Conformational studies of hexapeptides containing two dehydroamino acid residues in positions 3 and 5 in peptide chain

Article abstract of DOI:10.1016/j.molstruc.2008.06.023

Synthesis and structural studies of hexapeptides containing two dehydroamino acid residues in positions 3 and 5 in a peptide chain were performed. All the investigated peptides adopted bent conformations, stabilized by intramolecular hydrogen bonding, and could exist as two different conformers in solution. Only in the case of the peptide containing ΔAla residues, expected 3₁₀-helical conformation was found.

Full text of DOI:10.1016/j.molstruc.2008.06.023

Journal of Molecular Structure 892 (2008) 446–451
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Conformational studies of hexapeptides containing two dehydroamino acid
residues in positions 3 and 5 in peptide chain
b
b
c
Rafal Latajka a, , Michal Jewginski , Maciej Makowski , Artur Krezel
*
a Faculty of Chemistry, Department of Bioorganic Chemistry, Wroclaw University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland
b Institute of Chemistry, University of Opole, Oleska 48, 45-052 Opole, Poland
c Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and structural studies of hexapeptides containing two dehydroamino acid residues in positions
3 and 5 in a peptide chain were performed. All the investigated peptides adopted bent conformations,
stabilized by intramolecular hydrogen bonding, and could exist as two different conformers in solution.
Received 7 February 2008
Received in revised form 6 June 2008
Accepted 16 June 2008
Only in the case of the peptide containing
D
Ala residues, expected 310-helical conformation was found.
Available online 2 July 2008
Ó 2008 Elsevier B.V. All rights reserved.
Keywords:
Conformation
NMR
CD spectroscopy
Dehydroalanine
Isomers of dehydrophenylalanine
1. Introduction
hydrogen bond of (i + 3) ? I type, and a-helix by (i + 4) ? i, this
mixed conformation is stabilized by both these types of bonds
[12]. Moreover, folded conformation present in relatively nonpolar
solvent (CHCl3) is destabilized in DMSO and peptides adopt largely
extended conformation in this solvent [13]. Taken together, litera-
ture data suggested that dehydroamino acid residues exert a power-
ful conformational influence, independent on other constraints.
Thus, introduction of dehydroamino acid residues into bioactive
peptide sequences has become a useful tool to study structure–
function relationship and to provide new analogues of enhanced
activity.
Cathepsin C (dipeptidyl dipeptidase I; EC 3.4.14.1) belongs to
papain family of proteases [14] and sequentially removes dipep-
tides from the free N-termini of proteins and peptides. It has a
broad substrate specificity being able to hydrolyse out nearly every
possible dipeptide unit, with the exception of those containing ba-
sic amino acids (Arg or Lys) at N-terminal position or Pro on either
side of the scissile bond. It is also quite unusual that it has a
requirement for the presence of halide ions for its activity. The
main function of cathepsin C is protein degradation in lysosymes,
but it is also found to participate in the activation of cytotoxic T
lymphocytes and natural killer cells (granzymes A and B), mast
cells (tryptase and chymase), and neutrophils (cathepsin G and
elastase) by removing their N-terminal activation dipeptides [15–
17]. Loss of function mutations in the cathepsin C gene result in
periodontal disease and palmoplantar keratosis [18]. Since dehyd-
roamino acids are quite reactive and various thiol nucleophiles are
known to add to their double bonds [15–18], we speculated that
Rational interpretation of the results of biological tests and ra-
tional designing of new compounds require the detailed knowl-
edge about spatial structure–activity relationship. One of the
main streams in drug discovery has focused attention on the syn-
thesis of small mimetics of potent therapeutic peptides, both nat-
ural and synthetic ones. For this reason, the search for new factors,
which could be used as modifiers of peptide chain conformation,
has become increasingly important.
Introduction of dehydroamino acid residues into peptide chain
has been found to influence the three dimensional structure of both
main chain and side chain dramatically, due to the presence of
a
C @Cb double bond [1]. For example, dehydroalanine adopts a
roughly planar conformation with trans orientation for the
w and
u torsions and induces an inverse -turn in the preceding residue
c
[2]. (Z)-Dehydrophenylalanine exerts a b-turn conformation in
short peptides [3] and 310-helical conformation in the case of pep-
tides with a longer main chain [4–6]. For dehydropeptides contain-
ing
[7–10]. In the case of peptides containing two dehydrophenylala-
nines separated by more than one saturated residue, besides 310
helix, -helix could also be found. This tendency is stronger while
DX-Y-DX motif, the most favorable conformation was 310-helix
-
a
the saturated sequence between two dehydroamino acids is longer
[11]. If 310-helix conformation is stabilized by intramolecular
* Corresponding author. Tel.: +48 713203344; fax: +48 713284064.
E-mail address: rafal.latajka@pwr.wroc.pl (R. Latajka).
0022-2860/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2008.06.023

R. Latajka et al. / Journal of Molecular Structure 892 (2008) 446–451
447
short dehydropeptide mimetics of artificial substrates of cathepsin
C might act as alkylating inhibitors of the enzyme. However, our
Gly-DAla-Phe-pNA). Blocking groups were removed with trifluoro-
acetic acid according to standard procedure [21], yielding peptides
no. 1 and no. 3 with 96–99% yield.
former studies of tetra- and pentapeptides containing
DAla and
D
ZPhe residues showed that they acted only as substrates or weak
inhibitors of cathepsin C [19–21]. For this reason, in the case of
longer chains, such as the hexapeptides presented in this paper,
we focused only on structural investigations.
2.2. Structural and conformational studies
NMR and CD studies were done in order to determine the struc-
tures and conformational preferences of dehydropeptides shown in
Fig. 1.
Conformational studies on pentapeptides containing two
DPhe
residues (Z and E isomers) in positions 2 and 4 in the peptide chain
showed that all the investigated peptides adopted bent conforma-
tion and majority of them could exist as two different low-ener-
getic conformers in solution [21]. In the case of hexapeptides
containing two dehydroamino acid residues located in positions
2 and 5, only for the peptide with free N-termini, rigid 310-helical
conformation was observed. For the rest of the examined peptides
extended and ‘‘zig-zag” conformers were predominant [22]. The
next step of our investigations, presented in this paper, is devoted
to the conformational studies of hexapeptides, where dehydroami-
no acids are isolated only by one saturated residue and located in
positions 3 and 5 in peptide chain (see Fig. 1).
2.3. CD spectroscopy
CD measurements were performed in two solvents (chloroform
and methanol) for the region down to 200 nm because at shorter
wavelengths there are overlapping contributions of the peptide,
aromatic and unsaturated chromophores, which makes the analy-
sis very complex. In the near-UV region, Cotton effects associated
with
DPhe and pNA groups were observed. This is strongly depen-
dent on the peptide conformation and therefore very useful in this
kind of conformational studies [23].
Analysis of CD spectra obtained in methanolic solution (Fig. 2a)
shows that the most unordered conformation is observed in the
case of saturated peptide no5. On the other hand, strong negative
2. Results and discussion
band detected at 220 nm for peptide no4 (Boc-Gly-Gly-
Gly-
EPhe-Phe-pNA) and strong positive band at value 305 nm
for peptide no3 suggest that compounds which possess Phe in
D
EPhe-
2.1. Synthesis
D
D
Peptides were obtained analogously to previously described
procedure [21,22], via 1 + 5 approach, by reacting Boc-GlyOH with
Gly-X-Gly-X-Phe-pNA in acetonitrile using TBTU as a coupling re-
agent. Products were purified by repetitive precipitation from
isopropanol by addition of hexane. They were of amorphous state.
Yields of Boc-protected peptides were: 71% (peptide no. 2), 76%
the chain adopt a more ordered conformation. Comparison of CD
spectra obtained for chloroform solutions of two structural isomers
(Fig. 2b) shows that the peptide containing Z-isomers of phenylal-
anine adopts a more ordered conformation. This result is surprising
in the sense of our former studies [20]. However, during the stor-
age of this class of peptides containing
D
EPhe, we observed ten-
(peptide no. 4), 83% (peptide no. 5) and 74% (Boc-Gly-Gly-
D
Ala-
dency for them to isomerise to Z-isomer, which could influence
the obtained results. Due to low solubility in CHCl3 it was not pos-
sible to measure the CD spectra of the rest of the investigated
peptides.
R-Gly-Gly-X-Gly-X-Phe-pNA
2.4. NMR spectroscopy
Peptide
R-
-X-
1
2
3
4
5
H
ΔAla
In order to find some evidence of the presence of intramolecular
hydrogen bonds in the studied peptides, we have performed mea-
surements of the influence of temperature on the chemical shifts of
their amide protons. The experiments were done in DMSO contin-
uously increasing temperature. The obtained results are presented
in Table 1. The main parameter indicating the presence of hydro-
gen bond is the value of dd/dT [ppm/K]. It is well established that
the existence of hydrogen bond is reflected in the value of this
coefficient being lower than 0.004 [ppm/K]. Results obtained from
Boc
ZPhe
Δ
Δ
H
ZPhe
Boc
Boc
ΔEPhe
Phe
Fig. 1. Investigated peptides.
a
b
20
20
10
0
10
0
-10
-20
-10
-20
240
270
300
330
360
390
240
270
300
330
360
390
Wavelength [nm]
Wavelength [nm]
Fig. 2. CD spectra obtained for the investigated peptides (a) in methanol – peptide no. 1 (blue), peptide no. 2 (red), peptide no. 3 (green), peptide no. 4 (olive), peptide no. 5
(magenta), (b) in chloroform – peptide no. 2 (red), peptide no. 4 (olive).

448
R. Latajka et al. / Journal of Molecular Structure 892 (2008) 446–451
Table 1
Temperature dependence of chemical shifts of amide protons of the investigated peptides in DMSO solution (no. 2a – CDCl3 solution) – temperature coefficient dd/dT (ppm Kꢀ1
)
Peptide
Gly[1]
Gly[2]
D
Phe[3]/
D
Ala[3]
Gly[4]
D
Phe[5]/
D
Ala[5]
Phe[6]
0.0055
HN pNA
a
No. 1
No. 2
No. 2a
No. 3
No. 4
0.0021
0.0043
0.0050
0.0068
0.0045
0.0004
0.0051
0.0051
0.0047
0.0055
0.0046
0.0066
0.0069
0.0055
0.0067
0.0058
0.0063
0.0032
0.0029
0.0030
0.0028
0.0067
0.0007
0.0036
0.0052
0.0060
0.0047
a
a
0.0021
0.0069
0.0047
0.082
a
Overlapping of the signals.
the experiments (see Table 1) showed that in every investigated
peptide, conformation could be stabilised by intramolecular hydro-
gen bonding. Except in peptide no. 1 (H2N-Gly-Gly-
DAla-Gly-
D
Ala-Phe-pNa), in all other cases amide proton of p-nitroanilide
moiety could be involved in such an interaction. This tendency
was observed also in the case of change of solvent from DMSO to
CDCl3 (see results for peptide 2a in Table 1). Contrary to hexapep-
tides containing two dehydroamino acids isolated by two satu-
rated residues [22], the influence of free N-termini on the ability
to create hydrogen bonding is not observed.
The most informative data about conformational preferences of
the investigated peptides were obtained from NOESY and ROESY
experiments. Based on the distance constraints and contacts ob-
tained from these spectra optimization of 50 of the most stable
conformers was calculated by using X-PLOR [24] programme (Figs.
3–8). The average values obtained for dihedral angles are pre-
sented in Table 2.
Fig. 5. Superposition of the most stable conformations of peptide no. 3 (H2N-Gly-
Gly-
ZPhe-Gly- ZPhe-Phe-pNA) proposed based on X-PLOR calculation.
D
D
ble conformations (of lower and higher energy) in solution. In con-
trast to their saturated analogue (peptide no. 5 – see Fig. 7), they
adopt ordered, stable conformation in solution. In the case of pep-
The results of spectroscopic studies and calculations suggest
that all the investigated dehydropeptides could exist in two possi-
tide no. 1 (H2N-Gly-Gly-DAla-Gly-DAla-Phe-pNA) values of the
dihedral angles (Table 2) are typical for 310-helical conformation,
which is in good agreement with literature data [11,13]. For the
rest of the studied compounds, obtained values of dihedral angles
are not similar. Comparison of two analogous peptides no. 2 and
no. 4, where the only difference in the structure is the type of dehy-
drophenylalanine isomer, confirmed the strong influence of this
factor on conformational preferences of main chain – peptides
adopt different conformations with totally different values of dihe-
dral angles. What is also interesting is that peptide no. 4 (Boc-Gly-
Gly-
cal conformations, where the only differences are observed for
the values of u [4], [4], u [5] and [5].
D D
EPhe-Gly- EPhe-Phe-pNA) could exist in two almost identi-
W
w
A very interesting result has been obtained after the comparison
of conformational preferences of peptide no. 2 (Boc-Gly-Gly-
D
Fig. 8). The conformations of the main chains are similar in both
the cases (Fig. 8c), the difference could be observed for the location
of phenyl rings – in the case of a non-polar solvent they create
dome kind of shield over the more polar parts of the peptide
(Fig. 8b).
ZPhe-Gly- ZPhe-Phe-pNA) in DMSO and CDCl3 solutions (see
D
Fig. 3. Superposition of the most stable conformation of peptide no. 1 (H2N-Gly-
Gly-DAla-Gly-DAla-Phe-pNA) proposed based on X-PLOR calculation.
3. Conclusions
All the investigated peptides adopted bent conformation,
which is stabilized by intramolecular hydrogen bonding, and they
could exist as two different conformers in solution. Only in the
case of peptide containing two
DAla residues expected 310-helical
conformation was found, whereas for the rest of the dehydropep-
tides various bent conformations were observed. In contrast to
the previously studied hexapeptides, containing dehydroamino
acids separated by two saturated residues, in this group of com-
pounds the influence of free N-termini on the conformation of
peptide was not significant.
Fig. 4. Superposition of the most stable conformation of peptide no. 2 (Boc-Gly-
Gly-
ZPhe-Gly- ZPhe-Phe-pNA) proposed based on X-PLOR calculation.
D
D

R. Latajka et al. / Journal of Molecular Structure 892 (2008) 446–451
449
Fig. 6. The most stable conformations obtained for peptide no. 4 (Boc-Gly-Gly-D D
EPhe-Gly- EPhe-Phe-pNA), conformers 4a (left) and 4b (right) proposed based on X-PLOR
calculation.
wise stated. Column chromatography was performed on silica gel
H60 (70–230 mesh).
4.2. NMR spectroscopy
NMR spectra were recorded on Bruker Avance DRX300, Bru-
ker AMX600 and Bruker AMX400 instruments in deuterated
DMSO, chemical shifts are given in relation to SiMe4. In all
the cases 15 mM peptide solutions were prepared. NMR spectral
signal assignment and integration were carried out with SPAR-
KY [25]. Where it was possible, the separation between two
geminal protons in –CH2– group was used as a reference in dis-
tance calculations, in the other cases to calculate distance were
taken from the intensity of the cross and diagonal peaks by
using the correction factor [26]. Conformational calculations
were carried out with X-PLOR [24]. In view of the investigated
peptides containing unnatural aminoacid residues, it was neces-
sary to modify the topology file available in distribution. To
Fig. 7. The most stable conformations obtained for peptide no. 5 (Boc-Gly-Gly-Phe-
Gly-Phe-Phe-pNA) proposed based on X-PLOR calculation.
4. Experimental
4.1. General
build the required topology for D D
ZPhe and EPhe, the available
topology for Phe was used. In addition, a big force constant
warranting planarity of the dehydroaminoacid residues was
added.
Materials were obtained from commercial suppliers (Sigma–Al-
drich, Fluka, Merck) and used without purification unless other-
Fig. 8. Dependence of solvent on the conformation of Boc-Gly-Gly-
D
ZPhe-Gly-
D
ZPhe-Phe-pNA. (a) DMSO solution, (b) CDCl3 solution, (c) superposition of conformations in
both solvents.

450
R. Latajka et al. / Journal of Molecular Structure 892 (2008) 446–451
Table 2
Average values of dihedral angle [°] obtained on basis of X-PLOR calculations (2a0 and 2b0–conformers observed in CDCl3 solution)
Pep
u[1]
w
[1]
u[2]
w
[2]
u[3]
w
[3]
U[4]
w
[4]
u[5]
w
[5]
u[6]
w[6]
1a
1b
2a
2b
2a’
2b’
3a
3b
4a
4b
5
–
–
155.39
64.51
89.40
162.41
ꢀ135.6
76.05
ꢀ86.80
ꢀ125.7
121.5
ꢀ95.47
100.34
ꢀ102.0
125.30
ꢀ60.2
44.9
ꢀ57.47
55.49
67.35
ꢀ72.88
7.3
ꢀ20.33
24.05
93.21
ꢀ94.77
ꢀ44.1
42.8
47.46
ꢀ46.83
77.31
81.54
158.42
67.51
ꢀ63.03
74.89
43.30
ꢀ72.50
58.38
32.42
ꢀ2.31
39.1
ꢀ33.7
11.18
ꢀ74.84
82.47
ꢀ88.73
36.82
53.07
ꢀ6.11
34.07
110.45
53.1
ꢀ70.4
87.69
ꢀ99.51
ꢀ26.18
33.35
77.55
54.33
61.24
93.92
99.47
91.03
89.33
54.4
ꢀ60.98
ꢀ75.56
73.14
105.12
102.15
162.9
ꢀ133.7
–
ꢀ91.37
ꢀ84.95
ꢀ100.9
ꢀ37.3
ꢀ112.4
ꢀ53.6
44.1
165.85
ꢀ172.87
ꢀ95.36
ꢀ98.13
ꢀ4.55
ꢀ20.86
66.4
30.3
23.8
ꢀ61.1
ꢀ75.23
70.73
ꢀ43.80
ꢀ28.40
42.50
ꢀ36.9
14.76
ꢀ82.80
113.48
ꢀ48.75
ꢀ12.52
ꢀ9.5
ꢀ52.97
87.39
ꢀ78.13
76.83
99.09
93.10
61.51
ꢀ167.03
68.28
93.97
86.13
ꢀ40.73
ꢀ43.91
95.51
–
89.70
ꢀ83.78
51.71
51.20
7.27
81.35
79.63
ꢀ36.28
72.86
ꢀ68.27
ꢀ113.20
136.36
74.21
20.11
4.2. CD spectroscopy
4.5. Peptide no. 3
1H NMR (DMSO, TMS): 3.15 ppm (HbPhe[6]); 3.61 ppm
CD spectra were recorded on Jasco J-715 spectropolarimeter,
at room temperature. Spectra were measured in MeOH and
CHCl3. Pathlength of 1 mm was used. Concentrations of the
solutions were ꢁ0.3 mg/mL. Each spectrum represents the
average of at least four scans. The data are presented as molar
ellipticity [h].
a
a
a
(H Gly[1]); 3.99 ppm (H Gly[4]); 4.05 ppm (H Gly[2]); 4.73 ppm
a
D
D
(H Phe[6]); 6.97 ppm (Hb ZPhe[5]); 7.16 ppm (Hb ZPhe[3]);
7.24–7.43 ppm (aromatic ring); 7.99 ppm (Hd#pNA); 8.07 ppm
(HNGly[1]); 8.22 ppm (H #pNA); 8.41 ppm (HNPhe[6]), 8.81 ppm
e
(HNGly[4]); 8.72 ppm (HNGly[2]); 9.80 ppm (HN
D
ZPhe[3]);
10.02 ppm (HN
D
ZPhe[5]); 10.08 ppm (HNpNA) 13C NMR (DMSO,
a
4.3. Peptide no. 1
TMS): 36.3 ppm (CbPhe[6]); 40.1 ppm (C Gly[1]); 42.3 ppm
a
a
a
(C Gly[2]);
43.4 ppm
(C Gly[4]);
55.9 ppm
(C Phe[6]);
1H NMR (DMSO, TMS): 2.97 ppm (HB1, Phe[6]); 3.12 ppm (HB2,
Phe[6]); 3.63 ppm (HA, Gly[1]); 3.84 ppm (HA, Gly[4]); 3.88 ppm
(HA, Gly[2]); 4.69 ppm (HA, Phe[6]); 7.19–726 ppm (aromatic
119.1 ppm
(Cd#pNA);
124.8 ppm
(C #pNA);
128.2 ppm
e
D
(Cb ZPhe[5]); 128.5–144.6 ppm (aromatic ring); 128.8 ppm
D
aD
(Cb ZPhe[3]); 133.2 ppm, 133.3 ppm (C ZPhe[3],
D
ZPhe[5]);
Phe[6]+HN
D
Ala[3]+HB#
D
Ala[3]); 7.76 ppm (HB#,
D
Ala[5]);
164.7 ppm (CO
D
ZPhe[5]); 165.9 ppm (CO ZPhe[3]); 166.4 ppm
D
7.89 ppm (HD#pNA); 8.10 ppm (HNGly[1]); 8.22 ppm (HE#pNA);
(COGly[1]); 168.6 ppm (COGly[2]); 170.5 ppm (COGly[4]);
170.7 ppm (COPhe[6]).
8.38 ppm
(HNGly[4]);
8.46 ppm
D
(HN,Phe[6]);
8.71 ppm
(HNGly[2]); 10.72 ppm (HN
Ala[5]); 10.75 ppm (HN,pNA); 13C
NMR (DMSO, TMS): 37.1 ppm (CbPhe[6]); 40.1 ppm (C Gly[1]);
4.6. Peptide no. 4
a
a
a
a
41.9 ppm (C Gly[2]); 42.1 ppm (C Gly[4]); 55.2 ppm (C Phe[6]);
119.0 ppm (Cd#pNA); 119.1 ppm (Cb Ala[3],
D
Ala[5]); 124.8 ppm
1H NMR (DMSO, TMS): 1.36 ppm (CH3 Boc); 2.91 ppm
D
e
a
(C #pNA); 126.6–137.1 ppm (aromatic ring, Phe[6]); 142.8 ppm
(Hb2Phe[6]); 3.14 ppm (HbPhe[6]); 3.59 ppm (H Gly[1]);
(CfpNA); 144.7 ppm (C pNA); 166.6 ppm (COGly[1]); 168.9 ppm
3.95 ppm (H Gly[4]); 3.97 ppm (H Gly[2]); 4.76 ppm (H Phe[6]);
c
a
a
a
D
D
(CO Gly[2]); 170.6 ppm (COPhe[6]). Elemental analysis for
C27H30N8O8 calculated: 54.54% C, 5.08% H and 15.08% N, found:
52.25% C, 4.31% H and 13.10% N.
6.54 ppm (Hb EPhe[5]); 6.58 ppm (Hb EPhe[3]); 6.99–7.45 ppm
(aromatic ring); 7.03 ppm (HNGly[1]); 7.98 ppm (Hd#pNA);
e
8.19 ppm (H #pNA); 8.21 ppm (HNGly[2]); 8.50 ppm (HNGly[4]);
8.75 ppm (HNPhe[6]); 9.79 ppm (HN
(HN
EPhe[5]); 10.17 ppm (HNpNA), 13C NMR (DMSO, TMS):
28.1 ppm (CH3 Boc); 36.4 ppm (CbPhe[6]); 42.5 ppm (C Gly[4]);
D
EPhe[3]); 9.94 ppm
4.4. Peptide no. 2
D
a
a
a
a
Obtained in % yield by crystallization from ethyl acetate–hex-
42.6 ppm (C Gly[2]); 43.2 ppm (C Gly[1]); 55.2 ppm (C Phe[6]);
ane mixture. Mp 197–200 °C. 1H NMR (DMSO, TMS): 1.37 ppm
78.2 ppm (C Boc); 118.21 ppm (Cb EPhe[3]); 118.22 ppm
c
D
a
D
e
(CH3Boc); 3.14 ppm (Hb1Phe[6]); 3.59 ppm (H #Gly[1]);
(Cb EPhe[5]); 119.2 ppm (Cd#pNA); 124.8 ppm (C #pNA); 128.4–
a
a
a
aD
3.92 ppm (H #Gly[2]); 4.02 ppm (H #Gly[4]); 4.72 ppm (H P-
144.7 ppm (aromatic ring); 131.2 ppm, 131.4 ppm (C EPhe[3],
EPhe[5]); 155.9 ppm (CO Boc); 164.5 ppm (CO EPhe[3]);
164.8 ppm (CO
EPhe[5]); 165.3 ppm (COPhe[6]); 168.4 ppm
he[6]); 6.99 ppm (Hb ZPhe[5]); 7.04 ppm (HNGly[1]); 7.14 ppm
D
D
D
(Hb ZPhe[3]); 7.19–7.62 ppm (aromatic ring); 7.99 ppm (Hd#pNA);
D
D
8.13 ppm (HNGly[2]); 8.22 ppm (H #pNA); 8.38 ppm (HNPhe[6]);
(COGly[4]); 170.1 ppm (COGly[2]); 170.4 ppm (COGly[1]); Elemen-
tal analysis for C44H46N8O10 calculated: 62.4% C, 5.47% H and
13.23% N, found: 59.72% C, 5.39% H and 12.79% N.
e
8.72 ppm (HNGly[4]); 9.66 ppm (HN
(HN
ZPhe[5]); 10.06 ppm (HNpNA), 13C NMR (DMSO, TMS):
28.0 ppm (CH3 Boc); 36.3 ppm (CbPhe[6]); 42.4 ppm (C Gly[2]);
D
ZPhe[3]); 9.97 ppm
D
a
a
a
c
43.2 ppm (C Gly[1]); 55.9 ppm (C Phe[6]); 78.0 ppm (C Boc),
4.7. Peptide no. 5
e
119.1 ppm
(Cd#pNA);
124.8 ppm
(C #pNA);
128.4 ppm
(Cb ZPhe[5]); 128.2–137.7 ppm (aromatic ring); 128.8 ppm
1H NMR (DMSO, TMS): 1.37 ppm (CH3 Boc); 2.75 ppm
(Hb1Phe[3]); 2.77 ppm (Hb#Phe[5]); 3.01 ppm (Hb1Phe[6]);
D
(Cb ZPhe[3]); 133.2 ppm (C Phe[3]); 133.4 ppm (C ZPhe[5]);
D
a
D
aD
142.3 ppm (CfpNA); 144.5 ppm (C pNA); 155.7 ppm (COBoc);
3.02 ppm
(Hb2Phe[3]);
3.14 ppm
(Hb2Phe[6]);
3.55 ppm
c
a
a
a
164.4 ppm (C)O
D
ZPhe[5]); 165.9 ppm (CO
D
ZPhe[3]); 169.0 ppm
(H Gly[1]); 3.74 ppm (H Gly[2]); 3.77 ppm (H Gly[4]); 4.50 ppm
a
a
a
(COGly[2]); 170.1 ppm (COGly[1]); 170.2 ppm (COGly[4]);
170.5 ppm (CPhe[6]). Elemental analysis for C44H46N8O10 calcu-
lated: 62.4% C, 5.47% H and 13.23% N, found: 60.50% C, 5.10% H
and 12.73% N.
(H Phe[3]); 4.55 ppm (H Phe[5]); 4.70 ppm (H Phe[6]); 7.02 ppm
(HNGly[1]); 7.1–8.26 ppm (aromatic ring); 7.89 ppm (Hd#pNA);
8.24 ppm (H #pNA); 7.92 ppm (HNGly[2]); 8.12 ppm (HNPhe[6]);
e
8.19 ppm (HNGly[2]); 8.32 ppm (HNGly[4]); 8.61 ppm (HNPhe[6]);

R. Latajka et al. / Journal of Molecular Structure 892 (2008) 446–451
451
10.76 ppm (HNpNA), 13C NMR (DMSO, TMS): 28.1 ppm (CH3 Boc);
37.3 ppm (CbPhe[6]); 37.45 ppm (CbPhe[5]); 37.48 ppm (CbPhe[3]);
[7] O. Pieroni, A. Fissi, C. Pratesi, P. Temussi, F. Ciardelli, Biopolymers 33 (1993) 1.
[8] M.R. Ciajolo, A. Tuzi, C.R. Pratesi, A. Fissi, O. Pieroni, Biopolymers 32 (1992)
717.
[9] M. R Ciajolo, A. Tuzi, C.R. Pratesi, A. Fissi, O. Pieroni, Int. J. Peptide Protein Res.
38 (1999) 539.
[10] R. Jain, M. Singh, V.S. Chauhan, Tetrahedron 50 (1994) 907.
[11] A. Tuzi, M.R. Ciajolo, D. Pictone, O. Crescenzi, P.A. Temussi, A. Fissi, O. Pieroni, J.
Pept. Sci. 2 (1996) 47.
[12] K.R. Rajashankar, S. Ramakumar, M.R. Jain, V.S. Chauhan, Biopolymers 42
(1998) 373.
[13] P. Mathyr, U.A. Ramagopal, S. Ramakumar, N.R. Jagannathan, V.S. Chauhan,
Biopolymers 84 (2006) 298.
[14] Y. Inai, S. Kurashima, T. Hirabayashi, K. Yokota, Biopolymers 53 (2000) 482.
[15] B. Turk, I. Dolenc, V. Turk, in: A.J. Barrett (Ed.), Handbook of Proteolytic
Enzymes, Academic Press, 1998, pp. 631–634.
[16] R. Butler, A. Michel, W. Kunz, M.O. Klinkert, Protein Pept. Lett. 2 (1995) 313.
[17] C. Toomes, J. James, A.J. Wood, C.L. Wu, D. McCormick, N. Lench, C. Hewitt, L.
Moynihan, E. Roberts, C.G. Woods, A. Markham, M. Wong, R. Widmer, K.A.
Ghaffa, M. Pemberton, I.R. Hussein, S.A. Temtamy, R. Davies, A.P. Sloan, M.J.
Dixon, N.S. Thakkern, Nat. Genet. 23 (1999) 421.
[18] A.O. Breitholle, C.H. Stammer, Tetrahedron Lett. 28 (1975) 2381.
[19] M. Makowski, M. Pawełczak, R. Latajka, K. Nowak, P. Kafarski, J. Peptide Sci. 7
(2001) 141.
a
a
a
41.80 ppm (C Gly[4]); 41.81 ppm (C Gly[2]); 43.1 ppm (C Gly[1]);
a
a
53 93 ppm (C Phe[3]); 53 94 ppm (C Phe[5]); 55.3 ppm
(C Phe[6]); 78.1 ppm (C Boc); 118.8 ppm (Cd#pNA); 124.7 ppm
a
c
(C #pNA); 126.3–129.2 ppm (aromatic ring); 137.2–137.9 ppm
e
(aromatic ring);; 142.2 ppm (CfpNA); 144.8 ppm (C pNA);
c
155.7 ppm (COBoc); 168.3 ppm (COGly[4]); 168.4 ppm (COGly[2]);
169.6 ppm (COGly[1]); 170.9 ppm (COPhe[5]); 170.8 ppm,
171.0 ppm (COPhe[3], COPhe[6]).
Acknowledgements
The molecular modeling was carried out using hardware and
software resources of The Supercomputing and Networking Centre
in Wroclaw. This project was financed by Ministry of Science and
Higher Education, Grant No. 2 P04B 02029. Michal Jewginski is a
recipient of Ph.D. fellowship from the project founded by the Euro-
pean Social Found and the government of Poland.
[20] R. Latajka, M. Makowski, M. Jewginski, M. Pawelczak, H. Koroniak, P. Kafarski,
New J.Chem. 30 (2006) 1009.
[21] R. Latajka, M. Jewginski, M. Makowski, M. Pawelczak, T. Huber, N. Sewald, P.
Kafarski, J. Peptide Sci., 2008, DOI: 10.1002/psc1054.
[22] R. Latajka, M. Jewginski, M. Makowski, A. Krezel, S. Paluch, Biopolymers 89
(2008) 691.
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