Introduction of non-natural amino acid residues into the substrate-specific P1 position of trypsin inhibitor SFTI-1 yields potent chymotrypsin and cathepsin G inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.03.045

A series of trypsin inhibitor SFTI-1compounds modified in substrate-specific P₁ position was synthesized by the solid-phase method. Lys5 present in the wild inhibitor was replaced by Phe derivatives substituted in para position of the phenyl ri

Full text of DOI:10.1016/j.bmc.2009.03.045

Bioorganic & Medicinal Chemistry 17 (2009) 3302–3307
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Introduction of non-natural amino acid residues into the substrate-specific
P1 position of trypsin inhibitor SFTI-1 yields potent chymotrypsin
and cathepsin G inhibitors
*
Anna Łe˛gowska , Dawid De˛bowski, Adam Lesner, Magdalena Wysocka, Krzysztof Rolka
´
´
Faculty of Chemistry, University of Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of trypsin inhibitor SFTI-1compounds modified in substrate-specific P1 position was synthesized
by the solid-phase method. Lys5 present in the wild inhibitor was replaced by Phe derivatives substituted
Received 7 November 2008
Revised 18 March 2009
Accepted 23 March 2009
Available online 27 March 2009
in para position of the phenyl ring,
with bovine -chymotrypsin and cathepsin G were estimated by determination of association equilib-
rium constants (Ka). All analogues inhibited bovine -chymotrypsin. The highest inihbitory activity dis-
L-pyridylalanine and N-4-nitrobenzylgycine. Their inhibitory activities
a
a
played peptides with the fluorine, nitro and methyl substituents. They were 13–15-fold more active than
[Phe5]SFTI-1 used as a reference. They are the most potent chymotrypsin inhibitors of this size. Substitu-
tion of Lys5 by Phe did not change the cathepsin G inhibitory activity. Introduction of Phe(p-F), Phe
(p-NH2) and Phe(p-CH3) in this position retained the affinity towards this proteinase, whereas Phe(p-gua-
nidine) gave an inhibitor more than twice as active, which appeared to be stable in human serum. On the
other hand, a peptomeric analogue with N-4-nitrobenzylglycine failed to inhibit cathepsin G. Despite the
fact the introduced amino acids were non-coded, the peptide bonds formed by them were hydrolyzed by
chymotrypsin. We postulate that additional interaction of para-substitutents with the enzyme are
responsible for the enhanced inhibitory activity of the analogues.
Key words:
Inhibitors, Peptides
Chemical synthesis
Cathepsin G
Chymotrypsin
Phenylalanine derivatives
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
of turkey ovomucoid third domain5 and bovine pancreatic trypsin
inhibitor.6 Since both inhibitors are proteins, the appropriate
Almost one-third of all proteases have been classified as serine
proteinase.1 They are responsible for a broad spectrum of biologi-
cal activity. Uncontrolled activity of these enzymes can be very
dangerous for the organism. Therefore a very strict mechanism of
their control (in both time and site) is required. One of the predom-
inant mechanisms rests on ubiquitous presence of inhibitors of
these proteinases. It turned out that many, but not all serine pro-
teinase inhibitors, display the same so-called standard mechanism
of association with their target enzymes.2 They interact with en-
zymes in a substrate-like fashion through an exposed fragment
named binding loop which is characterized by unique canonical
conformation.3 A hyper exposed amino acid residue located in
the centre of this loop, named after Schechter and Berger4 P1 resi-
due, interacts with S1 cavity of the enzyme accounting for up to
50% of the inhibitor–enzyme contacts.5,6 P1 residue determines
the inhibitor specificity. In many cases, a single substitution of an
amino acid residue in this position in naturally occurring serine
proteinase inhibitor changes dramatically their specificity as
shown by systematic studies on analogues modified in P1 position
changes were mainly introduced using site-directed mutagenesis
(few non-coded amino acids were inserted into the amino acid se-
quence by enzymatic semisynthesis5) and therefore focused on
coded amino acids. Extensive studies on the influence of amino
acid residue present in the position on inhibitor specificity were
also performed on shorter compounds like peptides based on the
binding loop of Bowman-Birk inhibitor.7 But also in this case,
mainly proteinogenic amino acids were investigated. One of the
main goals behind such investigations is to develop a drug candi-
date to treat several diseases including arthritis, rheumatoid
arthritis, hemophilia, emphysema, neurodegenerative disorders,
inflammatory and allergic disorders (including asthma) and many
others. For several reasons, including poor bioavailability and sus-
ceptibility to degradation by proteinases, application of proteins
and peptides as drugs is rather limited. One of the obvious ap-
proaches to overcome these obstacles is introduction of non-coded
amino acids into the peptide chain. In our previous works we have
shown that despite a very strict structural requirements for inhib-
itor–enzyme P1–P0 interactions,8 some of the artificial amino acid
1
residues are well tolerated.
In past years, we focused our attention on trypsin inhibitor
* Corresponding author. Tel.: +48 585235359.
E-mail address: legowska@chemik.chem.univ.gda.pl (A. Łe˛gowska).
SFTI-1 isolated from sunflower seeds.9 This dicyclic 14-amino acid
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.045

A. Łe˛gowska et al. / Bioorg. Med. Chem. 17 (2009) 3302–3307
3303
Phe(p-guanidine)] introduced into the position of the SFTI-1 ana-
logues display both aromatic and basic character and therefore
they are likely to facilitate the interaction of these analogues with
cathepsin G.
Figure 1. Primary structure of trypsin inhibitor SFTI-1.
2. Results and discussion
residues inhibitor (Fig. 1) contains Lys5 in P1 position and therefore
exhibits affinity towards trypsin-like proteinases. By the synthesis
of active SFTI-1 analogues modified in position P1 with hydropho-
The determined molecular weights and the results of HPLC
analysis of the synthesized peptides, together with their inhibitory
activities, are summarized in Table 1. All the synthesized SFTI-1
analogues are monocyclic with free C-terminal carboxyl group.
Conversion of this group to amide reduced their inhibitory activity.
In our previous work15 we have shown that one of the cyclic ele-
ments can be removed from SFTI-1 without significant loss of the
trypsin inhibitory activity. Replacement of Lys5 only, present in
inhibitory P1 position, by Phe in monocyclic SFTI-1 with a disulfide
bridge, increased 400-times the inhibitory activity against bovine
bic
a
-hydroxymethylamino acids, we have demonstrated10 that
the ion pair formation between the enzymic b-carboxyl group of
Asp189 and the positively charged side chain group of an amino
acid residue present in P1 position of the inhibitor, the main source
of the enzyme–inhibitor interaction can be successfully replaced
by a network of hydrogen bonds. Recently, we have synthesized
SFTI-1 analogues which contained in this position N-(4-aminobu-
tyl)-glycine and N-benzylglycine that mimic Lys and Phe, to obtain
trypsin and chymotrypsin inhibitors, respectively.11
It is well documented that aromatic residues in inhibitor P1 are
optimal for interactions with chymotrypsin. Enhanced activity of
chymotrypsin inhibitors having Tyr in position P1, as compared
to those with Phe5,6 suggests that additional p-hydroxyl group sta-
bilizes the chymotrypsin-inhibitor complex. Unfortunately, there
are no X-ray data available supporting this statement. Taking into
consideration these results, we decided to synthesize and deter-
mine inhibitory activity of a series of SFTI-1 monocyclic analogues
(with disulfide bridge only) modified in substrate-specific P1 posi-
tion by Phe residues substituted in the benzene ring and, in addi-
tion, one peptomeric analogue with N-4-nitrobenzylglycine
(Nphe(p-NO2). Our purpose was to determine the influence of the
para-substituent on enzyme–inhibitor interaction. Chemical for-
mulas of the Phe derivatives are shown in Figure 2.
a
-chymotrypsin, reaching an association equilibrium constant as
high as 2.0 ꢀ 109 Mꢁ1. [Phe5]SFTI-1 will be used as a reference
compound. All the synthesized SFTI-1 analogues exhibited affinity
towards bovine a-chymotrypsin. Substitution of Phe by Tyr yielded
about a 10-fold more potent chymotrypsin inhibitor. This is in
good agreement with the results obtained by other teams working
on proteinous inhibitors: bovine pancreatic trypsin inhibitor
(BPTI)6 and turkey ovomucoid third domain.5 Nevertheless, in both
cases the impact of the additional OH group on inhibitory activity
was less pronounced. Among the synthesized analogues, the high-
est chymotrypsin inhibitory activity, 15 times as high as that of the
reference compound, was determined for the one with Phe(p-F).
Almost equipotent activity was displayed by peptides with un-
charged substituents introduced into para position of the phenyl
ring, [Phe(p-NO2)5]SFTI-1 and [Phe(p-CH3)5]SFTI-1. Introduction
in this position of a positively charged guanidine group dramati-
cally reduced the affinity towards the experimental enzyme. Inter-
estingly, amino acid residues Pal and Phe(p-NH2) with less basic
aromatic amines on their side chains produced potent chymotryp-
sin inhibitors. It is also worth noting that high chymotrypsin inhib-
itory activity was determined for peptomeric analogue [Nphe(p-
NO2)5]SFTI-1. Also in this case, the nitro substituent increased
The other enzyme which was the subject of our investigations
was cathepsin G. It combines both chymotrypsin-like and tryp-
sin-like specificities which is not very common among proteinases.
Duodenase and a crab fiddler collagenase are two examples of such
enzymes.12,13 Glu226 of cathepsin G divides the bottom of sub-
strate pocket into sub-compartments that can accommodate posi-
tively charged side chains of Lys or Arg or phenyl ring of Phe or Tyr.
As shown in Figure 3, some of the Phe derivatives [Phe(p-NH2),
Figure 2. Chemical formulas and abbreviations of Phe derivatives introduced into P1 position of monocyclic trypsin inhibitor SFTI-1.

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A. Łe˛gowska et al. / Bioorg. Med. Chem. 17 (2009) 3302–3307
mAU
150
time 0
24 h
2
125
100
75
1
50
25
1
0
-10
5
10
15
20
25
30
35
40 minutes
Figure 3. Proteolytic susceptibility of analogue 4 (Gly-Arg-Cys(&)-Thr-Phe(p-NO2)-Ser-Ile-Pro-Pro-Ile-Cys(&)-Phe-Pro-Asp) (nomenclature of cyclic peptides as recom-
mended by Spengler et al.18) in the presence of
-chymotrypsin, at pH 8.3: peak 1—intact peptide, peak 2—peptide with hydrolyzed Phe(p-NO2)—Ser peptide bond. Peaks with
a
retention times above 30 min come from Triton X-100.
inhibitory activity by almost one order of magnitude. The Ka values
determined for analogues 4, 5 and 7 with chymotrypsin indicate
that they are the most potent chymotrypsin inhibitors of this size
reported to date.
bond between amino acid residues in position P1 and P0 underwent
1
gradual hydrolysis until the equilibrium between the intact and
hydrolyzed forms of the inhibitor (peaks 1 and 2, respectively)
was reached. No further progress in the proteolysis was detected,
which indicates that the peptides belong to the classical canonical
group of inhibitors, where the equilibrium between the intact and
hydrolyzed form of the peptide is observed. This phenomenon is
not observed when the amino acid residue in position P1 of the
inhibitor is replaced by a peptoid residue. Substitution of Phe(p-
NO2) by Nphe(p-NO2) in this position of the peptide results in a
proteolytically resistant analogue (see Fig. 4).
Surprisingly enough, our research has shown that native SFTI-1
does not have such high activity against cathepsin G as that
claimed by Luckett et al. (Ki ꢂ 0.15 nM).9 When we calculated the
Ki using our experimental data collected for the determination of
Ka, we obtained a value of 570 nM which is approximately three
orders of magnitude higher. Unfortunately, the cited report does
not contain any details about the measurement of Ki value. Thus,
it is impossible to find the origins of these discrepancies. Since
It is well documented that in canonical inhibitors, the peptide
bond formed by P1 and P01, called the reactive site, is selectively
cleaved by a cognate enzyme. In neutral pH, the hydrolysis con-
stant between the intact and hydrolyzed inhibitor is close to
unity.16 Unlike substrates, standard-mechanism inhibitors are
characterized by slow hydrolysis of the P1–P0 reactive site. It is rea-
1
sonable to assume that introduction of non-coded amino acids in
P1 position should increase proteolytic stability of the reactive site
of the inhibitor and might increase inhibitory activity. In our recent
work,17 we have shown that introduction of the Nphe residue in P1
position decreased the hydrolysis rate of the reactive site of inhib-
itors based on the SFTI-1 structure. Hydrolysis patterns for the
three compounds: two most potent inhibitors [Phe(p-NO2)5]SFTI-
1 (4) (Fig. 3), [Phe(p-F)5]SFTI-1 (5) and the reference compound
[Phe5]SFTI-1 (not shown) were similar. In all cases the peptide
Table 1
Physicochemical properties and association equilibrium constants (Ka) of SFTI-1 analogues with bovine
a-chymotrypsin and cathepsin G
Inhibitora
MW calcd (found)
HPLCb rt (min)
Bovine
a-chymotrypsin
Cathepsin G
Ka (Mꢁ1
)
Relative activity
Ka (Mꢁ1
)
Relative activity
1.54
SFTI-1 (dicyclic)
SFTI-1
(5.2 1.5) ꢀ 106 [14]
(5.0 1.4) ꢀ 106 [14]
2.0 ꢀ 109
(5.7 0.3) ꢀ 106
n.d.
[Phe5]SFTI-1
1550.7 (1550.6)
1550.7 (1550.8)
1549.8 (1549.5)
1595.8 (1595.2)
1566.8 (1566.6)
1552.8 (1552.2)
1595.8 (1595.6)
1568.8 (1568.4)
1565.8 (1565.5)
1564.8 (1564.3)
1607.9 (1607.5)
19.6
19.5
19.7
19.8
18.9
17.0
20.3
20.6
17.7
21.1
18.9
1.00
0.19
Below 0.01
1.20
9.50
(3.7 0.23) ꢀ 106
1.00
[Nphe5] SFTI-1
(3.8 0.3) ꢀ 108 [11]
(1.0 0.1) ꢀ 107
(2.4 0.4) ꢀ 109
(1.9 0.2) ꢀ 1010
(1.1 0.1) ꢀ 1010
(2.6 0.3) ꢀ 1010
(3.0 0.6) ꢀ 1010
(1.9 0.3) ꢀ 1010
(2.5 0.3) ꢀ 1010
(1.8 0.1) ꢀ 108
N/A
N/A
N/A
[Nphe5] SFTI-1(NH2)
[Nphe(p-NO2)5]SFTI-1 (1)
[Tyr5] SFTI-1 (2)
Below 105
Below 105
Below 105
(3.8 0.2) ꢀ 106
(3.6 0.2) ꢀ 106
(2.8 0.2) ꢀ 106
(8.1 0.4) ꢀ 107
[Pal5]SFTI-1 (3)
5.50
[Phe(p-NO2)5]SFTI-1 (4)
[Phe(p-F)5]SFTI-1 (5)
[Phe(p-NH2)5]SFTI-1 (6)
[Phe(p-CH3)5]SFTI-1 (7)
[Phe(p-guanidine)5]SFTI-1 (8)
13.00
15.00
9.50
12.50
0.09
1.02
0.97
0.76
2.19
a
Except wild SFTI-1, all inhibitors are monocyclic with disulfide bridge only.
Conditions for HPLC analysis are described in Section 3; N/A—not active, n.d.—not determined.
b

A. Łe˛gowska et al. / Bioorg. Med. Chem. 17 (2009) 3302–3307
3305
mAU
100
1
1
time 0
24 h
75
50
25
0
-5
5
10
15
20
25
30
35
40 minutes
a-chymotrypsin, at pH 8.3.
Figure 4. Proteolytic susceptibility of analogue 1 (Gly-Arg-Cys(&)-Thr-Nphe(p-NO2)-Ser-Ile-Pro-Pro-Ile-Cys(&)-Phe-Pro-Asp) in the presence of
all our experiments were carried out under the same conditions,
we will use our Ka value as a reference in further discussion. As a
result of substitution of Lys5 by Phe, the inhibitory activity against
this enzyme was retained. This is in line with the substrate speci-
ficity of cathepsin G which can accommodate in its S1 cavity the
side chains of both aromatic and positively charged amino acids.
As reported by Polanowska et al.19 cathepsin’s G inhibitory activity
of BPTI with Lys and Phe present in position P1 was almost the
same and the measured Ka values were of the same order of mag-
nitude as those determined for SFTI-1 and [Phe5]SFTI-1. Only four
of the synthesized SFTI-1 analogues exhibited a considerable inhib-
itory activity. The most potent, twice as active as the reference
anine in P1 position. Introduction of fluorine, amino or methyl sub-
stituents in para position of the phenyl ring did not affect
association of the SFTI-1 analogue with cathepsin G. On the other
hand, the hydroxyl and nitro groups or introduction of a nitrogen
atom into the phenyl ring (analogue 3 with Pal in P1) gave SFTI-1
analogues with low cathepsin G inhibitory activity. All three ana-
logues with peptoid monomers did not inhibit this proteinase. It
is worth emphasizing that the peptoid monomers introduced in
inhibitor’s P1 position are well tolerated by trypsin, chymotrypsin
and elastase.11,20
HPLC analysis of analogue 8 (with Phe(p-guanidine) in P1 posi-
tion) incubated with human serum (Fig. 5) reveal that peptide re-
mains stable up to 24 h. It is worth noting that after incubation of
inhibitor, appeared to be analogue 8 with p-guanidine-
L-phenylal-
mAU
time 0
800
20 minutes
24 h
700
600
500
400
300
200
100
1
1
1
0
-86
10
20
30
40 minutes
Figure 5. HPLC analysis of analogue 8 (Gly-Arg-Cys(&)-Thr-Phe(p-guanidine)-Ser-Ile-Pro-Pro-Ile-Cys(&)-Phe-Pro-Asp) after 20 h of incubation with human serum. Peaks with
retention times above 18 min come from human serum.

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A. Łe˛gowska et al. / Bioorg. Med. Chem. 17 (2009) 3302–3307
the serum with each substrate, an increasing absorption of ANB-
NH2 was observed indicating the presence of active form of cathep-
sin G, proteinase 3 and human leukocyte elastase, respectively.
To the best of our knowledge, analogue 8 is one of the most
potent inhibitors of cathepsin G that has been reported to date.
Taking into account its high serum stability, similar compounds
could be candidates for designing a novel class of therapeutic
agents.
peptides was checked on an RP Kromasil 100, C8, 5 lm column
(4.6 ꢀ 250 mm) (Knauer, Germany). The solvent systems were
0.1% TFA (A) and 80% acetonitrile in A (B). Linear gradient from
10% to 90% B for 30 min, flow rate 1 mL/min, monitored at
226 nm. The mass spectrometry analysis was carried out on a MAL-
DI MS (a Biflex III MALDI-TOF spectrometer, Bruker Daltonics, Ger-
many) using a-CCA matrix.
The results discussed above correlate well with our previous
data obtained on chromogenic substrates.21,22 We synthesized a
series of short-chain peptides containing as a C-terminal residue
the same Phe derivatives with attached 5-amino-2-nitrobenzoic
acid amide (ANB-NH2) to their carboxyl function. In such designed
substrates, Phe(X) derivatives were located in P1 position and ANB-
NH2 was released upon interaction with enzyme. The highest spec-
3.2. Determination of the association equilibrium constants
The bovine b-trypsin (Sigma Chem. Co. USA) concentration was
determined by spectrophotometric titration with 4-nitrophenyl-
40-guanidinobenzoate (NPGB) at an enzyme concentration oscillat-
ing around 10ꢁ6 M.26 A standardized trypsin solution was used to
titrate ovomucoid from turkey egg whites, which in turn served
ificity constants (kcat/KM) with bovine
a-chymotrypsin, used as a
to determine the solution concentration of the bovine a-chymo-
measure of substrate activity, were obtained for peptides with
Phe(p-NO2), Tyr and Phe and Phe(p-NH2).22 Also the peptide with
Phe(p-guanidine) appeared to be the most active substrate of
cathepsin G.21 As already mentioned, canonical inhibitors interact
with cognate enzyme in a substrate-like fashion. The good correla-
tion between substrate and inhibitory activity of peptides modified
in P1 position by Phe derivatives suggests that introduction into the
peptide chain of non-natural amino acids did not influence the
mechanism of inhibition of the SFTI-1 analogues studied.
In summary, we would like to point out that synthetic Phe
derivatives introduced in the inhibitor P1 position are well toler-
ated and in the case of chymotrypsin some of the modifications
gave extremely potent inhibitors.
trypsin (Sigma Chem. Co. USA). A bovine b-trypsin standardized
solution of sunflower trypsin inhibitor (SFTI-1) was used for the
determination of the cathepsin G (Biocentrum Sp. z o.o., Kraków,
Poland) active form. The concentrations of the SFTI-1 analogues
were determined by titration of their stock solutions with stan-
dardized bovine
a-chymotrypsin and cathepsin G, with Suc-Ala-
Ala-Pro-Leu-4-nitroanilide and Ac-Phe-Ala-Thr-Phe(4-guanidine)-
ANB-NH2 as substrates,21 respectively. The association constants
were measured by the method developed in the laboratory of Las-
kowski, Jr.27,28 Enzyme–inhibitor interactions were determined in
the 0.1 M Tris–HCl (pH 8.3) buffer containing 20 mM CaCl2 and
0.005% Triton X-100 at room temperature. Increasing amounts of
the inhibitor, varying from 0 to 2E0 (E0—the initial enzyme concen-
tration), were added to a fixed amount of the enzyme. After a 3-h
incubation, the residual enzyme activity was measured on a Cary
3E spectrophotometer (Varian, Australia) using the turnover sub-
strate. The measurements were carried out at initial enzyme con-
centrations over the ranges 5.3 and 8.0 nM for chymotrypsin and
3. Materials and methods
3.1. Peptide synthesis
cathepsin G, respectively. The residual enzyme activity was mea-
29
All the peptides were synthesized by the solid-phase method
using Fmoc chemistry. The following amino acid derivatives were
used: Fmoc-Gly, Fmoc-Abu, Fmoc-Arg(Pbf), Fmoc-Cys(Trt), Fmoc-
Thr(tBu), Fmoc-Ser(tBu), Fmoc-Ile, Fmoc-Pro, Fmoc-Phe, Fmoc-As-
p(OtBu), Fmoc-Phe(p-NO2), Fmoc-Phe(p-F), Fmoc-Phe(p-NH2),
Fmoc-Phe(p-CH3), Fmoc-Phe(p-guanidine) and Fmoc-Pal. The C-
terminal amino acid residue, Fmoc-Asp(OtBu), was attached to
the 2-chlorotritylchloride resin (substitution of Cl 1.46 mequiv/g)
(Calbiochem-Novabiochem AG, Switzerland) in the presence of
an equimolar amount of DIPEA based on the amino acid in anhy-
drous condition in DCM solution. Peptide chains were elongated
in the consecutive cycles of deprotection and coupling. Deprotec-
tion was performed with 20% piperidine in DMF/NMP (1:1, v/v)
with addition of 1% Triton X-100, whereas the chain elongation
was achieved with standard DIC/HOBt chemistry; 3 equiv of pro-
tected amino acid derivatives were used. Nphe(p-NO2) was intro-
duced into the peptide chain by the submonomer approach.23 In
the first step, bromoacetic acid (5 equiv) was attached to the pep-
tidyl-resin using DIC/HOBt method, followed by nucleophilic
replacement of bromine with p-nitrobenzylamine (8 equiv). After
completing the syntheses, the peptides were cleaved from the re-
sin simultaneously with the side chain deprotection in a one-step
procedure, using a TFA/phenol/triisopropylsilane (88:5:2.5, v/v/v)
mixture.24 In the last step, the disulfide bridge formation was per-
formed using a 0.1 M methanolic iodine solution and the proce-
dure described elsewhere.25 The crude peptides were purified by
HPLC on a Beckman Gold System (Beckman, USA) using an RP Kro-
sured with Z-Phe-Ala-Thr-Tyr-ANB-NH2
and Ac-Phe-Ala-Thr-
Phe(4-guanidine)-ANB-NH2 as chromogenic substrates for chymo-
trypsin and cathepsin G inhibitors, respectively. In all cases the ini-
tial substrate concentration was below 0.1 KM. The experimental
points were analysed by plotting the residual enzyme concentra-
tion [E] versus the initial inhibitor concentration [I0]. The experi-
mental data were fitted to the theoretical values using the GRAFIT
software package.30
3.3. Proteolytic susceptibility
The SFTI-1 analogues were incubated in 100 mM Tris–HCl buf-
fer (pH 8.3) containing 20 mM CaCl2 and 0.005% Triton X-100 using
catalytic amounts of the enzymes (1 mol %).31 The incubation was
carried out at room temperature and sample aliquots of the mix-
ture were taken out periodically and submitted to RP-HPLC analy-
sis. The analysis were performed on an HPLC Gold System
(Beckman, USA) using a Vydac Protein & Peptide, C18, 10 lm col-
umn (4.6 ꢀ 250 mm) (Grace, USA). The solvent system was 0.1%
TFA (A) and 80% acetonitrile in A (B). Linear gradient from 10% to
90% B for 40 min, flow rate 1 mL/min, monitored at 226 nm.
3.4. Serum stability assay
Human serum (500 lL) collected from healthy volunteer donors
(kindly provided by Dr. Julia Kulczycka, Laboratory of Clinical
Immunology Medical University of Gdansk) was mixed with
masil-100, C8, 5
l
m column (8 ꢀ 250 mm) (Knauer, Germany). The
500
tube, followed by addition of 10
1 h, 4 h and 24 h the 50- L aliquots of the mixture were analyzed
by HPLC. To confirm the proteolytic activity, the serum was incu-
l
L of 0.2 M Tris–HCl buffer (pH 8.3), placed into Eppendorf
solvent systems were 0.1% TFA (A) and 80% acetonitrile in A (B).
Either isocratic condition or linear gradient were applied (flow rate
3.0 mL/min, monitored at 226 nm). The purity of the synthesized
l
L of peptide (2 mg/mL). After
l

A. Łe˛gowska et al. / Bioorg. Med. Chem. 17 (2009) 3302–3307
3307
10. Zabłotna, E.; Kret, A.; Jas´kiewicz, A.; Olma, A.; Leplawy, M. T.; Rolka, K. Biochem.
Biophys. Res. Commun. 2006, 340, 823.
11. Stawikowski, M.; Stawikowska, R.; Jas´kiewicz, A.; Zabłotna, E.; Rolka, K.
ChemBioChem 2005, 6, 1057.
12. Zamolodchikova, T. S.; Vorotyntseva, T. I.; Antonov, V. K. Eur. J. Biochem. 1995,
227, 866.
bated with chromogenic substrates recently developed by our
group. Ac-Phe-Val-Thr-Phe(4-guanidine)-ANB-NH2,21 Abz-Tyr-
Tyr-Abu-ANB-NH232 and Z-Phe-Phe-Pro-Val-ANB-NH229 were used
to determine cathepsin G, proteinase 3 and human leukocyte elas-
tase, respectively. The analysis was performed on an HPLC Gold
13. Tsu, C. A.; Perona, J. J.; Schellenberger, V.; Turck, C. W.; Craik, C. S. J. Biol. Chem.
1996, 269, 19565.
System (Beckman, USA) using a Vydac Protein & Peptide, C18
10
was 0.1% TFA (A) and 80% acetonitrile in A (B). Linear gradient from
,
´
´
l
m column (4.6 ꢀ 250 mm) (Grace, USA). The solvent system
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oratory of Clinical Immunology, Medical University of Gdansk, for
providing the human serum.
This work was supported by the University of Gdansk (BW/
8000-5-0125-8) and Ministry of Science and Higher Education
(Grant No. 2889/H03/2008/34).
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