide the NMR results combined with the
the peptides studied could be detected. Trivial, intraresidue CD ones obtained not for DMSO but for MeOH, TFE, and
contacts are present in each investigated peptide. Nontrivial MeCN, were used for the conformational calculations. The
contacts were observed only for Z-OMe, Z-p-NA, and conformation of Z-p-NA in DMSO and the way the calcula-
E-p-NA. They are presented in Table IV. Such contacts are tions were performed for that peptide will be discussed in the
absent in E-OMe, confirming the lack of intramolecular
hydrogen bonds in that peptide. These contacts were used as
the input data for calculations of the lowest-energy conform-
ers of Z-OMe, Z-p-NA, and E-p-NA. Besides them, the calcu-
lations were based only on the unambiguous results obtained
at the same time by the NMR and CD. For example, if the
NMR spectrum of a peptide indicated that two amide pro-
tons are involved in intramolecular hydrogen bonds, suggest-
ing the presence of two b-turns, and the CD spectrum
showed that there are two b-turns, one of a given type, at
specific residues and stabilized by a hydrogen bond formed
by one of those amide protons detected by NMR as hydro-
Table III Dependence of Amide Protons Chemical Shifts on the
Solvent Polarity for Z-OMe and E-OMe
Dd 5 dNH(DMSO-d6) 2 dNH(CDCl3)
Residue
Z-OMe
E-OMe
Gly1
1.47
1.53
0.72a
0.80a
1.61
1.30
2.24
1.22
DPhe2
Gly3
Phe4
a
Protons forming hydrogen bonds.
Biopolymers

1060
Lisowski et al.
Table IV Interatomic Nontrivial Contacts in Z-OMe, Z-p-NA, and E-p-NA Detected from the ROESY Spectra
Interatomic Contacts in:
Z-OMea
Z-p-NAb
E-p-NAb
CaH2 Gly1 $ NH DZPhe2
CaH2 Gly1 $ NH Gly3
NH DZPhe2 $ NH Gly3
NH Gly3 $ NH Phe4
CaH2 Gly3 $ NH Phe4
NH DZPhe2 $ CaH2 Gly1
NH DZPhe2 $ NH Gly1
NH DZPhe2 $ NH Gly3
NH DZPhe2 $ aromatic H’s Phe4
NH Gly3 $ CaH2 Gly1
NH Gly3 $ CbH DZPhe2
NH Gly3 $ NH Phe4
NH DEPhe2 $ CaHa1 Gly1
NH DEPhe2 $ CaHa2 Gly1
NH DEPhe2 $ NH Gly3
NH Gly3 $ NH Phe4
Aromatic H’s Phe4 $ CbH DEPhe2
NH p-NA $ CbH DEPhe2
NH p-NA $ CaH Phe4
CaHa1 Gly3 $ NH Gly1
NH Phe4 $ CaH2 Gly1
NH Phe4 $ CaHa2 Gly3
NH p-NA $ CaH Phe4
NH p-NA $ NH Phe4
NH p-NA $ NH Phe4
a
In CDCl3.
b
In DMSO-d6.
next section. The calculated lowest-energy conformers of
A large intensity of the negative DZPhe CD bands of Z-
Z-OMe, Z-p-NA, and E-p-NA are presented in Figures 3a–3c OMe at 280 nm in MeOH, TFE, and MeCN (see Figure 1)
and the values of their dihedral angles are given in Table V. shows that the peptide adopts a chiral, rigid structure of the
Figures 3d–3f also show the ensembles of 50 lowest-energy same type in each solvent. This structure must involve the
structures of those peptides, chosen from the 500 calculated Phe4 residue because otherwise two enantiomeric forms of
peptide conformers.
that conformation would be present in the conformational
equilibrium of Z-OMe resulting in a very small intensity of
the DZPhe band. It has been found that a negative DZPhe
band at 280 nm is typical of dehydropeptides adopting a type
II b-turn, with a DZPhe residue at the i 1 2 position.42 The
presence of such a band in the CD spectra of Z-OMe suggests
that there is a type II b-turn in that peptide at the Gly1 and
DZPhe2 residues, stabilized by the intramolecular hydrogen
bond between the amide proton of Gly3 and the carbonyl ox-
ygen of the Boc group. But this conformation does not
involve the Phe4 residue whereas the CD spectra show that
this residue must also be a part of the ordered structure of Z-
OMe. It suggests the presence of another b-turn in that pep-
tide, at the DZPhe2 and Gly3 or Gly3 and Phe4 residues. Since
the b-turn at Gly3 and Phe4 could not be stabilized by the
DISCUSSION
The CD spectra of dehydropeptides in the far-UV region are
difficult to analyze since there are overlapping contributions
of the peptide, aromatic, and unsaturated chromophores. In
this connection, we limited our analysis of the spectra of four
peptides studied to the near-UV region only. The peptides
contain a DPhe residue that gives a large CD band in that
region, at about 280 nm, which is very sensitive to a dehydro-
peptide conformation.18,19,27–44 Thus, this residue is a very
good conformational probe in the studies on dehydropepti-
des. Moreover, Z-p-NA and E-p-NA contain the p-NA group
that has also been found to be a useful tool in the conforma-
tional studies on peptides.63–66
Table V Torsion Angles Values of Z-OMe, Z-p-NA, and E-p-NA Calculated on the Basis of NMR and CD Parameters with an X-PLOR
Program
Z-OMe
Z-p-NA
E-p-NA
Residue
u
C
u
C
u
C
Gly1
2488 (668)
498 (6118)
778 (6368)
2418 (6878)
1148 (6258)
188 (6218)
108 (6348)
—
2568 (628)
308 (628)
398 (648)
1148 (658)
308 (628)
538 (6118)
278 (6908)
1438 (6898)
2268 (638)
2548 (638)
2658 (648)
21168 (61018)
2348 (628)
278 (638)
DPhe2
Gly3
Phe4
2618 (6128)
2408 (638)
Standard deviations for 50 lowest-energy structures are given in brackets.
Biopolymers

Effect of the DPhe Residue Configuration
1061
FIGURE 3 The lowest-energy conformers of Z-OMe (a), Z-p-NA (b), and E-p-NA (c), and
ensembles of the 50 lowest-energy structures of Z-OMe (d), Z-p-NA (e), and E-p-NA (f).
intramolecular 4?1 hydrogen bond due to the lack of a pro- there are positive ellipticities in that region, except the spec-
ton donor, the b-turn at DPhe2 and Gly3 stabilized by the trum in MeCN. The intensities of the negative DZPhe band
hydrogen bond between the amide proton of Phe4 and the at 280 nm suggest that the ordered conformation of Z-OMe
carbonyl group of Gly1 is much more probable. These con- is the most highly populated in chloroform and MeOH,
clusions were confirmed by the NMR studies on Z-OMe. whereas in MeCN and TFE its population is decreased. The
They show the presence of two intramolecular hydrogen lowest-energy conformation of Z-OMe and a comparison of
bonds, formed by the amide protons of Gly3 and Phe4 its 50 lowest-energy conformers are shown in Figures 3a and
(Tables II and III). The dihedral angles of successive residues 3d, respectively. The ensemble of 50 lowest-energy structures
of Z-OMe (Table V) indicate that these hydrogen bonds sta- indicates that Z-OMe is quite flexible in solution and it has a
bilize two overlapping b-turns, of type II at Gly1 and DZPhe2 substantial conformational freedom.
and type III0 at DPhe2 and Gly3. Such a sequence of b-turns
A small intensity of the DEPhe CD bands of E-OMe (see
occurs very often in the case of Aib-containing peptides67,68 Figure 1) at 270–280 nm in each solvent suggests that the
in which the N-terminal b-turn is followed by a left-handed peptide is unordered. The same result was obtained from the
3
10 helical conformation, that is, type III0 b-turns. It has also NMR studies, which do not show any interatomic contacts
been observed for Boc-L-Val-DZPhe-DZPhe-DZPhe-L-Val- that would suggest the ordered conformation of that peptide.
OMe.28 The type III0 b-turn in Z-OMe corresponds very well Positive CD bands of very small intensity at 280 nm have
with the CD spectra of that peptide. It has been found that been observed in water and MeOH for trifluoroacetate (TFA)
DZPhe-containing dehydropeptides in the type III b-turn of H-Tyr-D-Ala-DZPhe-Gly-NH2 for which the unordered
conformation give a positive CD band at about 280 nm and structure was postulated.37 The intensities of the positive
a negative band at about 235 nm.42 It shows that dehydro- DEPhe band of E-OMe in MeOH and TFE are about twice as
peptides in the type III0 b-turn conformation should give a large as those observed for TFA ꢀ H-Tyr-D-Ala-DZPhe-Gly-
negative CD band at 280 nm and a positive one at 235 nm. NH2. It does not allow to exclude the presence of some
This spectral pattern agrees with the CD spectra of Z-OMe in amounts of ordered structures in the conformational equili-
each solvent. Indeed no positive band at about 235 nm in bria of E-OMe. These structures could possibly account for
those spectra is observed, which may be due to its overlap the surprisingly small temperature coefficient of the Phe4
with a stronger positive band at shorter wavelengths, but amide proton (Table II) that suggests the presence of an
Biopolymers

1062
Lisowski et al.
intramolecular hydrogen bond. Still, the NMR and CD stud- Phe4, NH Gly3 $ CaH2 Gly1, CaHa1 Gly3 $ NH Gly1, and
ies show that an unordered structure is by far the dominating NH Phe4 $ CaH2 Gly1), are preserved in the final, lowest-
conformation of E-OMe in each solvent.
energy structure. It proves the correctness of our approach.
At the beginning of the discussion of the results obtained
The CD spectra of Z-p-NA (see Figure 2) in the near-UV
for Z-p-NA, the matter of whether the NMR results obtained in MeOH, TFE, and MeCN show that the peptide adopts the
for the peptide in DMSO may be directly compared with type II b-turn conformation at the Gly1 and DZPhe2 residues,
those obtained by CD in other solvents should be addressed. stabilized by the intramolecular hydrogen bond between the
The NMR studies on Z-p-NA in DMSO show that there are amide proton of Gly3 and the carbonyl oxygen of the Boc
two intramolecular hydrogen bonds in that peptide, formed group.18 The presence of such a conformation is consistent
by the Gly3 and Phe4 amide protons (Table II). It indicates with the NMR studies, which show that the Gly3 amide pro-
that Z-p-NA adopts the folded conformation of the b-turn ton is involved in a hydrogen bond (Table II) and by the con-
type. This conclusion is confirmed by a large number of non- formational calculations (Table V). The analogous reasoning
trivial interatomic contacts detected for Z-p-NA from the as in the case of Z-OMe indicates that there is another
ROESY spectra (Table IV), especially by contacts NH DZPhe2 b-turn, either at the DZPhe2 and Gly3 or Gly3 and Phe4 resi-
$ aromatic H’s Phe4, NH Gly3 $ CaH2 Gly1, CaHa1 Gly3 $ dues, stabilized by the corresponding 4?1 hydrogen bond. A
NH Gly1, and NH Phe4 $ CaH2 Gly1, which suggest the large intensity of the positive band of the p-NA group sug-
presence of a b-turn conformation. Table IV shows that the gests that this group should also be a part of the second
largest number of contacts was detected for that very peptide. b-turn of Z-p-NA, hence the b-turn at the Gly3 and Phe4 res-
At the same time the CD spectrum of Z-p-NA in DMSO idues, with a hydrogen bond between p-NA NH and DZPhe2
shows a very small, negative band of the DZPhe residue at CO, seems more probable than the one at DZPhe2 and Gly3.
282 nm and a positive band of the p-NA group that is dis- It is consistent with the solid state structure of Z-p-NA in
tinctly smaller than those observed in other solvents. It sug- which two type II b-turns, at Gly1-DZPhe2 and Gly3-Phe4, are
gests that the population of the ordered structures of Z-p-NA present.2 Meanwhile the NMR studies showed that Z-p-NA
in DMSO is markedly decreased as compared with MeOH, adopts the same conformation as Z-OMe. The second intra-
TFE, and MeCN. Still, these ordered structures seem to be of molecular hydrogen bond in Z-p-NA is formed not by the
the same type as in the other solvents. Their decreased popu- p-NA group, a good hydrogen bond donor, but by the Phe4
lation in DMSO is in some contradiction to the NMR results amide proton (Table II). This result was unexpected since in
that indicate an evidently ordered conformation of Z-p-NA such a case the p-NA group is not a part of any ordered
in DMSO. Since the NMR and CD must reflect the same structure. In this connection, it is difficult to explain a large
conformation of Z-p-NA in DMSO, then it should be intensity of its CD band. A distinctly low value of the tem-
assumed that the CD spectrum of the peptide corresponds to perature coefficient of the Phe4 amide proton indicates that a
its ordered structure seen by the NMR. It is difficult, how- hydrogen bond formed by that proton is very strong. The
ever, to explain in such a case this substantial decrease of the calculations (Table V) showed that the second b-turn at the
CD bands of Z-p-NA in DMSO. We can not give at the pres- DZPhe2 and Gly3 residues, stabilized by the Phe4 NH ꢀ ꢀ ꢀ Gly1
ent any reliable explanation for that. One can speculate that CO hydrogen bond, is of type III0. The privileged solution
the DZPhe side chain of Z-p-NA in DMSO adopts such a conformation of Z-p-NA is presented in Figure 3b. The cal-
conformation that its optical activity is strongly diminished culated lowest-energy conformers of that peptide (Figure 3e)
or that the peptide is present in two conformations with the show that its conformational flexibility is distinctly reduced
opposite dispositions of the DZPhe side chain whose CD con- as compared with Z-OMe.
tributions largely cancel each other. Since the CD spectra of
In the case of E-p-NA, its CD spectra (see Figure 2) in
Z-p-NA suggest the presence of the same ordered structures each solvent used are quite similar to each other, indicating
in each solvent, then it seems reasonable to assume that con- small solvent-dependence of its conformation. A large inten-
clusions drawn from the CD spectra of Z-p-NA in MeOH, sity of the DEPhe and especially p-NA CD bands at about
TFE, and MeCN correspond to the NMR results for Z-p-NA 280 and 310 nm, respectively, suggests that, like in the case of
in DMSO. It was confirmed by the conformational calcula- Z-OMe and Z-p-NA, E-p-NA adopts the conformation com-
tions performed for Z-p-NA on the basis of NMR results for posed of two b-turns, stabilized by intramolecular hydrogen
DMSO and the CD results for MeOH, TFE, and MeCN. As bonds. One of those b-turns should be located at the Gly3
can be seen in Figure 3b, all interatomic contacts detected for and Phe4 residues since otherwise the p-NA group would not
Z-p-NA, among them those of significant importance for the be a part of the ordered structure. The positive band of the
conformation of the peptide (NH DZPhe2 $ aromatic H’s DEPhe2 residue indicates that those b-turns may be both of
Biopolymers

Effect of the DPhe Residue Configuration
1063
type III or II0, or type III/II0.42 The temperature coefficients of a DPhe residue, it seems that the conformational differen-
of the amide protons of E-p-NA (Table II) show the presence ces between isomeric peptides result mainly from the differ-
of two hydrogen bonds formed by NH’s of Phe4 and p-NA. It ent steric interactions between the DPhe side chain and other
indicates that there are two overlapping b-turns in E-p-NA, parts of the peptides. The results presented also show the
located at the DEPhe2-Gly3 and Gly3-Phe4 residues. They are influence of the C-terminal blocking group on a dehydropep-
stabilized by 4?1 hydrogen bonds between Phe4 NH and tide conformation in relation to the configuration of a dehy-
Gly1 CO and between p-NA NH and DEPhe2 CO. The calcu- dropeptide. In the case of peptides containing the DZPhe res-
lated lowest-energy conformation of E-p-NA is presented in idue, the substitution of the methyl group with the p-NA one
Figure 3c. The values of dihedral angles of the peptide (Table does not bring about any conformational changes whereas
V) show that its both b-turns are of type III. A very large in- such a substitution causes very large conformational changes
tensity of the negative p-NA band in the CD spectrum of E- in dehydropeptides with the DEPhe residue. It shows that
p-NA shows that this group is much more fixed in that pep- in the latter case a DPhe residue of the E configuration alone
tide than in Z-p-NA. It is connected to the intramolecular is not sufficient to induce the ordered conformation in a
hydrogen bond formed by the p-NA group in E-p-NA that is peptide.
absent in the case of Z-p-NA. A comparable intensity of the
DPhe2 CD bands in the spectra of Z-p-NA and E-p-NA sug-
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Reviewing Editor: David E. Wemmer
Biopolymers