Analogues of trypsin inhibitor SFTI-1 with disulfide bridge substituted by various length of carbonyl bridges

Article abstract of DOI:10.2174/092986610792231483

Series of eight new monocyclic analogues of trypsin inhibitor SFTI-1 was synthesized by the solid phase method. In these analogues disulfide bridge Cys3 - Cys11 present in native inhibitor was replaced by different-sized car- bonyl bridges formed by the amino groups of the side chain of Lys, Orn, Dab or Dap located in positions 3 and/or 11. All analogues appeared to be potent trypsin inhibitors. The values of association equilibrium constants determined with bo- vine β-trypsin ranging 10⁸ - 10⁹ M^-1 with the highest (3.90 × 10⁹ M^-1) determined for analogue containing Lys and Dap in aforementioned positions. The obtained results clearly shown that this redox stable modification is well tolerated in the structure of proteinase inhibitor. It is worth stressing that the procedure of the introduction of carbonyl bridge into the pep- tide structure is straightforward and therefore beneficial for the design of new enzyme inhibitors. Bentham Science Publishers Ltd.

Full text of DOI:10.2174/092986610792231483

Protein & Peptide Letters, 2010, 17, 1223-1227
1223
Analogues of Trypsin Inhibitor SFTI-1 with Disulfide Bridge Substituted
by Various Length of Carbonyl Bridges
Anna ꢀ gowska*, Elꢁbieta Bulak, Anna Jaꢂkiewicz, Izabella Maꢃuch, Michaꢃ Sieracki,
Magdalena Wysocka, Adam Lesner and Krzysztof Rolka
Faculty of Chemistry, University of Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland
Abstract: Series of eight new monocyclic analogues of trypsin inhibitor SFTI-1 was synthesized by the solid phase
method. In these analogues disulfide bridge Cys3 – Cys11 present in native inhibitor was replaced by different-sized car-
bonyl bridges formed by the amino groups of the side chain of Lys, Orn, Dab or Dap located in positions 3 and/or 11. All
analogues appeared to be potent trypsin inhibitors. The values of association equilibrium constants determined with bo-
vine ꢄ-trypsin ranging 108 – 109 M-1 with the highest (3.90 x 109 M-1) determined for analogue containing Lys and Dap in
aforementioned positions. The obtained results clearly shown that this redox stable modification is well tolerated in the
structure of proteinase inhibitor. It is worth stressing that the procedure of the introduction of carbonyl bridge into the pep-
tide structure is straightforward and therefore beneficial for the design of new enzyme inhibitors.
Keywords: Serine proteinases, inhibitors, synthesis, carbonyl bridge.
INTRODUCTION
Circular trypsin inhibitor SFTI-1 isolated from the sun-
Figure 1. Primary structure of trypsin inhibitor SFTI-1.
flower seeds [1] attracted interest of several research groups
in different fields because of its small size (14-amino acid
residues) and potent inhibitory activity. Garcia Boy R. et al.
[2] evaluated SFTI-1 as a possible scaffold structure for the
development of peptide-based radiopharmaceuticals that
could bind to matriptase, overexpressed in a variety of tumor
and tumor cell lines. Pereira et al. [3] investigated the appli-
cation of SFTI-1 and its analogues as ligands for isolation of
serine proteases from various biological sources using affin-
ity chromatography. The Austin et al. [4] successfully intro-
duced the biosynthesis of cyclic peptides in E.coli using in-
tramolecular native chemical ligation with a modified protein
splicing.
we decided to use monocyclic (disulfide bridged) SFTI-1 as
a starting structure for designing trypsin, chymotrypsin and
cathepsin G inhibitors [8-10]. Very recently we focused our
interest on the role of the disulfide bridge in induction of
proteinase inhibitory activity. We synthesized the series of
SFTI-1 in which Cys3 – Cys11 disulfide bridge was replaced
by disulfide bridges formed by Pen (L-pencillamine), homo-
L-cysteine (Hcy), N-sulfanylethylglycine (Nhcy) or com-
bination of these residues with Cys. In substrate specificity
P1 position of the synthesized analogues Lys, Nlys (N-(4-
aminobutyl)glycine), Phe or Nphe (N-benzylglycine) were
present and therefore they were designed to inhibit trypsin or
chymotrypsin. The obtained results have shown that Pen and
Nhcy are not acceptable in position 3, yielding inactive ana-
logues, whereas second Cys11 residue can be substituted
without significant loss of affinity towards experimental pro-
teinases. The elongation of Cys3 side chain by one methyl-
ene group (introduction of Hcy) does not affect inhibitory
activity and analogue with Hcy – Hcy disulfide bridge is
more than twice as effective as the reference compound
([Phe5]SFTI-1) in inhibition of bovine ꢅ-chympotrypsin.
In this canonical inhibitor the inhibitor reactive site P1-
P1 (according to notation of Schechter and Berger [5]),
’
which is responsible for the specificity of this inhibitor, is
located between residues Lys5 and Ser6 Fig. (1). The struc-
ture of SFTI-1 is stabilized by the two cycles: disulfide
bridge and head-to-tail cyclization. In our earlier work [6]
we have shown that monocyclic analogues of SFTI-1 retain
inhibitory activity. Elimination of the disulfide bridge de-
creased trypsin inhibitory activity 2.5-fold (analogue with
head-to-tail cyclization only), whereas monocyclic SFTI-1
with disulfide bridge inhibited bovine ꢄ-trypsin with the
same strength (determined as association equilibrium con-
stant Ka) as the native inhibitor. Our findings were in a good
agreement with the work of Korsinczky et al. [7] who proved
that disulfide bridge has a greater impact on the inhibitor’s
structure than head-to tail cyclization. Based on these results,
Bearing all this in mind, we decided to continue these
investigations changing the length of cycle bridging amino
acid residues located in positions 3 and 11. Since not many
Cys derivatives with different lengths of a side chain are
commercially available, we decided to close the ring by oth-
er covalent linkage that can give much more options to
change its size. In 2006 Guo et al. [11] synthesized SFTI-1
in which the disulfide bond was replaced by the isosteric
diselenide bond. This analogue had the increased redox sta-
bility but its activity decreased by almost one order of mag-
*Address correspondence to this author at the Faculty of Chemistry, Univer-
sity of Gdansk, Sobieskiego 18, 80-952 Gdansk, Poland;
Tel: ++48585235359; Fax:++48585235472;
E-mail: legowska@chem.univ.gda.pl
0929-8665/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

1224 Protein & Peptide Letters, 2010, Vol. 17, No. 10
ꢀꢁgowska et al.
nitude compared to that of native SFTI-1. One year later Jing
et al. [12] designed and synthesized upon the basis of SFTI-1
potent and metabolically stable ethtylene-bridged matriptase
inhibitor. This fully redox stable peptide displayed compara-
ble inhibitory activity, however the synthesis of such peptide
is robust and difficult from synthetic point of view. In 1997
Pawlak et al. [13] described side-chain-to-side-chain cycliza-
tion via a carbonyl bridge. It was achieved by linking amino
side chains of dibasic amino acids to form ureido moiety. By
applying this approach they obtained cyclic analogues of
enkephalin [14], dermorphin [15-17] and deltorphin [18].
Extensive studies on carbonyl bridge of the mentioned
opioid peptides showed that disulfide bridge can be success-
fully replaced by a carbonyl one. Availability of dibasic ami-
no acids and straightforward method of carbonyl bridge for-
mation enabled a synthesis of cyclic peptides with different
size of the ring. It should be noted that such modification has
not been explored yet in the case of proteinase inhibitors.
(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 protected amino acid derivatives were
used. After completing the syntheses, the allyl ester from the
C-terminal ꢀ-carboxyl group (peptide was attached to the
resin through ꢁ-carboxyl group) of analogue 1 was removed
using Pd(Ph3)P)4, according to the procedure described in the
literature [19]. This was followed by the deprotection of N-
terminal amino group. The head-to-tail cyclization of 1 was
performed by the method with PyBop/DIPEA (molar ratio
1:2) in DMF [20]. The progress of the reaction was moni-
tored by MS analysis using MALDI-TOF method. The cycli-
zation was completed within 6 hours. Dde and ivDde protect-
ing groups were removed from ꢂ-, ꢃ-, ꢄ- and ꢁ-amino func-
tions of Orn, Lys Dab, and Dap, respectively, peptidyl-resins
with 2% hydrazine solution in DMF [21]. In the next step,
the carbonyl bridge was introduced. This was performed in
DMF at room temperature by addition in few portions of
bis(4-nitrophenyl) carbonate (1 equiv.) [15] to the peptidyl-
resin. Progress of the reaction was monitored by the ninhy-
drin and chloranil tests and analyzed by MS. The reaction
was carried out until free amino groups were no longer de-
tected. The correctness of the product was confirmed by the
mass spectrometry (MS) method. The cyclization reaction of
the synthesized peptides was completed within nine hours.
Finally, the peptides were cleaved from the resin simultane-
ously with the side chain protecting groups in a one-step
Taking all into the consideration, we synthesized the se-
ries of monocyclic SFTI-1 analogues in which a ring forma-
tion was achieved via a ureido group incorporating the side-
chain amino groups see Fig. (2) of L-2,3-diaminopropionic
acid (Dap), L-2,4-diaminobutyric acid (Dab), Orn and Lys
placed in positions originally occupied by Cys residues. The
objective of the introduction of the set of dibasic amino acid
residues starting with Dap (one CH2 group) up to Lys (four
CH2 groups) in positions 3 and 11 was the determination of
the optimal size of the SFTI-1 cycle/ring for the inhibitor –
trypsin interaction. In addition, unlike disulfide bridge, N-
(ureidoethyl)amide moieties are redox stable. This is a very
important factor in case of introducing such peptides into the
biological system.
procedure, using
a
TFA/phenol/triisopropylsilane/water
(88:5:2:5; v/v/v/v) mixture [22].The crude peptides were
purified by HPLC on a Beckman Gold System (Beckman,
USA) using an RP Kromasil-100, C8, 5 μm column (8 ꢅ 250
mm) (Knauer, Germany). The solvent system was 0.1% TFA
(A) and 80% acetonitrile in A (B). Isocratic condition or
linear gradient was applied, flow rate 3.0 mL/min, monitored
at 226 nm. The purity of the synthesized peptides was
checked on an RP Kromasil 100, C8, 5 μm column (4.6 ꢅ
250 mm) (Knauer, Germany). The solvent system was 0.1%
TFA (A) and 80% acetonitrile in A (B). Linear gradients
from 10 to 90% B in 30 min; flow rate 1 mL/min, monitored
at 226 nm, were applied. The mass spectrometry analysis of
all peptides synthesized was carried out on a MALDI MS (a
Biflex III MALDI-TOF spectrometer, Bruker Daltonics,
Germany) using ꢀ-cyano-4-hydroksycy-namic acid matrix.
Figure 2. Chemical structure of SFTI-1 analogues with carbonyl
bridge, (x,y = 1 for Dap, 2 for Dab, 3 for Orn and 4 for Lys).
MATERIALS AND METHODS
Peptide Synthesis
Determination of Association Equilibrium Constants
Bovine ꢀ-trypsin was standardized by burst kinetics with
4-nitrophenyl-4’-guanidinobenzoate (NPGB) at an enzyme
concentration of 10-6 M [23]. The concentration of SFTI-1
analogues was determined by titration of their stock solu-
tions with standardised bovine ꢁ-trypsin with Bz-DL-Arg-
pNA (BAPNA) as substrate. The association constants were
measured by the method developed in the laboratory of M.
Laskowski, Jr., [24, 25]. The interaction between bovine ꢀ-
trypsin and the inhibitor was determined in 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 in-
hibitor, varying from 0 to 2E0 (E0 - the total enzyme concen-
tration), were added to the constant amount of the enzyme.
After a 3-hour incubation, the residual enzyme activity was
measured using Bz-Val-Gly-Arg-pNA as a chromogenic
substrate on a Cary 3E spectrophotometer (Varian, Austra-
All peptides were synthesized by the solid-phase method
using Fmoc chemistry. The following amino acid derivatives
were used: Fmoc-Gly, Fmoc-Arg(Pbf), Fmoc-Thr(tBu),
Fmoc-Ser(tBu), Fmoc-Ile, Fmoc-Pro, Fmoc-Phe, Fmoc-Asp
(OtBu), Fmoc-Asp-OAll, Fmoc-Lys(Boc), Fmoc-Lys(ivD-
de), Fmoc-Orn(Dde), Fmoc-Dab(ivDde), and Fmoc-Dap
(ivDde). The C-terminal amino acid residue Fmoc-Asp
(OtBu) or Fmoc-Asp-OAll (in the case of analogue 1) was
attached to 2-chlorotrityl chloride resin (substitution of Cl
1.46 meq/g) (Calbiochem-Novabiochem AG, Switzerland) in
the presence of an equimolar amount of DIPEA (in relation
to the amino acid derivative) in anhydrous condition in DCM
solutions. Peptide chains were elongated in the consecutive
cycles of deprotection and coupling. Deprotection was per-
formed with 20% piperidine in the mixture of DMF/NMP

Analogues of Trypsin Inhibitor SFTI-1 with Disulfide Bridge Substituted
Protein & Peptide Letters, 2010, Vol. 17, No. 10 1225
lia). In all cases the total substrate concentration was below
0.1 KM. The measurements were carried out at total enzyme
concentration of 3 nM. The experimental points were ana-
lysed by plotting the residual enzyme concentration [E] ver-
sus the total inhibitor concentration [I0]. The experimental
data were fitted to the theoretical values using the GraFit
software package [26].
Cys3 and Cys11). The shortest equivalent fragment in ana-
logues 1 and 2 (with Dap residues in positions 3 and 11)
comprises 6 chemical bonds. Taking into the account that S –
S and C – S chemical bonds are significantly longer (2.05 Å
and 1.83 Å, respectively) than C – C and C – N (1.54 Å and
1.47 Å, respectively), the size of the ring formed by Dap
residues is almost the same as in SFTI-1. Both analogues are
less potent inhibitors than the reference SFTI-1 (mono- and
dicyclic), which is probably the result of the different con-
formation flexibility of disulfide and carbonyl bridges. The
activity of dicyclic analogue 1 is more than 13-fold lower
compared to that of the native SFTI-1. Elimination of the
head-to tail cyclization (analogue 2) decreased the activity.
Also the activity of monocyclic SFTI-1 was lower than for
the native inhibitor. Interestingly, the size of the rings closed
by carbonyl bridges did not have a significant impact on the
trypsin inhibitory activity of the synthesized analogues. All
values of Ka were in the range 108 – 109 M-1 with the highest
(3.90ꢀ109 M-1) determined for analogue 8 containing Lys and
Dap in positions 3 and 11, respectively. As linear SFTI-1 is
unable to inhibit trypsin activity [5], these results suggest
that any type of cyclic element present in SFTI-1 (including
the carbonyl bridge) can induce a conformation that allows
the inhibitor to interact with enzyme.
RESULTS AND DISCUSSION
Trypsin inhibitory activity along with HPLC and MS
analyses of new SFTI-1 analogues containing carbonyl
bridges is summarized in Table 1. In the series of analogues
1 – 9 disulfide bridge was replaced by another cyclic element
– carbonyl bridge formed by the amino groups of the side
chains of Dap, Orn or Lys. These residues were introduced
in the places originally occupied by two Cys. In all ana-
logues the urea bridges were formed with high yields and the
reaction time never exceeded nine hours. An example of MS
analysis of linear and cyclic [Dap3,11]SFTI-1 (2) is shown in
Fig. (3) whereas inhibition curves of some of the synthesized
analogues are presented in Fig. (4). The presented results
clearly show that disulfide bridge naturally present in occur-
ring SFTI-1 can be successfully replaced by a carbonyl
bridge. All obtained analogues displayed inhibitory activity
of the same order of magnitude (the Ka values varied from
3.46x108 to 3.89x109 M-1) as the reference monocyclic SFTI-
1 (Ka = 9.9x109 M-1). The values of Ka determined for these
peptides with bovine ꢀ-trypsin indicate that they all display
strong inhibitory activity. Interestingly, the length of the
formed cycle did not have significant impact on the inhibi-
tory activity. The length of the disulfide bridge in SFTI-1
consists of 5 chemical bonds (counting between ꢁ-carbons of
CONCLUSIONS
All the synthesized SFTI-1 analogues in which the disul-
fide bridge was replaced by a carbonyl bridge of a different
size have high trypsin inhibitory activity, indicating that the
carbonyl bridge can be successfully utilized in the design of
proteinase inhibitors. It was proved that this redox stable
modification can be introduced into the peptide.
Table 1. Physicochemical Properties and Association Equilibrium Constants (Ka) with Bovine ꢁ-Trypsin of SFTI-1 Analogues
Containing Carbonyl Bridges
Analoguea
Ka [M-1]
Number of Chemical Bonds
in the Side-Chain Ring
MW
Calc. (Found)b
RTc
[min]
SFTI-1 native [3]
SFTI-1 [3] – ref. comp.
([Dap3,11]SFTI-1)dicyclic (1)
[Dap3,11]SFTI-1 (2)
5
5
1513.8 (1513.4)
1531.8 (1531.8)
1507.8 (1507.9)
1525.8 (1526.2)
1581.8 (1582.0)
1609.9 (1609.9)
1595.9 (1596.0)
1595.9 (1596.4)
1581.9 (1581.9)
1566.8 (1568.5)
1567.8 (1567.8)
16.7
15.4
20.9
15.6
17.0
18.3
17.6
17.5
17.4
17.3
17.4
(1.1±0.2)ꢀ1010
(9.9±1.1)ꢀ109
6
(8.07±0.81)ꢀ108
(3.46±0.27)ꢀ108
(5.42±0.52)ꢀ108
(5.91±0.51)ꢀ108
2.06±0.19)ꢀ109
(1.03±0.12)ꢀ109
(8.85±1.19)ꢀ108
(3.89±0.71)ꢀ109
(3.52±0.73)ꢀ108
6
[Orn3,11]SFTI-1 (3)
10
12
11
11
10
9
[Lys3,11]SFTI-1 (4)
[Lys3,Orn11]SFTI-1 (5)
[Orn3,Lys11]SFTI-1 (6)
[Lys3,Dab11]SFTI-1(7)
[Lys3,Dap11]SFTI-1 (8)
[Orn3,Dab11]SFTI-1(9)
9
a Except native SFTI-1 and analogue 1, the remaining analogues are monocyclic with carbonyl bridge only.
bMolecular weights of the peptides were determined on a Bruker Biflex III MALDI-TOF spectrometer (Bruker, Germany). The average masses were given.
c HPLC was performed as described in Materials and methods.

1226 Protein & Peptide Letters, 2010, Vol. 17, No. 10
ꢀꢁgowska et al.
Fig. (3). MS analysis of linear and cyclic [Dap3,11]SFTI-1 (2) and its synthetic intermediates: A) linear analogue with ivDde on ꢀ-amino group
of Dap; B) linear analogue after removal of both ivDde; C) spectrum recorded 3 hours after the start of cyclization reaction; peaks m/z 1499.7
and m/z 1525.7 correspond to linear and cyclic peptide, respectively; D) analogue (2) with carbonyl bridge.
ACKNOWLEDGMENTS
This work was supported by Ministry of Science and
Higher Education (grant no. 2889/H03/2008/34).
REFERENCES
[1]
[2]
Luckett, S.; Santiago Garcia, R.; Barker, J. J.; Konarev, A.V.;
Shewry, P.R.; Clarke, A.R.; Brady, R.L. High-resolution structure
of a potent, cyclic proteinase inhibitor from sunflower seeds. J.
Mol. Biol., 1999, 290, 525-533.
Garcia Boy, R.; Mier, W.; Nothelfer, E.M.; Altmann, A.; Eisenhut,
M.; Kolmar, H.; Tomaszowski, M.; Krämer, S.; Haberkorn, U.
Sunflower Trypsin Inhibitor 1 Derivatives as Molecular Scaffolds
for the Development of Novel Peptidic Radiopharmaceutcals. Mol.
Imaging. Biol., 2009, (in press).
[3]
[4]
Pereira, H.J.V.; salgado, M.C.O.; Oliveira, E.B. Immobilized ana-
logues of sunflower trypsin inhibitor-1 constitute a versatile group
of affinity sorbents for selective isolation of serine proteases. J.
Chromatogr. B, 2009, 877, 2039-2044.
Austin, J.; Kimura, R.H.; Woo, Y-H.; Camarero, J.A. In vivo bio-
synthesis of an Ala-scan library based on the cyclic peptide SFTI-1.
Amino Acids, 2009, [Epub ahead of print].
Fig. (4). Inhibition curves of bovine ꢀ-trypsin by: A)
[Lys3,Dap11]SFTI-1, B) [Lys3,Dab11]SFTI-1.
The equation used for calculation the association constant (Ka) is
given below:
[5]
[6]
Schechter, I.; Berger, A. On the size of the active site in proteases.
I. Papain. Biochem. Bophys. Res. Commun., 1967, 27, 157-162.
Zabꢁotna, E.; Kaꢂmierczak, K.; Jaꢃkiewicz, A.; Stawikowski, M.;
Kupryszewski, G.; Rolka, K. Chemical Synthesis and Kinetic
Study of the Smallest Naturally Occurring Trypsin Inhibitor SFTI-1

Analogues of Trypsin Inhibitor SFTI-1 with Disulfide Bridge Substituted
Protein & Peptide Letters, 2010, Vol. 17, No. 10 1227
Isolated from Sunflower Seeds and Its Analogues. Biochem.
Biophys. Res. Commun., 2002, 292, 855-859.
[16]
[17]
[18]
Filip, K.; Oleszczuk, M.; Wójcik, J.; Chung, N.N.; Schiller, P.W.;
Pawlak, D.; Zieleniak, A.; Parci ska, A.; Witkowska, E.; Izdebski,
J. Cyclic enkephalin and dermorphin analogues containing a car-
bonyl bridge. J. Pept. Sci., 2005, 11, 347-52.
[7]
[8]
Korsinczky, M. L.J.; Schirra, H.J.; Craik, D.J. Sunflower trypsin
inhibitor-1. Curr. Protein Pept. Sc., 2004, 5, 351-364.
ꢀꢁgowska, A.; Bulak, E.; Wysocka, M.; Jaꢂkiewicz, A.; Lesner, A.;
Dꢁbowski, D.; Rolka, K. Peptomeric analogues of trypsin inhibitor
SFTI-1 isolated from sunflower seeds. Bioorg. Med. Chem., 2008,
16, 5644-5652.
ꢀꢁgowska, A.; Dꢁbowski, D.; Lesner, A.; Wysocka, M.; Rolka, K.
Introduction of non-natural amino acid residues into the substrate-
specific P1 position of trypsin inhibitor SFTI-1 yields potent chy-
motrypsin and cathepsin G inhibitors. Bioorg. Med. Chem., 2009,
17, 3302-3307.
ꢀꢁgowska, A.; Dꢁbowski, D.; Lesner, A.; Wysocka, M.; Rolka, K.
Selection of peptomeric inhibitors of bovine ꢃ-chymotrypsin and
cathepsin G based on trypsin inhibitor SFTI-1 using combinatorial
chemistry approach. Mol. Divers., 2010, 14, 51-58.
Guo, X.; Shi, J.; Tang, Z.; Cui, D. Synthesis and Biological Activ-
ity of Seleno Sunflower Trypsin Inhibitor Analog. Chem. Biol.
Drug. Des., 2006, 68, 341-344.
Jiang, S.; Li, P.; Lee, S-L.; Lin, C.Y.; Long, Y-Q.; Johnson, M.D.;
Dickson R.B.; Roller, P.P Design and Synthesis of Redox Stable
Analogues of Sunflower Trypsin Inhibitors (SFTI-1) on Solid Sup-
port, Potent Inhibitors of Matriptase. Org. Lett., 2007, 9, 9-11.
Pawlak, D.; Chung, N.N.; Schiller, P.W.; Izdebski, J. Synthesis of a
novel side-chain to side-chain cyclized enkephalin analogue con-
taining a carbonyl bridge. J. Pept. Sci., 1997, 3, 277-281.
Pawlak, D.; Oleszczuk, M.; Wójcik, J.; Pachulska, M.; Chung,
N.N.; Schiller, P.W.; Izdebski, J. Highly potent side-chain to side-
chain cyclized enkephalin analogues containing a carbonyl bridge:
synthesis, biology and conformation. J. Pept. Sci., 2001, 7, 128-
140.
Witkowska, E.; Nowakowski, M.; Oleszczuk, M.; Filip, K.;
Ciszewska, M.; Chung, N.N.; Schiller, P.W.; Wójcik, J.; Izdebski,
J. Ureido group containing cyclic dermorphin(1-7) analogues: syn-
thesis, biology and conformation. J. Pept. Sci., 2007, 13, 519-528.
Ciarkowski, J.; Zieleniak, A.; Rodziewicz-Motowidꢄo, S.; Rusak,
ꢀ.; Chung, N.N.; Czaplewski, C.; Witkowska, E.; Schiller, P.W.;
Izdebski, J. Deltorphin analogs restricted via a urea bridge: struc-
ture and opioid activity. Adv. Exp. Med. Biol., 2009, 611, 491-502.
Kates, S.A.; Daniels, S.B.; Albeticio, F. Automated allyl cleavage
for continuous-flow synthesis of cyclic and branched peptides.
Anal. Biochem., 1993, 212, 303-310.
Coste, J.; Le-Nguyen, D.; Castro, B. PyBOP®: A new peptide
coupling reagent devoid of toxic by-product. Tetrahedron Lett.,
1990, 31, 205.
Rohwedder, B.; Mutti, Y.; Dumy, P.; Mutter, M. Hydrazinolysis of
Dde: Complete orthogonality with Aloc protecting groups. Tetra-
hedron Lett., 1998, 39, 1175-1178.
Sole, N.A.; Barany G. Optimization of solid-phase synthesis of
[Ala8]-dynorphin. J. Org. Chem. 1992, 57, 5399-5403.
Chase, T.; Schaw, E.M. Jr. p-Nitrophenyl-p'-guanidinobenzoate
HCl: a new active site titrant for trypsin. Biochem. Biophys. Res.
Commun., 1967, 29, 508-514.
Park, S.J. Effect of Amino Acid Replacement in Ovomucoid Third
Domains upon Their Association with Serine Proteinase. PhD The-
sis, Purdue University, IN, USA 1985.
Empie, M.W.; Laskowski, M.Jr. Thermodynamics and kinetics of
single residue replacements in avian ovomucoid third domains: ef-
fect on inhibitor interactions with serine proteinases. Biochemistry,
1982, 21, 2274-2284.
Leatherbarrow, R.J. GraFit Version 5. Erithacus Software, Horley,
2001.
[9]
[19]
[20]
[21]
[10]
[11]
[12]
[22]
[23]
[13]
[14]
[24]
[25]
[15]
Filip, K.; Oleszczuk, M.; Pawlak, D.; Wójcik, J.; Chung N.N.;
Schiller, P.W.; Izdebski, J. Potent side-chain to side-chain cyclized
dermorphin analogues containing a carbonyl bridge. J. Pept. Sci.,
2003, 9, 649-657.
[26]
Received: January 29, 2010
Revised: April 19, 2010
Accepted: May 10, 2010