Peptide p-nitrophenylanilides containing (E)-dehydrophenylalanine - Synthesis, structural studies and evaluation of their activity towards cathepsin C

Article abstract of DOI:10.1039/b601634k

Tetrapeptide p-nitroanilides containing (E)-dehydrophenylalanine were synthesized and evaluated as inhibitors and substrates of cathepsin C. Peptides containing a free, unblocked amino group appeared to be quite good substrates of the enzyme, whereas full

Full text of DOI:10.1039/b601634k

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PAPER
www.rsc.org/njc | New Journal of Chemistry
Peptide p-nitrophenylanilides containing (E)-dehydro-
phenylalanine—synthesis, structural studies and evaluation of their
activity towards cathepsin Cw
a
b
a
b
c
´
R. Latajka,* M. Makowski, M. Jewginski, M. Pawe$czak, H. Koroniak and
P. Kafarskiab
Received (in Montpellier, France) 1st February 2006, Accepted 8th May 2006
First published as an Advance Article on the web 5th June 2006
DOI: 10.1039/b601634k
Tetrapeptide p-nitroanilides containing (E)-dehydrophenylalanine were synthesized and evaluated
as inhibitors and substrates of cathepsin C. Peptides containing a free, unblocked amino group
appeared to be quite good substrates of the enzyme, whereas fully protected peptides acted as
very weak inhibitors. Structural studies by means of NMR and CD, alongside with molecular
modelling, have proved that these peptides are hydrolysed in one step by direct removal of
p-nitroaniline from the tetrapeptide.
3.4.14.1) belongs to the papain family of proteases8 and sequen-
Introduction
tially removes dipeptides 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 basic amino acids (Arg or Lys)
at the N-terminal position or Pro on either side of the scissile
bond. It is also quite unusual in that it requires 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 in cytotoxic T lymphocytes and
natural killer cells (granzymes A and B), mast cells (tryptase and
chymase), and neutrophils (cathepsin G and elastase) by remov-
ing their N-terminal activation dipeptides.9,10 Loss of function
mutations in the cathepsin C gene result in periodontal disease
and palmoplantar keratosis.11
Dehydroamino acids contribute in a catalytic role in the active
sites of some yeast and bacterial enzymes,1 as well as occuring
in a variety of peptide antibiotics of bacterial origin, including
the lantibiotics2 (nisin, subtilin, epidermin, gallidermin) and
more highly modified peptides.
Dehydroamino acid residues in peptides have been found to
influence the main-chain and side-chain dramatically, due to
the presence of the CaQCb double bond.3 For example,
dehydroalanine adopts a roughly planar conformation with
trans orientation for the c and j torsions and induces an
inverse g-turn in the preceding residue.1 (Z)-Dehydrophenyl-
alanine exerts a b-turn conformation in short peptides4 and
310-helical conformation in the case of peptides with longer
main-chain.5–7 This suggests that dehydroamino acid residues
exert a powerful 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 relationships and to provide
new analogues of enhanced activity.
Since dehydroamino acids are quite reactive and various
thiol nucleophiles are known to add to their double bonds9,12
we speculated that short dehydropeptide mimetics of artificial
substrates of cathepsin C might act as alkylating inhibitors of
the enzyme. Quite surprisingly, however, tri- and tetrapeptides
containing DAla and DZPhe residues acted only as substrates
of cathepsin C with activity comparable to their classic
counterparts.13 The results of structural and conformational
investigations have shown that in majority of these peptides
we have observed conformations similar to these found for
model peptides.14
Cathepsins form quite a large family of lysosomal proteases
involved in many physiological functions in the human body.
Elevated activity of these enzymes in serum or the extracellular
matrix often signifies a number of gross pathological conditions.
Cathepsin-mediated diseases include: Alzheimer’s disease, nu-
merous types of cancer, autoimmune related diseases such as
arthritis and the accelerated breakdown of bone structure seen
with osteoporosis. Cathepsin C (dipeptidyl dipeptidase I; EC
In order to understand better the observed phenomenon we
have synthesized a set of analogous peptides (Fig. 1) contain-
ing DEPhe in place of DZPhe and determined their conforma-
tions in solution, evaluated their influence on cathepsin C and
modelled their interactions with the enzyme by means of
quantum chemistry methods.
a Department of Bioorganic Chemistry, Faculty of Chemistry,
Wroc!aw University of Technology, Wybrze(e, Wyspianskiego 27,
50-370 Wroclaw, Poland. E-mail: rafal.latajka@pwr.wroc.pl; Fax:
0048713284064; Tel: 0048713203344
b Institute of Chemistry, University of Opole, Oleska 48, 45-052
Opole, Poland
Results and discussion
c Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6,
Chemistry
´
60-780 Poznan, Poland
The synthesis of dehydropeptide mimetics containing DEPhe is
not trivial and is still challenging. According to the literature,
w Electronic supplementary information (ESI) available: CD spectra.
See DOI: 10.1039/b601634k
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Scheme 1 Synthesis of investigated peptides.
shown in Fig. 1 with those exhibited by their counterparts
containing either DZPhe or Phe in the place of DEPhe.
CD spectroscopy. CD measurements were carried out in two
solvents (methanol and acetonitrile) for the region down to
200 nm because at shorter wavelengths there are overlapping
contributions of the peptide, aromatic and unsaturated chro-
mophores which makes the analysis very complex. In the near-
UV region Cotton effects associated with the (E)-dehydrophe-
nylalanine and p-nitroanilide groups were observed. They are
strongly dependent on the peptide conformation and therefore
very useful in this type of conformational studies. The ob-
tained CD spectra are presented in Fig. 2. In the case of
methanolic solutions of Boc-Gly-DEPhe-Phe-pNA and Gly-
DEPhe-Gly-Phe-pNA a very strong and large positive band at
280 nm and negative band at 309 nm were observed with
spectra of both compounds being very similar. This suggests
that they adopt very similar and quite rigid conformations in
methanol. Quite oppositely, in the case of Gly-Gly-DEPhe-
Phe-pNA CD bands of very low intensity were observed,
which suggests that conformation of this peptide in methanol
is completely unordered and that (E)-dehydrophenylalanine
most likely exerts no influence on peptide chain conformation.
When using acetonitrile as a solvent quite similar spectra have
been obtained for all three studied compounds. In every case a
strong positive band in the range 280–290 nm and negative
band at 310 nm were observed, which suggests that peptides
adopt ordered conformations in this solvent.
Fig. 1 Structures of investigated peptides.
the only way of their synthesis relies on photoisomerization of
DZPhe to the desirable (E)-isomer.8 The strategy applied in
this work is briefly illustrated in Scheme 1. Dehydropeptides
were obtained by a standard solution method using a mixed
anhydride procedure. The starting substrate, fully protected
dehydrotripeptide-Boc-Gly-DEPhe-Gly-OMe, was obtained
according to the literature as a mixture containing nearly
equivalent quantities of both DZPhe and DEPhe isomers.15
Successful resolution of these isomeric dehydrodipeptides by
column chromatography followed by deprotection of either
N- or C-termini under mild conditions enabled to elongate the
peptide chain and finally to obtain the desired dehydrotetra-
peptides shown in Fig. 1. It is of note that DEPhe is far less
thermodynamically stable than its (Z) counterpart and it has a
tendency to isomerise under the reaction conditions. Thus
upon the condensation step leading to Boc-Gly-DEPhe-Phe-
pNA the product was contaminated with Boc-Gly-DZPhe-Phe-
pNA. Resolution of these peptide isomers was achieved by
column chromatography and fractional crystallization.
NMR spectroscopy. In order to find some evidence of the
presence of intramolecular hydrogen bonds in the studied
peptides we have performed measurements of the dependence
of chemical shifts of their amide protons on solvent polarity.
The experiments have been made in chloroform and in mix-
tures of CDCl3–DMSO of various concentration. The ob-
tained results are presented in Tables 1 and 2.
Generally, if an amide proton is involved in creation of
hydrogen bonding its chemical shift is independent of the
polarity of the solvent and would remain at the same position
invarient of the concentration of DMSO in chloroform. The
obtained results suggest that in this group of peptides there are
no observed strong hydrogen bonds—in all cases chemical
shifts of amide protons increase with the increase of the
solvent polarity. However, the problems with solubility of
the studied compounds and observed overlapping of amide
Structural and conformational studies
NMR and CD studies were undertaken in order to compare
structures and conformational preferences of dehydropeptides
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Fig. 2 CD spectra of peptides obtained in (a) methanol and (b) acetonitrile solution.
signals with signals derived from aromatic protons made this
experiment not easy to interpret and therefore of limited
usefulness.
creation of hydrogen bonding. The main parameter indicating
the presence of a hydrogen bond is the value of dd/dT [ppm
K
À1]. It is well established that the existence of a hydrogen
It is well known that an increase of temperature results in
changing values of chemical shifts of amide protons. The lack
of such a change suggests that the amide proton is involved in
bond is reflected in the value of this coefficient being lower
than 0.004 ppm KÀ1. Observed chemical shifts of amide
protons vs. temperature recorded in DMSO solutions are
shown in Table 3.
Table 1 Dependence of solvent polarity on amide proton chemical
shifts for Gly-DEPhe-Gly-Phe-pNA
Table 2 Dependence of solvent polarity on amide protons chemical
shifts for Boc-Gly-DEPhe-Gly-Phe-pNA
d (ppm)
% DMSO in
CDCl3
d (ppm)
NH pNA
NH DEPhe
NH Gly[3]
NH Gly[1]
% DMSO
in CDCl3 NH DEPhe NH pNA NH Gly[3] NH Phe NH Gly[1]
CDCl3
5
—
—
—
10.50
10.49
10.64
10.75
10.75
—
—
—
9.95
9.97
9.96
9.94
10.30
—
—
—
—
—
—
—
CDCl3
5
10
15
20
30
40
50
9.02
9.83
9.99
10.09
10.16
10.22
10.23
10.22
8.66
9.31
9.41
9.47
9.54
9.68
9.76
9.85
—
—
—
—
—
10
15
20
30
40
50
a
5.68
5.97
6.18
6.37
6.63
6.78
6.87
8.13
8.42
8.39
8.61
8.74
8.84
8.89
8.90
8.27
8.32
8.34
8.36
8.36
8.39
7.79
—
a
a
—
—
a
8.59
8.59
a
—
Overlapping with signals of aromatic protons.
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Based on the data presented in Table 3 it might be assumed
that in solutions of Gly-DEPhe-Gly-Phe-pNA the only hydro-
gen bonding is formed by N-terminal glycine, because in the
case of NHGly(1) the observed dd/dT was equal to 0.002 ppm
Table 3 Temperature dependence on chemical shifts of amide
protons in Gly-DEPhe-Gly-Phe-pNA
dd/dT (ppm KÀ1
G-DEF-G-F-pNA G-G-DEF-F-pNA Boc-G-DEF-F-pNA
)
K
À1 and thus it is lower than the indicative value of 0.004. Far
more interesting results were obtained for Gly-Gly-DEPhe-
Phe-pNA where recorded values of dd/dT clearly indicate
involvement of two amide moieties in hydrogen bonding.
These are amide groups derived from: p-nitroanilide (dd/dT =
0.0035) and N-terminal glycine (dd/dT = 0.0018 ppm KÀ1).
These results were compared with those obtained for the
substrate of both tetrapeptides, namely Boc-Gly-DEPhe-Phe-
pNA tripeptide. In this case only one amide group, derived
from the nitroanilide part of the molecule, is involved in
hydrogen bond formation (for NHpNA the observed dd/dT
was 0.0036 ppm KÀ1). The results, although limited to only
NH pNA 0.0057
NH DEPhe 0.0052
0.0035
0.0071
0.0104
0.0018
0.0050
0.0036
0.0101
0.0122
0.0114
—
NH Phe
NH Gly(1) 0.0020
NH Gly 0.0078
0.0080
three examples, enable to notice that elongation of the peptide
chain towards N-termini resulted in a more ordered structure
of the molecule. Anyway, comparison of the abilities of
hydrogen bond formation by dehydrotetrapeptides containing
DEPhe, DZPhe and Phe have shown, that the peptides contain-
Fig. 3 Two-dimensional ROESY spectra of Boc-Gly-DEPhe-Gly-Phe-pNA in DMSO.
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ing the (Z) isomer of dehydrophenylalanine more closely
resemble those containing phenylalanine than do peptides
of DEPhe.
Table 4 Substrate activity of dehydrotetrapeptides towards cathe-
psin C
Michaelis constant,
KM/mM
Vmax/mmol
minÀ1
The most useful information about conformational prefer-
ences of investigated peptides had been obtained from
NOESY and ROESY experiments. Two-dimensional spectra
of Boc-Gly-DEPhe-Gly-Phe-pNA, for which the most interest-
ing results were obtained, are shown in Fig. 3. Analysis of
nondiagonal signals recorded in DMSO allowed the following
contacts to be detected: (a) NH DEPhe 2 CbH DEPhe, (b) NH
Gly[3] 2 NH Phe, (c) NH pNA 2 NH Phe, (d) NH pNA 2
CaH Phe, (e) NH Phe 2 CaH Phe, (f) CH3 Boc 2 CbH2 Phe.
Based on these data (especially the contact between CH3 Boc
2 CbH2 which suggests turned conformation of peptide
chain) the conformation of this dehydropeptide was proposed
(Fig. 4). The most significant, from a conformational point of
view, are contacts between amide protons of neighbouring
amino acid residues (b and c), which clearly indicate the
existence of a turned conformation. These results, reinforced
by molecular modeling, also confirm the possibility of forma-
tion of intramolecular hydrogen bonds between hydrogen of
p-nitroanilide and carboxylate oxygen of glycine(3), and thus
in some manner confirm the results from temperature studies.
Peptide
Gly-DEPhe-Gly-Phe-pNA
Gly-DZPhe-Gly-Phe-pNA
Gly-Phe-Gly-Phe-pNA
Gly-Gly-DEPhe-Phe-pNA
Gly-Gly-DZPhe-Phe-pNA
Gly-Gly-Phe-Phe-pNA
Gly-Phe-pNA
3.2
7.8
22.5
4.3
0.008
0.224
0.075
0.060
0.003
7.8
Insoluble in water
0.23
0.014
mined for DZPhe-containing dehydrotetrapeptides, as well as
those found for the model tetrapeptides. As expected, they are
an order of magnitude more weakly bound than the standard
substrate—p-nitroanilide of glycylphenylalanine. There is no
possibility to draw any meaningful relationship between the
structure of the peptide and the rate of its hydrolysis repre-
sented by the Vmax value (the higher the Vmax value, the better
is the substrate).
Generally, all the studied peptides were quite efficiently
hydrolysed by the enzyme, as measured by the release of
p-nitroaniline. One may consider that the hydrolysis of the
tetrapeptide is a sequential reaction involving release of the
dipeptidyl fragment from the N-terminus of the tetrapeptide
followed by hydrolysis of the formed dipeptidyl p-nitroanilide.
However, also a possibility exists that these peptides are
complexed with the enzyme in a non-typical manner and the
hydrolysis is simply a one-step process. In order to test these
hypotheses we have used molecular modelling to establish the
mode of binding of these peptides to the active site of
cathepsin C. There were no evidence of hydrolysis of the
amide bond of dehydrophenylalanine in aqueous solution.
Enzymatic studies
All four dehydrotetrapeptides were evaluated as substrates
and inhibitors of cathepsin C. Those which possess an un-
blocked N-terminal moiety (studied as trifluoroacetates) ex-
erted quite good substrate activity, whereas Boc-peptides
acted as very weak inhibitors of the enzyme causing around
5% inhibition of its activity. In Table 4, the substrate activities
of the studied peptides are compared with those determined
earlier13 for their counterparts containing DZPhe and Phe and
additionally with that obtained for the standard artificial
substrate of the enzyme—p-nitroanilide of glycylphenyl-
alanine.
Molecular modelling
The affinity of inhibitors to proteins is determined by the
intermolecular interactions in the ligand–receptor system. The
understanding of the physical nature of these interactions is
vital for understanding the molecular assembly of the enzyme–
inhibitor complex and for understanding the mode of inhibi-
tory action.
As seen from Table 4 affinities of the dehydropeptides
containing DEPhe towards cathepsin C are significantly higher
(which is represented by lower KM values) than those deter-
Optimal conformations of the inhibitory dehydrotetrapep-
tides were obtained by using Gaussian03 at the HF/6-31g (d,p)
level16 in the gas phase and they were docked to the active site
of cathepsin C available from Protein Data Bank EC
3.4.14.117 by using the AutoDock program.18,19 We chose
these structures for the docking process, because they were
in good agreement with the structures predicted from the
NMR experiment. Moreover, use of AutoDock to the docking
process required good assignment of charge, which we ob-
tained from the ab initio calculations with the
Merz–Singh–Kollman scheme.20 It was possible to use these
structures, because in the next step the complexes of dehy-
dropeptides with enzyme were optimized using Accelrys’s
DISCOVER program with the cff97 force field. After the
docking process, for each peptide we obtained a variety of
structures, where each showed a different binding mode. In the
next stage we selected the most probable docked structures,
Fig. 4 Schematic conformation of Boc-Gly-DEPhe-Gly-Phe-pNA in
DMSO solution.
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Fig. 6 Binding of Gly-Gly-DEPhe-Phe-pNA (top) and Gly-DEPhe-
Gly-Phe-pNA (bottom) in the active site of cathepsin C.
Fig. 5 Binding of Gly-Gly-DEPhe-Phe-pNA (top) and of Gly-DEPhe-
Gly-Phe-pNA (bottom) in the active site of cathepsin C with shown
intermolecular hydrogen bonds (white dashed lines).
geometry for reaction. This reasoning finds some confirmation
when the dependence of rate of p-nitroaniline formation vs.
time (Fig. 7), which exhibits some lag-phase and non-typical
course (all the other curves are linear), is analysed.
Molecular modelling also enabled to propose the origin of
the observed very low inhibitory activity of totally protected
dehydrotetrapeptides. As shown in Fig. 8 for a representative
example (Boc-Gly-DEPhe-Gly-Phe-pNA) these peptides ad-
here to the surface of the enzyme and form a ‘‘cap’’ covering
the entrance to its active site.
and from these, after optimisation, we chose those which had
the best ligand–enzyme interaction and thus the lowest inter-
action energies. As seen from Fig. 5 the docked peptides are
stabilised by intermolecular hydrogen bonds. The arrange-
ment of Gly-Gly-DEPhe-Phe-pNA (Fig. 5, top) is stabilised by
five intermolecular hydrogen bonds and the interaction energy
is equal to À115.8 kcal molÀ1, while the arrangement of Gly-
DEPhe-Gly-pNA (Fig. 5, bottom) is stabilised by only one
intermolecular hydrogen bond and the interaction energy is
equal to À84.17 kcal molÀ1. As is shown in Fig. 6 both
peptides are able to bind to the active site in such a manner
that allows direct hydrolysis of p-nitrophenylamide if consid-
ering both placement of the active site residues and proximity
of the hydrolysed amide bond to the thiol fragment of active
site cysteine 234. However, the binding modes differ substan-
tially between these two peptides. As seen from Fig. 6, Gly-
Gly-DEPhe-Phe-pNA is submerged into the binding cavity of
cathepsin much more deeply than is Gly-DEPhe-Gly-Phe-
pNA. Thus, although both compounds are bound with nearly
identical affinity the hydrolysis of Gly-Gly-DEPhe-Phe-pNA
requires its removal from this cavity in order to reach proper
Fig. 7 Dependence of concentration of p-nitrophenylaniline vs. time
upon hydrolysis of Gly-Gly-DEPhe-Phe-pNA by cathepsin C.
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Experimental
General
Materials were obtained from commercial suppliers (Sigma-
Aldrich, Fluka, Merck) and used without purification unless
otherwise stated. Column chromatography was performed on
silica gel H60 (70–230 mesh). NMR spectra were recorded on
Bruker Avance DRX300 and Bruker AMX600 instruments in
CDCl3, deuterated DMSO and CDCl3–DMSO mixtures and
chemical shifts are given relative to SiMe4. In all cases 15 mM
peptide solutions were prepared. CD spectra were recorded on
a Jasco J-715 spectropolarimeter at room temperature. Spectra
were measured in methanol and acetonitrile with concentra-
tions of the solutions being 0.15–0.25 mg mlÀ1; a pathlength of
1 mm was used.
Syntheses
Boc-Gly-DEPhe-Gly-OMe. This was obtained similarly as
described in the literature using the mixed carboxylic acid–
carbonic anhydride approach. Resolution of (Z) and (E)
isomers was achieved by column chromatography using silica
gel H60 (Merck) and solutions of ethyl acetate and benzene of
varying concentration (from 5 to 60% v/v of EtOAc) as eluent.
The desired product was precipitated from benzene–ethyl
actetate (4 : 1 v/v) with hexane and was obtained as a white
powder. Yield 34%; mp 133.5–135 1C. Isolated Boc-Gly-
DZPhe-Phe-OMe was purified by precipitation with hexane
from its ethyl acetate solution. Yield 38%, mp 168–170 1C.
C19H25N3O6 (391.41): calc.: C 58.39, H 6.44, N 10.73; found:
C 58.12, H 6.66, N 10.65%.
Fig. 8 Binding of Boc-Gly-DEPhe-Gly-Phe-pNA over the active site
of cathepsin C; the upper panel shows the intermolecular hydrogen
bonds.
Boc-Gly-DEPhe-Phe-pNA. This was obtained starting from
Boc-Gly-DPheEOH prepared according to the literature pro-
cedure.21 Thus, triethylamine (1.39 mL, 10.0 mmol) was added
to a stirred solution of Boc-Gly-DEPheOH (1.60 g, 5.0 mmol)
in tetrahydrofuran (12 mL) and the solution was cooled to
À10 1C. Then isobutylchloroformate (0.654 mL, 5.0 mmol)
was added and the mixture left for 1.5 min. Finally,
L-phenylalanine p-nitrophenylanilide trifluoroacetate (1.997
g, 5.0 mmol) was added to this mixture and the reaction was
carried out for 24 h at room temperature. The formed
precipitate was filtered off and solvent was removed under
reduced pressure. The resulting oil was dissolved in ethyl
acetate (25 mL) and washed successively with 2 M hydro-
chloric acid (2 Â 10 mL), saturated potassium bicarbonate (2
 10 mL) and brine (10 mL). The organic layer was dried over
anhydrous MgSO4, drying agent was removed by filtration
and solvent was evaporated in vacuo. This procedure provided
the mixture of (Z) and (E) isomers, which were resolved by
means of column chromatography using silica gel H60
(Merck) and a gradient of ethyl acetate in benzene as eluent
(from 15 to 45% of ethyl acetate). The desired product was
slowly precipitated with hexane from a mixture of methanol
and diethyl ether (15 : 1 v/v). Yield: 0.520 g (18%), mp
201–203 1C. Unfortunately, the (Z) isomer appeared to be
the main product of reaction. It was purified by precipitation
with hexane from an ethyl actetate–methanol mixture (4 : 1
v/v). Yield: 1.54 g (52%), mp 198.5–201.5 1C.
The position of the ligand is stabilised by four intermole-
cular hydrogen bonds (Fig. 8, top) and the interaction energy
is equal to À110.5 kcal molÀ1
.
Therefore the activity of the studied dehydrotetrapeptides
does not result from similarity to peptidyl substrates of
cathepsin C, for which the free, unblocked N-terminal amino
group is required, but from simple adhesion to the part of the
enzyme forming an entrance to its active site. This adhesion is
governed mainly by weak van der Waals forces, which implies
weak inhibitory activity of the studied compounds.
Conclusions
The comparison of results obtained for peptide p-nitrophenyl-
anilides have showed that this group of compounds adopts a
more ordered conformation then their saturated counterparts
and analogues which contain DAla or DZPhe residues. More-
over, peptides which contain a free, unblocked amino group
appeared to be quite good substrates of cathepsin C, whereas
fully protected peptides acted as very week inhibitors. The
correlation of spectroscopic data (NMR and CD) with mole-
cular modelling results suggested that these peptides are
hydrolysed in one step by direct removal of p-nitroaniline
from the peptide chain.
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Elemental analysis: calc. for C31H33O7N5 (587.5): C 63.36,
H 5.66; found: C 63.49, H 5.61%. 1H NMR (DMSO): 1.38 (s,
9H, CH3 Boc), 3.09 + 2.87 (m, 2H, CbH2 Phe), 3.86 + 3.73
(m, 2H, Ca H2Gly), 4.76 (q, 1H, CaH Phe), 6.47 (s, 1H, CbH
DEPhe), 7.28 + 7.02 (m, aromatic rings + NH Gly), 8.08 +
8.22 (2 Â d, 4H, aromatic ring of pNA), 8.74 (d, 1H, NH Phe),
10.19 (s, 1H, NH DEPhe), 10.21 (s, 1H, NH pNA); 13C NMR
(DMSO): 28.2 (CH3 Boc), 36.4 (CbPhe), 43.0 (CaGly), 55.1
(CaPhe), 78.1 (C Boc), 117.9 (CbDEPhe), 119.3 + 124.6
(aromatic ring of pNA), 123.0 + 126.9 (aromatic rings),
133.8 (CaDEPhe), 137.4 (CgDEPhe), 144.6 + 142.1 (aromatic
ring of pNA), 155.7 (CO Boc), 164.4 (CO DEPhe), 169.0 (CO
Gly), 170.2 (CO Phe).
Boc-Gly-Gly-DEPhe-Phe-pNA. This was obtained by two-
step procedure, namely removal of Boc from Boc-Gly-DEPhe-
Phe-pNA followed by acylation of the product with BocGly.
Thus, Boc-Gly-DEPhe-Phe-pNA (0.584 g, 1.0 mmol) was dis-
solved in anhydrous trifluoroacetic acid (3.0 mL) and stirred at
room temperature for 30 min. Then dichloromethane (40 mL)
was added and the volatile components of reaction mixture
removed in vacuo. The glassy product was purified by dissol-
ving in diethyl ether (20 mL) and evaporation of volatiles. This
procedure was repeated three times. The resulting trifluoroa-
cetate was dissolved in isopropanol and precipitated with
hexane. Yield: 0.601 g (B100%).
TBTU (0.337 g, 1.05 mmol) was added to a stirred solution
of Boc-Gly (0.176 g, 1.0 mmol), crude trifluoroacetate of Gly-
DEPhe-Phe-pNA (0.601 g, 1.0 mmol) and triethylamine (0.286
mL, 2.05 mM) in acetonitrile (4.0 mL) and the reaction was
carried out for 24 h at room temperature. Then solvent was
removed under reduced pressure and the resulting oil dissolved
in ethyl acetate (60 mL). The organic layer was washed
successively with: 2 M hydrochloric acid (4 Â 3 mL), saturated
potassium bicarbonate (4 Â 3 mL) and brine (3 mL) and dried
over anhydrous MgSO4. The drying agent was removed by
filtration and solvent evaporated in vacuo. The oily product
was precipitated from isopropanol with hexane. Yield: 0.620 g
(96%). Elemental analysis: calc. for C33H36O8N6 (664.6): C
61.48, H 5.63; found: C 61.32, H 5.57%.
1H NMR (DMSO): 1.36 (s, 9H, CH3 Boc), 3.10 + 2.9 (m,
2H, CbH2 Phe), 3.67 (d, 2H, Ca H2Gly(1)), 3.94 (d, 2H, Ca
H2Gly(2)), 4.72 (q, 1H, CaH Phe), 6.49 (s, 1H, CbH DEPhe),
7.15 (m, 10H, aromatic rings), 7.92 + 8.19 (m, 5H, aromatic
ring of pNA + NH Gly(1)), 8.63 (d, 1H, NH Phe), 8.34 (d, 1H,
NH Gly(2)), 10.02 (s, 1H, NH DEPhe); 10.14 (s, 1H, NH
pNA); 13C NMR (DMSO): 28.16 (CH3 Boc), 36.4 (CbPhe),
41.6 (CaGly(1)), 43.2 (CaGly(2)), 55.2 (CaPhe), 78.18 (C Boc),
119.11 (CbDEPhe), 119.3 + 124.9 (aromatic ring of pNA),
126.5 + 129.1 (aromatic rings), 129.1 (CaDEPhe), 134.1 (aro-
matic ring of DEPhe), 137.45 (CgPhe), 142.5 + 144.7 (aromatic
ring of pNA), 164.4 (CO DEPhe), 168.5 (CO Gly(1)), 169.9
(CO Gly(2)), 170.4 (CO Phe), 170.99 (CO Boc).
Boc-Gly-DEPhe-Gly-Phe-pNA. This was obtained by alka-
line deprotection of Boc-Gly-DEPhe-Gly-OMe followed by
coupling with Phe-pNA trifluoroacetate. Thus, 1 M sodium
hydroxide solution (1.1 mL) was added to a solution of Boc-
Gly-DEPhe-Gly-OMe (0.380 g, 1.0 mmol) in a mixture of 2.0
mL MeOH and 1.0 mL water. The reaction mixture was
stirred at room temperature for 2 h and then acidified with 1
M hydrochloric acid to pH 4. To the resulting solution, brine
(50 mL) was added, and the aqueous phase extracted with
ethyl acetate (5 Â 5 mL). Combined organic layers were then
washed successively with 0.5 M hydrochloric acid (2 Â 3 mL)
and brine (2 Â 3 mL). The organic layer was dried over
anhydrous MgSO4 (1 h only!) and the drying agent was filtered
off. The product was precipitated with hexane from an ethyl
acetate–methanol mixture (29 : 1 v/v). Yield: 0.744 g (98%),
mp 127–130 1C.
This product was used directly for p-nitrophenylanilide
preparation. Thus, TBTU (0.337 g; 1.05 mM) was added to
a stirred solution of Boc-Gly-DEPhe-Gly-OH (0.377 g, 1.0
mM), trifluoroacetate of L-phenylalanine p-nitroanilide
(0.399 g, 1.0 mmol) and triethylamine (0.286 mL, 2.05 mmol)
in acetonitrile (4.0 mL). The reaction was carried out for 28 h
at room temperature. Then the solvent was removed under
reduced pressure and resulting oil dissolved in ethyl acetate
(60 mL). The organic layer was washed successively with: 2 M
hydrochloric acid (3 Â 3 mL), saturated potassium bicarbo-
nate (3 Â 3 mL) and brine (3 mL) and dried over anhydrous
MgSO4; drying agent was removed by filtration and solvent
was evaporated under reduced pressure. Pure product was
precipitated from isopropanol with hexane. Yield: 0.527 g
(82%). Elemental analysis: calc. for C33H36O8N6 (664.6): C
61.48, H 5.63; found: C 61.60, H 5.42%.
1H NMR (DMSO): 1.27 (s, 9H, CH3 Boc), 3.12 + 3.14 (m,
2H, CbH2 Phe), 3.63 (d, 2H, Ca H2Gly(3)), 3.72 (t, 2H, Ca
H2Gly(1)), 4.76 (m, 1H, CaH Phe), 6.55 (s, 1H, CbH DEPhe),
7.10 (t, 1H, NH Gly(1)), 7.32 + 7.18 (m, 10H, aromatic rings),
7.88 + 8.23 (2 Â d, 4H, aromatic ring of pNA), 8.40 (d, 1H, NH
Phe), 8.93 (s, 1H, NH Gly(3)), 10.10 (s, 1H, NH pNA), 10.20 (s,
1H, NH DEPhe); 13C NMR (DMSO): 28.0 (CH3 Boc), 36.8
(CbPhe), 42.9 (CaGly(3)), 43.1 (CaGly(1)), 55.9 (CaPhe), 78.0 (C
Boc), 117.8 (CbDEPhe), 119.1 + 124.8 (aromatic ring of pNA),
126.5 + 129.2 (aromatic rings), 131.5 (CaDEPhe), 137.6 +
134.2 (aromatic rings), 144.8 + 142.5 (aromatic ring of pNA),
155.8 (CO Boc), 166.0 (CO DEPhe), 168.8 (CO Gly(3)), 169.0
(CO Gly(1)), 170.0 (CO Phe).
Gly-DEPhe-Gly-Phe-pNA. Boc-protected dehydrotetrapep-
tide p-nitroanilide (0.5 mmol) was dissolved in anhydrous
trifluoroacetic acid (1.5 mL) and stirred at room temperature
for 30 min. Then dichloromethane (20 mL) was added and
volatile components of reaction mixture removed under re-
duced pressure. The obtained solid substance was purified by
dissolving in diethyl ether (10 mL) and evaporation. This step
was repeated three times, the resulting trifluoroacetate was
dissolved in isopropanol (1 mL) and pure product precipitated
by slow addition of hexane. Yield: 0.319 g (97%). Elemental
analysis: calc. for C28H37O6N6 (553.52): C 60.7, H 6.73; found:
C 50.96, H 6.75%.
1H NMR (DMSO): 3.15 + 3.93 (m, 2H, CbH2 Phe), 3.68 +
3.82 (d, 4H, Ca H2Gly(1) and Gly(3)), 4.68 (t, 1H, CaH Phe),
6.73 (s, 1H, CbH DEPhe), 7.15 + 7.34 (m, 10H, aromatic
rings), 7.82 + 8.20 (2 Â d, 4H, aromatic ring of pNA), 8.04 (s,
2H, NH2 Gly(1)), 8.34 (d, 1H, NH Phe), 8.54 (t, 1H, NH
Gly(3)) 10.23 (s, 1H, NH DEPhe); 10.53 (s, 1H, NH pNA); 13
C
NMR (DMSO): 37.3 (CbPhe), 40.7 (CaGly(3)), 43.8
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(CaGly(1)), 55.2 (CaPhe), 118.7 (CbDEPhe), 119.0 + 124.8
(aromatic ring of pNA), 129.1 + 126.4 (aromatic rings), 130.8
(CaDEPhe), 137.2 + 134.0 (aromatic rings), 144.7 + 142.4
(aromatic ring of pNA), 162.54 (CO DEPhe), 164.9 (CO
Gly(3)), 168.4 (CO Gly(1)), 170.9 (CO Phe).
the next steps the side chain atoms from active side were
unfrozen and all atoms of the dehydropeptide were analysed
using the conjugate gradient algorithm and tether constraints
on the unfrozen heavy atoms, which was decreased from a
value of 200 to 20 kcal AÀ2, also the maximum derivative was
decreased from a value of 30 to 1 kcal AÀ1. In the last step we
optimized the complex without any force constant using a
conjugate gradient algorithm with the maximum derivative
equal to 0.1 and then 0.01 kcal AÀ1. To decide which arrange-
ments were the best for each dehydropeptide, we calculated the
Gly-Gly-DEPhe-Phe-pNA. This was obtained in identical
manner as described above. Yield: 0.323 g (98%). Elemental
analysis: calc. for C28H37O6N6 (553.52): C 60.7, H 6.73; found:
C 50.98, H 6.62%.
1H NMR (DMSO): 3.15 + 2.9 (m, 2H, CbH2 Phe), 3.67 (s,
2H, Ca H2Gly(1)), 4.04 (d, 2H, Ca H2Gly(2)), 4.77 (q, 1H, CaH
Phe), 6.55 (s, 1H, CbH DEPhe), 7.05 + 7.30 (m, 10H, aromatic
rings), 7.97 + 8.25 (2 Â d, 4H, aromatic ring of pNA), 8.14 (s,
2H, NH2 Gly(1)), 8.72 (d, 1H, NH Phe), 8.86 (t, 1H, NH
energy of interaction Eint, which was defined as Eint = Ecomp
À
(Een + Elig), where Ecomp was the energy of the enzyme–ligand
complex excluding the water layer; Een was the energy of the
enzyme; Elig was the energy of the ligand.
Gly(2)) 10.24 (s, 1H, NH DEPhe); 10.26 (s, 1H, NH pNA); 13
C
NMR (DMSO): 36.6 (CbPhe), 40.3 (CaGly(1)), 42.2
(CaGly(2)), 55.3 (CaPhe), 118.6 (CbDEPhe), 119.3 + 124.9
(aromatic ring of pNA), 129.2 + 126.5 (aromatic rings), 131.4
(CaDEPhe), 134.2 (aromatic ring of DEPhe), 137.4 (CgPhe),
144.9 + 142.4 (aromatic ring of pNA), 164.5 CO (DEPhe),
166.6 (CO Gly(1)), 167.9 (CO Gly(2)), 170.4 (CO Phe).
Acknowledgements
The molecular modeling were carried out using hardware and
software resources of The Supercomputing and Networking
Centre in Wroclaw.
References
Assay of the enzymatic activity
1 D. E. Palmer, C. Pattaroni, K. Nunami, R. K. Chadha, M.
Goodman, T. Wakamiya, K. Fukase, S. Horimoto, M. Kitazawa,
H. Fujita, A. Kubo and T. Shiba, J. Am. Chem. Soc., 1992, 114,
5634.
2 H. Allgaier, G. Jung, R. G. Werner, U. Schneider and H. Zamer,
Angew. Chem., 1985, 24, 1051.
3 O. Pieroni, A. Fissi, R. M. Jain and V. S. Chauhan, Biopolymers,
1996, 38, 141.
4 C. M. Venkatachalam, Biopolymers, 1968, 6, 1425.
5 S. M. Patel, J. O. Currie, Jr. and R. K. Olsen, J. Org. Chem., 1973,
38, 126.
Cathepsin C was isolated according to the described proce-
dure.22 Enzymatic reaction was assayed at 37 1C in acetate
buffer (pH 5) containing NaCl (10 mM final concentration)
and 2-mercaptoethanol (5 mM final concentration). The assay
mixture contained dehydrotetrapeptide (1–8 mM final con-
centration) and the course of reaction was monitored by
following the change in absorbance at 400 nm. Michaelis
constants (KM) and maximal velocities (Vmax) were obtained
using the computer program kindly provided by Dr J. Hurek
(University of Opole).
6 K. R. Rajashankar, S. Ramakumar and V. S. Chauhan, J. Am.
Chem. Soc., 1992, 114, 9225.
7 M. R. Ciajolo, A. Tuzi, C. R. Pratesi, A. Fissi and O. Pieroni,
Biopolymers, 1990, 30, 911.
Molecular modelling
8 Y. Inai, S. Kurashima, T. Hirabayashi and K. Yokota, Biopoly-
mers, 2000, 53, 482.
9 B. Turk, I. Dolenc and V. Turk, Handbook of Proteolytic Enzymes,
ed. A. J. Barrett, Academic Press, New York, 1998, pp. 631–634.
10 R. Butler, A. Michel, W. Kunz and M. O. Klinkert, Protein Peptide
Lett., 1995, 2, 313.
11 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. Ghaffar, M. Pemberton, I.
R. Hussein, S. A. Temtamy, R. Davies, A. P. Sloan, M. J. Dixon
and N. S. Thakker, Nat. Genet., 1999, 23, 421.
12 A. O. Breitholle and C. H. Stammer, Tetrahedron Lett., 1975, 28,
2381.
13 M. Makowski, M. Pawe"czak, R. Latajka, K. Nowak and P.
Kafarski, J. Pept. Sci., 2001, 7, 141.
14 M. Lisowski, R. Latajka, M. Makowski, T. Lis, B. Picur, J. Panek
and P. Kafarski, J. Am. Chem. Soc., submitted.
15 M. Makowski, B. Rzeszotarska, Z. Kubica, G. Pietrzynski and J.
Hepter, Liebigs Ann. Chem., 1986, 980.
16 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A.
Robb, J. R. Cheeseman, J. A. Montgomery, Jr., T. Vreven, K. N.
Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V.
Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G. A.
Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R.
Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O.
Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian, J.
B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E.
Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J.
Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P. Salvador, J.
J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D. Daniels,
The structures of the studied dehydropeptides were optimized
in program Gaussian03 at the HF/6-31g (d,p) level16 in the gas
phase using the Merz–Singh–Kollman scheme20 in the deter-
mination of the atomic charges. The calculation of the docking
process were performed using the AutoDock program.18 The
starting geometry of the dehydropeptides was taken from the
ab initio calculations, and also we assigned the charges using
charges from the ab initio calculation. The structure of cathe-
psin C was taken from the structure of human dipeptidyl
peptidase I deposited EC 3.4.14. in the Protein Data Bank.17
During the docking process the main chain of the dehydro-
peptide was fixed, whereas side chains and the terminal groups
(–NH2, –pNA, –Boc) were left as flexible. The coordinates of
the SH proton from the Cys234 were taken as a grid center in
the docking process. Several possible structures ligand–
enzyme complexes for each dehydropeptide were obtained in
this manner, which were grouped in clusters. For the next
stage we chose structures from the most possible clusters. In
the next step the selected complex compounds were optimized
using Accelrys’s DISCOVER program with the cff97 force
field, at neutral pH and considering a 10 A water layer. In the
first step all heavy atoms were frozen and a steep descent
algorithm with maximum derivative equal to 0.1 was used. In
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M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K.
Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul,
S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P.
Piskorz, I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A.
Al-Laham, C. Y. Peng, A. Nanayakkara, M. Challacombe, P. M.
W. Gill, B. G. Johnson, W. Chen, M. W. Wong, C. Gonzalez and
J. A. Pople, GAUSSIAN 03 (Revision C.02), Gaussian, Inc.,
Wallingford, CT, 2004.
18 G. M. Morris, D. S. Goodsell, R. Huey, W. E. Hart, S. Halliday
and R. Belew, AutoDock (version 3.05).
19 G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W. E.
Hart, R. K. Belew and A. J. Olson, J. Comput. Chem., 1998, 19,
1639.
20 B. H. Besler, K. M. Merz, Jr and P. A. Kollman, J. Comput.
Chem., 1990, 11, 431.
21 M. Makowski, B. Rzeszotarska, Z. Kubica and P. Wieczorek,
Liebigs Ann. Chem., 1984, 920–928.
17 D. Turk, V. Janji, I. Stern, M. Podobnik, D. Lamba, S. Weis Dahl,
C. Lauritzen, J. Pedersen, V. Turk and B. Turk, EMBO J., 2001,
20, 6570.
22 J. K. McDonald, P. X. Callahan and P. Ellis, Methods Ezymol.,
1971, 25B, 272.
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1018 | New J. Chem., 2006, 30, 1009–1018