Efficient enzyme-catalyzed synthesis of peptide secondary amides for use as serine proteinase inhibitors

Article abstract of DOI:10.1016/j.molcatb.2012.04.019

An efficient and straightforward approach toward enzymatic synthesis of secondary amides of peptides was developed. A number of peptide derivatives containing amide moiety at C-terminus was obtained via subtilisin catalysis in good yields under mild conditions. Biological activity of some peptamides is evaluated.

Full text of DOI:10.1016/j.molcatb.2012.04.019

Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Efficient enzyme-catalyzed synthesis of peptide secondary amides for use as
serine proteinase inhibitors
Maxim E. Sergeeva,∗, Tatiana L. Voyushinaa, Olga A. Sergeevab, Galina G. Belozerskayab
a Laboratory of Protein Chemistry after V. M. Stepanov, Institute of Microbial Genetics, Moscow, Russia
b Laboratory for Pathology and Pharmacology of Hemostasis, Hematology Research Center, Russian Academy of Medical Sciences, Moscow, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient and straightforward approach toward enzymatic synthesis of secondary amides of peptides
was developed. A number of peptide derivatives containing amide moiety at C-terminus was obtained
via subtilisin catalysis in good yields under mild conditions. Biological activity of some peptamides is
evaluated.
Received 18 October 2011
Received in revised form 19 April 2012
Accepted 20 April 2012
Available online 3 May 2012
© 2012 Elsevier B.V. All rights reserved.
Keywords:
Peptides
Peptamides
Enzyme
Catalysis
Medicinal chemistry
1. Introduction
enzyme from an enzymatic mixture or a biological sample [11–16].
The peptidic ligands with better specificity to the enzyme are
required, for example peptidoaldehydes and peptide cyclic amides
[17–21]. Thus, until the present work there existed no satisfactory
1.1. Statement of significance
Cyclic amides of amino acids and peptides, which are inhibitors
of serine proteinases such as proteinases of haemostasis system
(thrombin, plasmin, etc.), can be used in medicine as anti-bleeding
or anti-thrombotic agents [1–3]. There is also a number of reports
covering the applicability of peptide amides for the treatment
of various conditions such as obesity and obesity-related dis-
eases (in which case they act as the ligands for melanocortin
receptors), hypertension, congestive heart failure and other blood
pressure-related disorders (renin inhibitors), cancer and immune
dysfunctions (tyrosine phosphatase inhibitors) [4–7]. Also, imi-
dazole derivatives of peptides and amino acids can be directly
converted into ␣-aminoaldehydes and peptidoaldehydes, which
in turn are inhibitors of plasmin and thrombin [8–10]. Affinity
chromatography is another important field where cyclic amides
of peptides are extensively used often offering many advantages
over other ligands. The affinity purification of proteins is based on
the functional characteristics of enzymes and their binding affinity
to the different ligands making it possible to separate the required
general procedure for the synthesis of such compounds, especially
cyclic amides, which are more stable than peptidoaldehydes.
1.2. Known synthetic procedures
Prior to our studies Kikumoto et al. prepared the N-protected
arginyl-piperidines by the mixed anhydrides method starting from
N-protected arginine and 4-methylpiperidine with subsequent
deprotection of the product [22]. However, this method includes
the steps of protection–deprotection of arginine side-group, which
procedure is pretty hard. Synthesis of morpholine derivatives of
leucine and arginine via coupling of N-protected amino acids and
morpholine in the presence of 1-hydroxybenzotriazole (HOBt) and
dicyclohexylcarbodiimide (DCC) at room temperature is described
earlier. The reaction yields were reasonably high (up to 80%) though
it took up to four days for the reaction to be completed [21]. The
piperidine derivatives of tryptophan-like compounds were pre-
pared in low yields (40–60%) by Reichert et al. via coupling of the
carboxylic acid derivatives and the piperidine with Mukayama’s
reagent (2-chloro-1-methylpyridinium iodide (CMPI)) as the cou-
pling agent [23]. Our procedure described earlier allows to obtain
secondary amides of amino acids with high yields [24], but in
further synthesis the yield of final tripeptide product decreases
dramatically when the conventional methods of peptide synthesis
∗
Corresponding author at: Laboratory of Protein Chemistry after V. M. Stepanov,
Institute of Microbial Genetics, 1st Dorozhny pr., 1, Moscow, 117545, Russia.
Tel.: +7 495 315 37 38; fax: +7 495 315 05 01.
E-mail address: m.e.sergeev@gmail.com (M.E. Sergeev).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.04.019

M.E. Sergeev et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66
59
Table 1
are used, such as activated-esters synthesis, or HOBt/DCC-coupling
Gradient elution for HPLC analysis.
of amino acid derivatives [25–27]. Synthesis of aspartic protease
inhibitors using Mukayama aldol condensation led to target com-
pounds in moderate yields of 20–60%. This procedure was also a
complicated one requiring multi-step synthesis [28].
Time, min
Flow rate, ml/min
%A
%B
0
10
15
20
1
1
1
1
80
40
40
80
20
60
60
20
Enzyme-catalyzed synthetic procedures spawned great appli-
cation field since it became now possible to obtain the desired
target compounds with excellent yields under mild conditions and
in most cases the purification procedures for reaction mixtures are
simple and short. Also, in case of enzymatic catalysis, the acylating
agents with non-protected side groups can be used and the optical
purity of the product can be preserved. Use of enzymes with broad
substrate specificity made it possible to carry out the experiments
with different nucleophiles, which in turn led to the synthesis of
a wide spectrum of potentially bioactive compounds with possible
use in various medical areas.
on thiopental-anaesthetized Shinshilla rabbits showed that bleed-
ing of liver surface wound (with a size approximately 2 cm × 2 cm)
was arrested in about 60–70 s vs 205 s with standard anti-bleeding
agent (␧-aminocaproic acid) [41].
2. Materials and methods
2.1. Enzyme and chemicals
A number of reports dealing with the enzyme-catalyzed syn-
thesis of bioactive compounds using different enzymes [29,30].
Maruyama et al. described the synthesis of sugar amino acid
esters using proteases (Subtilisin Carlsberg and others) [31]. It was
shown that conversion of starting material in organic media such
as pyridine at 40 ◦C did not exceed 50% after 24 h. Application
of enzyme–surfactant complex allows to achieve ∼65% conver-
sion at these conditions but the yields of target compounds are
still low. The chemoenzymatic synthesis of peptidomimetics based
on (R)-3,4-diaminobutanoic acid utilizing combination of solid-
phase synthesis and enzymatic catalysis with lipase B from Candida
antarctica was reported. The yield of title compound was found to
be about 76% after 3 days at 30 ◦C [32]. There are also some reports
about subtilisin-catalyzed peptide synthesis under high pressure,
but the yields of these reactions are still low (about 30%) [33,34].
Some papers dealt with enzyme-catalyzed synthesis of primary
amide derivatives of peptides deals with few primary amines only
and the yields of the acylation reaction catalyzed by subtilisin BPN’
were as high as 40–70% [40].
The research of enzyme-catalyzed synthesis of various sub-
strates and enzyme-inhibitors has been the most important
purpose of our laboratory for many years. We have reported a num-
ber of efficient methods for the synthesis of peptides and different
peptide-derivatives which utilized thermolysin, various subtilisins,
trypsin, ␣-chymotrypsin and others [35–39]. The most effective
among the enzymes used was subtilisin [41]. Highly aqueous media
are undesirable in enzymatic reactions due to easy hydrolysis of
acylating agent [42]. After having been bound on the solid-phase
carrier, the enzyme becomes potentially vulnerable to the carrier
hydrate water; but due to the lack of free water molecules in solvent
hydrolysis process was strongly reduced [43,44]. Earlier we demon-
strated that the usage of solid-phase-bound subtilisin-72 (serine
proteinase from Bacillus subtilis st. 72) distributed on porous carrier
Silochrom-C80 allowed to carry out the synthesis in organic media,
which were generally non-specific for enzymatic synthesis due to
enzyme inactivation by organic solvent. We have also proved the
feasibility of peptamide synthesis by acylation of amino acid amides
with peptide esters. The reaction between For-Ala-Phe-OMe and
LysPip afforded the tripeptide ForAlaPhe-LysPip in high yield [45].
By this procedure we synthesized the two novel syn-
thetic peptide amides, namely formyl-l-alanyl-l-phenylalanyl-
l-lysyl-piperidide (For-Ala-Phe-Lys-Pip) and formyl-l-alanyl-l-
phenylalanyl-l-lysyl-morpholide (For-Ala-Phe-Lys-Mrf) of formu-
lae 1 and 2 (Fig. 1), and tested them for the plasmin-inhibiting
activity in vitro and in vivo. In vitro experiments proved that these
peptides are good inhibitors of euglobulin factor XIIa-dependent
lysis (time of thrombi degradation about 21–23 s while 40–50 s
with tranexamic acid or ␧-aminocaproic acid which are standard
bleeding-terminating agents) and plasmin inhibition constants Ki
are 19.20 ␮M and 19.08 ␮M correspondingly. In vivo experiments
Subtilisin-72 was isolated and purified by common methods
from B. subtilis st. 72. All other chemicals used were of analytical
grade and purchased from Sigma–Aldrich and Serva.
2.2. Analytical methods
The aminolysis reaction was monitored by RP-HPLC analysis
carried out in a system (Beckman System Gold) composed of a col-
umn (Beckman C18 5 ␮M, 250 mm × 4.6 mm) and a UV/vis detector
(Beckman System Gold, model 167, ꢀ = 220 nm). The compounds
were eluted by gradient elution with an eluent system of 5%
acetonitrile/H2O (A) and 95% acetonitrile/H2O (B), both containing
0.05% of trifluoroacetic acid (see Table 1).
2.3. Determination of enzyme activity
Z-Ala-Ala-Leu-pNA solution in DMF (50 ml, 5 mg/mL) was added
to 2 ml of 0.05 M Tris/HCl buffer (pH 8.3) containing 1.5 mM CaCl2
and the mixture was incubated at 20 ◦C for 10 min. Then, immo-
bilized enzyme (20 mg subtilisin/250 mg silochrom) was added to
initiate the reaction and the suspension was incubated at 20 ◦C
with stirring for 1–4 d. Thereafter, the absorbance of reaction mix-
ture was measured at ꢀ 410 nm. Qualitative curves are plotted in
coordinates A410 – reaction time.
2.4. Enzymatic synthesis of peptide amides
␻
General procedure for the synthesis of N -(benzyloxycarbonyl)-
l-alanyl-l-alanyl-l-(O-methyl)glutamic acid (imidazol-1-yl)amide
(Cbz-AlaAlaGlu(OMe)-Im): subtilisin-72 (from B. subtilis, strain 72)
(20 mg, 0.69 ␮mol) was dissolved in phosphate buffer (pH 7.5,
600 ␮l). The obtained solution was deposited dropwise on the sur-
face of silochrom-C80 (250 mg), placed in flat-bottom flask as a
‘single-layer’. The flask was placed under high vacuum and dried
over phosphorous pentoxide for 3 days.
␻
N -(benzyloxycarbonyl)-l-alanyl-l-alanyl-l-(O-
methyl)glutamic acid methyl ester (46 mg, 100 ␮mol) was
dissolved in 1.5 ml of freshly distilled acetonitrile to prepare
solution A. Imidazole (68 mg, 1000 ␮mol) was dissolved in 1 ml
of freshly distilled acetonitrile to prepare solution B. The solu-
tions A and B were mixed together and the catalyst was added
immediately after being unvacuumed. The flask was sealed and
the reaction mixture was stirred by means of orbital shaker for
5 days at room temperature (about 18–22 ◦C). The catalyst was
filtered from acetonitrile and washed thoroughly with methanol
(3 × 10 ml) under sonication. The fraction with less product content
was discarded. The remaining fraction was filtered and evaporated.
The residue was dissolved in methanol (500 ␮l) and precipitated

60
M.E. Sergeev et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66
O
N
N
O
O
HN
HN
HN
O
O
HN
HN
O
O
HCl
HCl
NH2
NH2
HN
O
O
1
2
Fig. 1. Structures of synthetic peptide-amides formyl-l-alanyl-l-phenylalanyl-l-lysyl-piperidide hydrochloride (For-Ala-Phe-Lys-Pip*HCl)
phenylalanyl-l-lysyl-morpholide hydrochloride (For-Ala-Phe-Lys-Mrf*HCl) 2.
1 and formyl-l-alanyl-l-
with diethyl ether (5 ml). The resulted solid was filtered out and
dried in vacuum. Title compound was obtained as a cream solid,
yield 96%.
HRMS (ES+): 508.2277 found, 508.2282 calculated.
␻
N
-(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-leucine
(imidazol-1-yl)amide (Dnp-AlaAlaLeu-Im):
1H NMR (400 MHz, d6-DMSO): ı 1.2–1.3 (6H, 2*CH3-Ala),
2.1–2.3 (m, 4H, 2*CH2, Glu), 3.6 (s, 3H, GluOCH3), 4.3–4.4 (m, 2H,
2*CH-Ala), 4.7–4.8 (m, 1H, CH-Glu), 5.1 (s, 2H, CH2-Cbz), 6.9 (d, 1H,
CH-Im), 7.3–7.4 (m, 5H, C6H5-Cbz), 7.6 (s, 3H, 3*NH), 8.1–8.2 (d, 1H,
CH-Im), 8.7 (s, 1H, CH-Im) ppm.
Yellow solid.
1H NMR (400 MHz, d6-DMSO): ı 0.87–0.92 (d, 6H, 2*CH3-Leu),
1.05–1.15 (m, 2H, CH2-Leu), 1.3–1.35 (m, 1H, CH-Leu), 1.2/1.4 (dd,
6H, 2*CH3-Ala), 3.9/4.2/4.6 (m, 3H, CH-(Ala/Ala/Leu), 6.9 (1H, d, 4-
CH-Im), 8.08 (1H, d, 5-CH-Im), 7, 8 (dd, 2H, 5,6-CH-Dnp), 8.55 (s,
3H, 3*NH), 8.7 (1H, s, 3-CH-Im), 8.8 (s, 1H, 3-CH-Dnp) ppm.
MS (MALDI-TOF): 490.6 [M+H].
MS (MALDI-TOF): 474.5 [M+H].
HRMS (ES+): 473.1915 found, 473.1910 calculated.
Scaled-up experiment, utilizing 1.5 g (3.2 mmol) of peptide
ester, resulted in the same yield of desired product without sig-
nificant increase of hydrolysis product formation.
In a similar way, using appropriate N-protected peptidic esters
and cyclic amines as starting materials the following peptamides
HRMS (ES+): 489.2001 found, 489.1972 calculated.
␻
N
-(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-(O-methyl)
glutamic acid (piperidin-1-yl)amide (Dnp-AlaAlaGlu(OMe)-Pip):
Yellow solid.
1H NMR (400 MHz, d6-DMSO): ı 1.1–1.25 (6H, 2*CH3-Ala),
1.4–1.6 (m, 6H, 2*m-CH2-Pip + p-CH2-Pip), 2–2.2 (m, 4H, 2*CH2,
Glu), 3.1–3.3 (dd, 4H, 2*o-CH2-Pip), 3.55 (s, 3H, GluOCH3), 4.3–4.4
(m, 2H, 2*CH-Ala), 4.7–4.8 (m, 1H, CH-Glu), 7–8 (dd, 2H, 5,6-CH-
Dnp), 7.6 (s, 3H, 3*NH), 8.8 (s, 1H, 3-CH-Dnp) ppm.
MS (MALDI-TOF): 537.2 [M+H].
were obtained:
␻
N
-(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-leucine
(piperidin-1-yl)amide (Dnp-AlaAlaLeu-Pip):
Orange solid.
1H NMR (400 MHz, d6-DMSO):
ı 0.8–0.9 (d, 6H, 2*CH3-
Leu), 1–1.1 (m, 2H, CH2-Leu), 1.3–1.35 (m, 1H, CH-Leu), 1.4–1.6
(m, 6H, 2*m-CH2-Pip + p-CH2-Pip), 1.23/1.47 (dd, 6H, 2*CH3-Ala),
3.9/4.2/4.6 (m, 3H, CH-(Ala/Ala/Leu), 3.1–3.3 (dd, 4H, 2*o-CH2-Pip),
7, 8 (dd, 2H, 5,6-CH-Dnp), 8.55 (s, 3H, 3*NH), 8.8 (s, 1H, 3-CH-
Dnp) ppm.
HRMS (ES+): 536.2236 found, 536.2231 calculated.
␻
N
-(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-(O-methyl)
glutamic acid (morpholin-1-yl)amide (Dnp-AlaAlaGlu(OMe)-
Mrf):
Yellow solid.
MS (MALDI-TOF): 507.2 [M+H].
1H NMR (400 MHz, d6-DMSO): ı 1.1–1.25 (6H, 2*CH3-Ala), 2–2.2
(m, 4H, 2*CH2, Glu), 3.1–3.2 (dd, 4H, 2*o-CH2-Mrf), 3.5–3.6 (m, 4H,
2*m-CH2-Mrf), 3.55 (s, 3H, GluOCH3), 4.3–4.4 (m, 2H, 2*CH-Ala),
4.7–4.8 (m, 1H, CH-Glu), 7–8 (dd, 2H, 5,6-CH-Dnp), 7.6 (s, 3H, 3*NH),
8.8 (s, 1H, 3-CH-Dnp) ppm.
HRMS (ES+): 506.5528 found, 506.5521 calculated.
␻
N
-(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-leucine
(3-
carboxy-4-hydroxyphenyl)amide (Dnp-AlaAlaLeu-ASA):
Yellow solid.
1H NMR (400 MHz, d6-DMSO): ı 0.8–0.9 (d, 6H, 2*CH3-Leu),
1–1.1 (m, 2H, CH2-Leu), 1.3–1.35 (m, 1H, CH-Leu), 1.23/1.47 (dd, 6H,
2*CH3-Ala), 3.9/4.2/4.6 (m, 3H, CH-(Ala/Ala/Leu), 7, 8 (dd, 2H, 5,6-
CH-Dnp), 6.9–7.4 (2*d, 2H, 2*5,6-CH-ASA), 8.3 (s, 1H, 3-CH-ASA),
8.8 (s, 1H, 3-CH-Dnp), 9.0 (s, 3H, 3*NH) ppm. 4-OH- and 3-COOH-
protons overlapped by NH signals.
MS (MALDI-TOF): 579.3 [M+H].
HRMS (ES+): 578.2020 found, 578.2023 calculated.
␻
N
-(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-(O-methyl)
glutamic acid diethylamide (Dnp-AlaAlaGlu(OMe)-DEA):
Yellow solid.
1H NMR (400 MHz, d6-DMSO): ı 0.9–1 (m, 6H, 2*CH3-Et),
3.3–3.4 (m, 4H, 2*CH2-Et), 1.1–1.25 (6H, 2*CH3-Ala), 2–2.2 (m,
4H, 2*CH2, Glu), 3.55 (s, 3H, GluOCH3), 4.3–4.4 (m, 2H, 2*CH-Ala),
4.7–4.8 (m, 1H, CH-Glu), 7–8 (dd, 2H, 5,6-CH-Dnp), 7.6 (s, 3H, 3*NH),
8.8 (s, 1H, 3-CH-Dnp) ppm.
MS (MALDI-TOF): 575.6 [M+H].
HRMS (ES+): 574.2015 found, 574.2023 calculated.
␻
N
-(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-leucine
(morpholin-4-yl)amide (Dnp-AlaAlaLeu-Mrf):
Orange solid.
MS (MALDI-TOF): 525.3 [M+H].
1H NMR (400 MHz, d6-DMSO): ı 0.8–0.9 (d, 6H, 2*CH3-Leu),
1–1.1 (m, 2H, CH2-Leu), 1.3–1.35 (m, 1H, CH-Leu), 1.23/1.47 (dd,
6H, 2*CH3-Ala), 3.9/4.2/4.6 (m, 3H, CH-(Ala/Ala/Leu), 3.1–3.2 (dd,
4H, 2*o-CH2-Mrf), 3.5–3.6 (m, 4H, 2*m-CH2-Mrf), 7–8 (dd, 2H, 5,6-
CH-Dnp), 8.55 (s, 3H, 3*NH), 8.8 (s, 1H, 3-CH-Dnp) ppm.
MS (MALDI-TOF): 509.6 [M+H].
HRMS (ES+): 524.2033 found, 524.2031 calculated.
␻
N
-(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-(O-methyl)
glutamic acid (pyridin-4-yl)amide (Dnp-AlaAlaGlu(OMe)-APy):
Yellow solid.
1H NMR (400 MHz, d6-DMSO): ı 1.1–1.25 (6H, 2*CH3-Ala),
2–2.2 (m, 4H, 2*CH2, Glu), 3.55 (s, 3H, GluOCH3), 4.3–4.4 (m, 2H,

M.E. Sergeev et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66
61
2*CH-Ala), 4.7–4.8 (m, 1H, CH-Glu),7–8 (dd, 2H, 5,6-CH-Dnp), 8.8
(s, 1H, 3-CH-Dnp), 7.5 (s, 3H, 3*NH), 7.6–8.6 (2*dd, 4H, 4*CH-Py)
8.1 (d, 1H, 5-CH-Im) ppm.
HRMS (ES+): 537.2664 found, 537.2659 calculated.
N -(benzyloxycarbonyl)-l-alanyl-l-alanyl-l-leucine
(imidazol-1-yl)amide (Cbz-AlaAlaLeu-Im):
␻
MS (MALDI-TOF): 546.2 [M+H].
White solid.
HRMS (ES+): 545.1875 found, 545.1870 calculated.
1H NMR (400 MHz, d6-DMSO): ı 1.1–1.3 (m, 2H, ␻-CH2-Lys), 1.2
(m, 6H, 2*CH3-Ala), 1.4–2 (m, 3H, (CH2)3-Lys), 1.3 (m, 1H, CH-Lys),
2.8–3 (t, 2H, CH2-Phe), 4–4.1 (m, 1H, CH-Ala), 4.3 (t, 1H, CH-Phe),
4.7 (t, 1H, CH-Lys), 5.0 (s, 2H, CH2-Bn), 6.9 (d, 1H, 4-CH-Im), 7.7 (s,
5H, 3*NH), 7.3 (m, 5H, C6H5-Phe), 8.1 (d, 1H, 5-CH-Im), 8.8 (s, 1H,
2-CH-Im) ppm.
␻
N
-(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-(O-methyl)
glutamic acid (imidazol-1-yl)amide (Dnp-AlaAlaGlu(OMe)-Im):
Yellow solid.
1H NMR (400 MHz, d6-DMSO): ı 1.1–1.25 (6H, 2*CH3-Ala), 2–2.2
(m, 4H, 2*CH2, Glu), 3.55 (s, 3H, GluOCH3), 4.3–4.4 (m, 2H, 2*CH-
Ala), 4.7–4.8 (m, 1H, CH-Glu), 6.9 (d, 1H, CH-Im), 7–8 (dd, 2H, 5,6-
CH-Dnp), 8.8 (s, 1H, 3-CH-Dnp), 7.6 (s, 3H, 3*NH), 8.1 (d, 1H, 5-CH-
Im), 8.8 (s, 1H, 2-CH-Im) ppm.
MS (MALDI-TOF): 458.6 [M+H].
HRMS (ES+): 457.2321 found, 457.2325 calculated.
␻
N
-(benzyloxycarbonyl)-l-alanyl-l-alanyl-l-(O-methyl)
MS (MALDI-TOF): 520.1 [M+H].
glutamic acid (indol-1-yl)amide (Cbz-AlaAlaGlu(OMe)-Ind):
White solid.
HRMS (ES+): 519.1717 found, 519.1714 calculated.
␻
N
-(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-(O-methyl)
1H NMR (400 MHz, d6-DMSO): ı 1.2–1.3 (6H, 2*CH3-Ala),
2.1–2.3 (m, 4H, 2*CH2, Glu), 3.6 (s, 3H, GluOCH3), 4.3–4.4 (m, 2H,
2*CH-Ala), 4.7–4.8 (m, 1H, CH-Glu), 5.1 (s, 2H, CH2-Cbz), 6.9 (d, 1H,
CH-Im), 7.3–7.4 (m, 5H, C6H5-Cbz), 7.6 (s, 3H, 3*NH), 6.2–8.2 (m,
6H, Ind) ppm.
glutamic acid (indol-1-yl)amide (Dnp-AlaAlaGlu(OMe)-Ind):
Yellow solid.
1H NMR (400 MHz, d6-DMSO): ı 1.2–1.3 (6H, 2*CH3-Ala),
2.1–2.3 (m, 4H, 2*CH2, Glu), 3.6 (s, 3H, GluOCH3), 4.3–4.4 (m, 2H,
2*CH-Ala), 4.7–4.8 (m, 1H, CH-Glu), 6.2–8.2 (m, 6H, Ind), 7–8 (dd,
2H, 5,6-CH-Dnp), 7.6 (s, 3H, 3*NH), 8.8 (s, 1H, 3-CH-Dnp) ppm.
MS (MALDI-TOF): 569.3 [M+H].
MS (MALDI-TOF): 537.3 [M+H].
HRMS (ES+): 536.2272 found, 536.2271 calculated.
␻
N
-(benzyloxycarbonyl)-l-alanyl-l-alanyl-l-leucine diethy-
HRMS (ES+): 568.1815 found, 568.1819 calculated.
lamide (Cbz-AlaAlaLeu-DEA):
␻
N
-(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-arginine
Cream solid.
(morpholin-4-yl)amide (Dnp-AlaAlaArg-Mrf):
Yellow solid.
1H NMR (400 MHz, d6-DMSO): ı 0.9–1 (m, 6H, 2*CH3-Et),
3.3–3.4 (m, 4H, 2*CH2-Et), 1.1–1.3 (m, 2H, ␻-CH2-Lys), 1.2 (m, 6H,
2*CH3-Ala), 1.4–2 (m, 3H, (CH2)3-Lys), 1.3 (m, 1H, CH-Lys), 2.8–3
(t, 2H, CH2-Phe), 4–4.1 (m, 1H, CH-Ala), 4.3 (t, 1H, CH-Phe), 4.7
(t, 1H, CH-Lys), 5.0 (s, 2H, CH2-Bn), 7.7 (s, 5H, 3*NH), 7.3 (m, 5H,
C6H5-Phe) ppm.
1H NMR (400 MHz, d6-DMSO): ı 1.25–1.46 (m, 2H, ␣-CH2-Arg),
1.2/1.4 (m, 2*CH3-Ala), 1.7 (m, 1H, ␤-CH2-Arg), 3.26 (m, 1H, ␥-CH2-
Arg), 3.2–3.6 (m, 8H, 4-CH2-Mrf), 3.9 (m, 2H, 2*CH-Ala), 7.1 (d, 1H,
6-CH-Dnp), 7.7 (s, 7H, 3*NH-Ala-Ala-Arg + NH + NH + NH2-Arg), 8.5
(d, 1H, 5-CH-Dnp), 8.7 (s, 1H, 3-CH-Dnp) ppm.
MS (MALDI-TOF): 553.6 [M+H].
MS (MALDI-TOF): 495.5 [M+H].
HRMS (ES+): 494.2481 found, 494.2489 calculated.
␻
HRMS (ES+): 552.2444 found, 551.2452 calculated.
N
-(benzyloxycarbonyl)-l-alanyl-l-alanyl-l-glutamic acid
␻
N
-(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-arginine
(imidazol-1-yl)amide (Cbz-AlaAlaGlu(OH)-Im):
White solid.
(imidazol-1-yl)amide (Dnp-AlaAlaArg-Im):
Yellow solid.
1H NMR (400 MHz, d6-DMSO): ı 1.2–1.3 (6H, 2*CH3-Ala),
2.1–2.3 (m, 4H, 2*CH2, Glu), 4.3–4.4 (m, 2H, 2*CH-Ala), 4.7–4.8 (m,
1H, CH-Glu), 5.0 (s, 2H, CH2-Cbz), 6.9 (d, 1H, CH-Im), 7.3–7.4 (m,
5H, C6H5-Cbz), 7.6 (s, 3H, 3*NH), 6.9, 8.1, 8.8 (3*s, 3H, 3*CH-Im),
6.2–8.2 (m, 6H, Ind) ppm.
1H NMR (400 MHz, d6-DMSO): ı 1.25–1.46 (m, 2H, ␣-CH2-Arg),
1.2/1.4 (m, 2*CH3-Ala), 1.7 (m, 1H, ␤-CH2-Arg), 3.26 (m, 1H, ␥-CH2-
Arg), 3.9 (m, 2H, 2*CH-Ala), 6.9 (d, 1H, 4-CH-Im), 7.1 (d, 1H, 6-CH-
Dnp), 7.7 (s, 7H, 3*NH-Ala-Ala-Arg + NH + NH + NH2-Arg), 7.9 (d, 1H,
5-CH-Im), 8.5 (d, 1H, 5-CH-Dnp), 8.7 (s, 1H, 3-CH-Dnp), 8.7 (s, 1H,
2-CH-Im) ppm.
MS (MALDI-TOF): 491.6 [M+H].
HRMS (ES+): 490.2419 found, 490.2427 calculated.
␻
MS (MALDI-TOF): 533.5 [M+H].
N
-(formyl)-l-alanyl-l-phenylalanyl-l-lysine (morpholin-1-
HRMS (ES+): 532.2155 found, 532.2142 calculated.
yl)amide (For-AlaPheLys-Mrf):
␻
N
-(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-arginine
White solid.
(pyrazol-1-yl)amide (Dnp-AlaAlaArg-Pyr):
1H NMR (400 MHz, d6-DMSO): ı 1.4–1.8 (m, 3H, (CH2)3-Lys),
1,6 (s, 3H, CH3-Ala), 2.6 (m, 2H, ␻-CH2-Lys), 2.8–3 (t, 2H, CH2-Phe),
3.2–3.6 (m, 8H, 4*CH2-Mrf), 4–4.1 (m, 1H, CH-Ala), 4.3 (t, 1H, CH-
Phe), 4.7 (t, 1H, CH-Lys), 6.1 (s, 5H, 3*NH + NH2), 7.2 (m, 5H, C6H5-
Phe), 7.4 (s, 1H, For) ppm.
Yellow solid.
1H NMR (400 MHz, d6-DMSO): ı 1.25–1.46 (m, 2H, ␣-CH2-
Arg), 1.2/1.4 (m, 2*CH3-Ala), 1.7 (m, 1H, ␤-CH2-Arg), 3.26 (m,
1H, ␥-CH2-Arg), 3.9 (m, 2H, 2*CH-Ala), 6.2 (d, 1H, 4-CH-Pyr), 7.1
(d, 1H, 6-CH-Dnp), 7.4 (d, 1H, 3-CH-Pyr), 7.7 (s, 7H, 3*NH-Ala-
Ala-Arg + NH + NH + NH2-Arg), 8.5 (d, 1H, 5-CH-Dnp), 8.7 (s, 1H,
3-CH-Dnp), 8.7 (s, 1H, 5-CH-Pyr) ppm.
MS (MALDI-TOF): 461.6 [M+H].
HRMS (ES+): 461.5545 found, 461.5512 calculated.
␻
N
-(formyl)-l-alanyl-l-phenylalanyl-l-lysine (piperidin-1-
MS (MALDI-TOF): 533.3 [M+H].
yl)amide (For-AlaPheLys-Pip):
HRMS (ES+): 532.2147 found, 532.2142 calculated.
White solid.
␻
N
-(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-arginine diethy-
1H NMR (400 MHz, d6-DMSO): ı 1.35–1.9 (m, 3H, (CH2)3-Lys),
1.5 (s, 3H, CH3-Ala), 1.5–1.6 (m, 6H, 3*CH2-Pip), 2.5–2.6 (m, 2H, ␻-
CH2-Lys), 2.7–3 (t, 2H, CH2-Phe), 3.1–3.3 (m, 4H, 2*CH2-Pip), 4–4.1
(m, 1H, CH-Ala), 4.3 (t, 1H, CH-Phe), 4.7 (t, 1H, CH-Lys), 6.1 (s, 5H,
3*NH + NH2), 7.2 (m, 5H, C6H5-Phe), 7.4 (s, 1H, For) ppm.
MS (MALDI-TOF): 459.6 [M+H].
lamide (Dnp-AlaAlaArg-DEA):
Yellow solid.
1H NMR (400 MHz, d6-DMSO): ı 0.9–1 (m, 6H, 2*CH3-Et),
1.25–1.46 (m, 2H, ␣-CH2-Arg), 1.2/1.4 (m, 2*CH3-Ala), 1.7 (m, 1H,
␤-CH2-Arg), 3.26 (m, 1H, ␥-CH2-Arg), 3.3–3.4 (m, 4H, 2*CH2-Et),
3.9 (m, 2H, 2*CH-Ala), 7.1 (d, 1H, 6-CH-Dnp), 7.7 (s, 7H, 3*NH-
Ala-Ala-Arg + NH + NH + NH2-Arg), 8.5 (d, 1H, 5-CH-Dnp), 8.7 (s, 1H,
3-CH-Dnp) ppm.
HRMS (ES+): 459.2820 found, 459.2846 calculated.
␻
N
-(formyl)-l-alanyl-l-phenylalanyl-l-lysine
(indol-1-yl)
amide (For-AlaPheLys-Ind):
MS (MALDI-TOF): 538.6 [M+H].
White solid.

62
M.E. Sergeev et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66
1H NMR (400 MHz, d6-DMSO): ı 1.4–2 (m, 3H, (CH2)3-Lys), 1,6
O
acetonitrile, rt
(s, 3H, CH3-Ala), 2.6 (m, 2H, ␻-CH2-Lys), 2.8–3 (t, 2H, CH2-Phe),
4–4.1 (m, 1H, CH-Ala), 4.3 (t, 1H, CH-Phe), 4.7 (t, 1H, CH-Lys), 6.1
(s, 5H, 3*NH + NH2), 7.2 (m, 5H, C6H5-Phe), 7.4 (s, 1H, For), 7.1–8.4
(m, 6H, Ind) ppm.
R1
R2
Xbb
Xcc
+
Xaa
PG
O
N
H
Subtilisin-72/Silochrom-C80
3
4
O
MS (MALDI-TOF): 491.3 [M+H].
Xbb
R1
HRMS (ES+): 491.2525 found, 491.2533 calculated.
Xaa
PG
Xcc
␻
N
N
-(formyl)-l-alanyl-l-phenylalanyl-l-lysine
(DL-
homoprolin-1-yl)amide (For-AlaPheLys-hP):
R2
5
White solid.
1H NMR (400 MHz, d6-DMSO): ı 1.3–1.8 (mm, 9H, 3*CH2-
hP + (CH2)3-Lys), 1,6 (s, 3H, CH3-Ala), 2.6 (m, 2H, ␻-CH2-Lys), 2.8–3
(t, 2H, CH2-Phe), 4–4.1 (m, 1H, CH-Ala), 4.3 (t, 1H, CH-Phe), 4.7 (t,
1H, CH-Lys), 5.37 (d, 1H, CH-hP), 7.0 (s, 5H, 3*NH + NH2), 7.0 (s, 1H,
COOH-hP), 7.2 (m, 5H, C6H5-Phe), 7.4 (s, 1H, For) ppm.
MS (MALDI-TOF): 503.4 [M+H].
Scheme 1. Enzyme-catalyzed synthesis pf peptamides.
␧-aminocaproic acids were used as positive control (known and
widely used agents).
HRMS (ES+): 503.2661 found, 503.2744 calculated.
␻
N
-(formyl)-l-alanyl-l-phenylalanyl-l-lysine diethylamide
3. Results and discussion
(For-AlaPheLys-DEA):
White solid.
3.1. Novel route to the enzymatic synthesis of peptamides
1H NMR (400 MHz, d6-DMSO): ı 0.9, 3.4 (2*m, 6H, 2*Et), 1.4–2
(m, 3H, (CH2)3-Lys), 1,6 (s, 3H, CH3-Ala), 2.6 (m, 2H, ␻-CH2-Lys),
2.8–3 (t, 2H, CH2-Phe), 4–4.1 (m, 1H, CH-Ala), 4.3 (t, 1H, CH-Phe),
4.7 (t, 1H, CH-Lys), 6.1 (s, 5H, 3*NH + NH2), 7.2 (m, 5H, C6H5-Phe),
7.4 (s, 1H, For) ppm.
Now we wish to present the new simple, efficient and conve-
nient method for the synthesis of peptide cyclic amides. Following
our procedure, the catalyst was prepared by distributing of
subtilisin-72 on the solid base Silochrom-C80 in an amount corre-
sponding to the maximal value according to its adsorption isotherm
on this silica derivative [46]. The enzyme was deposited as the solu-
tion in phosphate buffer (pH 7.5). After distribution the sorbent was
thoroughly dried in a high vacuum over fresh phosphorus pen-
toxide. The catalyst could be efficiently used in the synthesis of
peptide cyclic amides. Earlier we have shown that the reactions
of subtilisin-catalyzed acylation of amino-acids could proceed bet-
ter with the acylating agent containing the ester moiety [47a,b].
We performed the enzyme-catalyzed acylation of free secondary
amine 4 with peptide esters 3 aiming to obtain N-protected peptide
cyclic amide derivatives 5 (Scheme 1, where PG stands for pro-
tecting group; Xaa, Xbb and Xcc stand for amino acid residues in
l-configuration; R1 and R2 stand for hydrocarbon radicals which
can be substituted or taken together with the nitrogen atom form
optionally substituted heterocycle).
Common 2,4-dinitrophenyl (Dnp-), benzyloxycarbonyl (Cbz-),
formyl (For-) and others can be used as protection groups. Amino
acids Xaa, Xbb and Xcc could be chosen from l-amino acids which
sequence is expected to manifest biological activity against desired
enzyme; on the other hand the sequence Xaa–Xbb–Xcc should not
contraverse the substrate specificity of the subtilisin. The secondary
amine 4 could be chosen from diethylamine, piperidine, imidazole,
indole, etc. (Table 3).
MS (MALDI-TOF): 447.4 [M+H].
HRMS (ES+): 447.2828 found, 447.2846 calculated.
2.5. Biological evaluation of synthesized compounds
Biological activity of some synthesized compounds was demon-
strated by in vivo and in vitro experiments. The haemostatic
aldehyde For-Ala-Phe-Lys-al was used to compare the activities of
peptamides and aldehydes.
2.5.1. Ki measurement
The inhibition constants for some synthesized compounds were
determined by standard procedure described by Drozd et al. [52].
The enzymes tested were plasmin and subtilisin.
2.5.2. In vitro experiments
The peptide was dissolved in distilled water at a concentration of
0.01 M. The solution was placed in a bulb in which 0.5 ml of human
blood plasma, 6.5 ml of water, 0.25 ml of caoline suspension and
0.18 ml of 1% acetic acid was added. The mixture was incubated
at 37 ◦C for 30 min. The reaction mixture was centrifuged and the
euglobulin clot was separated. The clot was dissolved in 0.5 ml of
Tris–HCl buffer with pH of 7.4. 0.5 ml of 0.27% CaCl2 solution was
added and the reaction mixture was incubated at 37 ◦C. The time
of full clot dissolution was registered. Distilled water was used as
control.
PGXaaXbbXccC(O)OCH3
[PGXaaXbbXccC(O)OCH3][E-H]
[PGXaaXbbXccC(O)][E]+ CH3OH
+
E-H
3
2.5.3. In vivo experiments
The haemostatic effect of new peptamides was carried out
in laboratory conditions using four ‘Shinshilla’ rabbits of 3–4 kg
weight. The animals were narcotized with thiopental (6–8 ml of 1%
solution intravenously and then 6–10 ml of 1,5% solution intraab-
dominally). Laparotomy was made using the dissection along the
abdomen white line. The intestine and the front liver surface were
elevated out of the wound and restricted by the tampons with warm
physiologic solution. The liver was dissected to produce the round
surface wound with square of about 1.5 cm2 and 0.3 cm depth. The
capillary-parenchymatous bleeding was stopped by the applica-
tion of tampon with peptamide solution directly on the wound.
The water-wet tampon was used as control. The bleeding-stop time
was measured and the blood loss was determined. Tranexamic and
H2O
[PGXaaXbbXccC(O)][E][H2O]
NHR1R2
4
PGXaaXbbXccC(O)OH
[PGXaaXbbXccC(O)][E][NHR1R2]
6
[PGXaaXbbXccC(O)]NR1R2]
5
Scheme 2. Mechanism of enzyme-catalyzed interaction between acyl-donor and
acyl-acceptor.

M.E. Sergeev et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66
63
Table 2
Model syntheses on subtilisin-72 catalyzed acylation of piperidine with Dnp-AlaAlaLeu-OMe, investigation of reaction conditions.
Run
Acyl donor 3a
Amine 4
Product 5
Solvent
pHb
Yield, %
Phase distribution %s/%c c
1
2
3
4
5
Dnp-AlaAlaLeu-OMe
Dnp-AlaAlaLeu-OMe
Dnp-AlaAlaLeu-OMe
Dnp-AlaAlaLeu-OMe
Dnp-AlaAlaLeu-OMe
Piperidine (Pip)
Piperidine (Pip)
Piperidine (Pip)
Piperidine (Pip)
Piperidine (Pip)
Dnp-AlaAlaLeu-Pip
Dnp-AlaAlaLeu-Pip
Dnp-AlaAlaLeu-Pip
Dnp-AlaAlaLeu-Pip
Dnp-AlaAlaLeu-Pip
MeCN
THF
Dioxane
MeCN
MeCN
7.5
7.5
7.5
8.5
9.5
80
78
67
77
74
14/66
13/65
14/53
13/64
12/62
Generally, enzyme-catalyzed reaction of amine acylation cor-
responds to the Scheme 2, where all the variable groups have the
meanings designated above and E stands for enzyme.
The model reactions were carried out using N-(2,4-
dinitrophenyl)-l-alanyl-l-alanyl-l-leucine methyl ester 3a
(Dnp-Ala-Ala-Leu-OMe) with piperidine 4a (Pip) (Scheme 3)
and N-(2,4-dinitrophenyl)-l-alanyl-l-alanyl-l-arginine methyl
ester 3c (Dnp-Ala-Ala-Arg-OMe) with morpholine 4b (Mrf)
(Scheme 4).
It is well known that in organic media the enzyme’s activity
can undergo significant changes [34]. Specific methods were devel-
oped to prevent the inactivation of enzyme-catalyst by organic
media except for enzyme sorption, for instance, the enzyme can
be lyophilized with amino-component or dispersed in solvent. Our
attempts to use these methods were not successful; it turned out
that the literature methods were not effective for the synthesis of
peptide cyclic amides. We also tried procedures described by Rich
and Montan˜ez-Clemente [48,49]. It was found that the suggested
method of lyophilization of amino component with the enzyme and
the reaction in organic medium (THF) at 40 ◦C did not lead to the
formation of the desired amides 5a and 5b. Ester hydrolysis result-
ing to formation of free peptides 6a and 6b was observed in this
case. The similar result was obtained when the reaction was car-
ried out using the dispersed enzyme in organic medium at 37–45 ◦C
[31,50].
Finally, the synthesis of compound 5a was successively car-
ried out using subtilisin-72 distributed on macroporous carrier
Silochrom-C80 with acetonitrile as solvent. It was shown that the
conversion of starting tripeptide 3a was almost quantitative and
the desired product 5a was obtained in 80% yield.
As can be seen from the kinetic curves presented in Fig. 2, the for-
mation of desired peptamide and the hydrolysis of starting peptide
ester begins immediately.
In about 3 days the peptide acid started to form the reaction
product with amino-component with the reaction rate increased
significantly. It was discovered that the enzyme-catalyzed acyla-
tion reaction is possible only in case of tri-membered and more
long peptides with the COOH moiety [39]. The optimal reaction
time was determined; all the reactions thereafter were carried out
for 5 days.
We have tried to investigate if the solvent choice influenced
target compound formation. According the literature data, tetrahy-
drofuran and dioxane could be used as appropriate reaction
solvents for subtilisin-catalyzed reactions. It was found that the
rate of product formation in THF was almost the same as in ace-
tonitrile, but when the reaction was carried out in dioxane, the
yields of target compounds decreased to about 40%. The formation
of peptide cyclic amide 5a did not depend on the concentration of
starting material in reaction mixture. Similar results were obtained
using different amounts of acetonitrile and THF; the reaction yields
were comparable (about 80–85%) in these cases. It was concluded
that varying ratio of liquid to solid phase in general did not gener-
ally have an impact on the reaction course. The investigation of
reaction optimal conditions is summarized in Table 2 using the
synthesis of Dnp-AlaAlaLeu-Pip as a model. As far as the catalyst
was prepared by depositing the enzyme as the solution in basic
buffer, the experiments varying the pH of the buffer for the sub-
tilisin distribution were conducted. It is a well-known fact that
pH-optimum for subtilisin is about 8.5 [41]. We have found that
usage of sodium phosphate (pH 7.5), Tris–Cl (pH 8.5) and sodium
borate (pH 9.5) buffers did not have any significant influence on
reaction course. The results were similar in all the cases, the yields
of target compounds varied by 1–2% only. The further experiments
were conducted using phosphate buffer (pH 7.5).
The results of further syntheses with different amines and pep-
tide esters are presented in Table 3.
Referring to Moree and co-workers, some comparative experi-
ments with primary amines were also carried out, but the reaction
yields were not as good as in case of secondary amines [39]. Acyla-
tion of p-aminopyridine and 5-aminosalicylic acid was performed
resulting in formation of the product in 56% and 6% yields, corre-
spondingly. The efforts to obtain the derivatives of p-nitroaniline
and 3-aminoquinoline failed; nothing but hydrolyzed peptide ester
was recovered.
100
80
60
40
20
0
O
NH
H
5a
NH
N
Dnp
O
N
H
O
N
4a
O
O
NH
hydrolysis
0
1
2
3
4
5
6
7
8
9
H
N
NH
NH
H
Time, d
Dnp
NH
N
6a
Dnp
DnpAlaAlaLeuOMe
DnpAlaAlaLeuPip
DnpAlaAlaLeuOH
O
3a
O
O
O
O
OH
Fig. 2. Kinetic curves of model reaction between Dnp-Ala-Ala-Leu-OMe and piperi-
dine. X-axis: reaction time in time in days; Y-axis: yield determined by HPLC.
Scheme 3. Synthesis of Dnp-Ala-Ala-Leu-Pip.

64
M.E. Sergeev et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66
NH2
Dnp
HN
O
NH
HN
NH
O
5b
H
N
NH2
N
H
4b
O
HN
NH
Dnp
HN
O
NH
O
N
O
H
N
hydrolysis
NH2
O
O
O
3b
HN
NH
Dnp
HN
O
NH
6b
H
N
O
O
OH
Scheme 4. Synthesis of Dnp-Ala-Ala-Arg-Mrf.
It was also found that in case of reactions with participation
to higher conformational flexibility of peptide in the presence of
dimethylsulfoxide. These results are in agreement with the work
of Yusupova and co-workers [50]. It should be pointed out that all
the reactions using arginine-containing peptide esters resulted in
moderate yields of the target amides as compared to the amine
acylation with other peptides. It could be explained by the strong
affinity of acidic arginine guanidine group to the silica-carrier,
of arginine-containing peptide esters 3 the addition of at least
25% of dimethylsulfoxide into acetonitrile media is required due
to low solubility of arginine derivatives 3 and 5 in acetonitrile
alone. The best results for arginine derivatives were obtained
using 35% DMSO/MeCN, when the decreased reaction time and
better yields were observed. We believe that this could be due
Table 3
Subtilisin-72 catalyzed acylation of amines with protected peptide esters.
Run
Acyl donor a
Amine
Product 5
Solvent
pHb
Yield, %
Phase distribution %s/%cc
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Dnp-AlaAlaLeu-OMe
Dnp-AlaAlaLeu-OMe
Dnp-AlaAlaLeu-OMe
Dnp-AlaAlaLeu-OMe
Morpholine (Mrf)
Diphenylamine (DPA)
5-Aminosalicylic acid (ASA)
Imidazole (Im)
3-Aminoquinoline (AQ)
Piperidine (Pip)
Morpholine (Mrf)
Diphenylamine (DPA)
Diethylamine (DEA)
4-Aminopyridine (APy)
Imidazole (Im)
Dnp-AlaAlaLeu-Mrf
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
25% DMSO/MeCN
35% DMSO/MeCN
35% DMSO/MeCN
35% DMSO/MeCN
35% DMSO/MeCN
35% DMSO/MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
8.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
7.5
66
0
14
87
0
74
78
0
12/54
0/0
6/8
81/6
0/0
18/56
14/64
0/0
57/12
9/47
Dnp-AlaAlaLeu-DPA
Dnp-AlaAlaLeu-ASA
Dnp-AlaAlaLeu-Im
Dnp-AlaAlaLeu-OMe
Dnp-AlaAlaLeu-AQ
Dnp-AlaAlaGlu(OMe)-OMe
Dnp-AlaAlaGlu(OMe)-OMe
Dnp-AlaAlaGlu(OMe)-OMe
Dnp-AlaAlaGlu(OMe)-OMe
Dnp-AlaAlaGlu(OMe)-OMe
Dnp-AlaAlaGlu(OMe)-OMe
Dnp-AlaAlaGlu(OMe)-OMe
Dnp-AlaAlaArg-OMe
Dnp-AlaAlaArg-OMe
Dnp-AlaAlaArg-OMe
Dnp-AlaAlaArg-OMe
Dnp-AlaAlaArg-OMe
Dnp-AlaAlaArg-OMe
Cbz-AlaAlaLeu-OMe
Cbz-AlaAlaGlu(OMe)-OMe
Cbz-AlaAlaLeu-OMe
Cbz-AlaAlaGlu(OMe)-OMe
Cbz-AlaAlaGlu(OH)-OH
For-Ala-Phe-Lys-OMe
For-Ala-Phe-Lys-OMe
For-Ala-Phe-Lys-OMe
For-Ala-Phe-Lys-OMe
For-Ala-Phe-Lys-OMe
Dnp-AlaAlaGlu(OMe)-Pip
Dnp-AlaAlaGlu(OMe)-Mrf
Dnp-AlaAlaGlu(OMe)-DPA
Dnp-AlaAlaGlu(OMe)-DEA
Dnp-AlaAlaGlu(OMe)-APy
Dnp-AlaAlaGlu(OMe)-Im
Dnp-AlaAlaGlu(OMe)-Ind
Dnp-AlaAlaArg-Mrf
Dnp-AlaAlaArg-Mrf
Dnp-AlaAlaArg-Mrf
Dnp-AlaAlaArg-Im
Dnp-AlaAlaArg-Pyr
Dnp-AlaAlaArg-DEA
Cbz-AlaAlaLeu-Im
Cbz-AlaAlaGlu(OMe)-Ind
Cbz-AlaAlaLeu-DEA
Cbz-AlaAlaGlu(OMe)-Im
Cbz-AlaAlaGlu(OH)-Im
For-Ala-Phe-Lys-Ind
For-Ala-Phe-Lys-hP
For-Ala-Phe-Lys-DEA
For-Ala-Phe-Lys-Pip
For-Ala-Phe-Lys-Mrf
69
56
73
70
45
31
35
46
49
36
82
75
83
96
88
76
68
89
77
64
51/22
3/67
Indole (Ind)
Morpholine (Mrf)
Morpholine (Mrf)
Morpholine (Mrf)
Imidazole (Im)
Pyrazole (Pyr)
Diethylamine (DEA)
Imidazole (Im)
Indole (Ind)
Diethylamine (DEA)
Imidazole (Im)
Imidazole (Im)
Indole (Ind)
19/26
10/21
11/24
19/27
18/31
5/31
56/26
27/48
64/19
71/25
70/18
16/60
22/46
81/8
Homoproline (hP)
Diethylamine (DEA)
Piperidine (Pip)
66/11
60/4
Morpholine (Mrf)
MeCN
a
Standard three-letter abbreviations used to designate l-amino acid residues; Dnp- stands for 2,4-dinitrophenyl; Cbz- stands for benzyloxycarbonyl; starting peptide esters
were obtained by conventional methods of peptide synthesis; such as EDCI/HOBt coupling and activated-esters coupling of amino acids. In case of tripeptide Ala-Ala-Glu;
the dimethyl ester of glutamic acid was used.
b
Phosphate buffer (pH 7.5), Tris buffer (pH 8.5) and borate buffer (pH 9.5) used.
Designates percentage of product in liquid (%s) and solid (%c) phases.
c

M.E. Sergeev et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66
65
Table 4
0.65
0.6
Inhibition data for synthesized compounds.
Run
Inhibitor
Ki plasmin, ␮M
Ki subtilisin, nM
0.55
0.5
1
2
3
4
5
For-Ala-Phe-Lys-ala,b
For-Ala-Phe-Lys-Pipb
For-Ala-Phe-Lys-Mrfb
Z-Ala-Ala-Leu-DEAc
Z-Ala-Ala-Leu-Mrfc
23
19.2
19.1
0.45
0.4
9.4
8
0.35
0.3
a
Lit. data by Potetinova et al. [37].
Chromogenic substrate ForAlaPheLyspNA used.
Chromogenic substrate ForAlaAlaLeupNA used.
b
c
0.25
0.2
Table 5
1
1.5
2
2.5
3
3.5
4
Euglobulin factor XIIa-dependent lysis time.
Time, days
Run
Inhibitor
Lysis time, min
Native enzyme
E/C-80+MeCN
E/C-80, dry
E/C-80+MeCN+inhibitor
1
2
3
4
5
6
7
For-Ala-Phe-Lys-al
For-Ala-Phe-Lys-Pip
For-Ala-Phe-Lys-Mrf
For-Ala-Phe-Lys-Ind
For-Ala-Phe-Lys-hP
For-Ala-Phe-Lys-DEA
Control
3.8 0.3
21.9 1.2
22.6 0.9
9.5 0.6
8.3 0.6
7.5 0.4
Fig. 3. Activity of native enzyme and enzyme under conditions of reaction. X-axis:
reaction time in days; Y-axis: absorbance at ꢀ 410 nm (A410).
when arginine-containing peptide bound to solid support and
became more sterically hindered for enzyme to be able to form
acyl–enzyme complex, which in turn led to lowered yields of the
final amides.
3
0.8
Table 6
Bleeding-stop time on liver surface wound.
It was found that the reaction product distributed non-
uniformly between the reaction phases (see Table 2 for details).
It was shown that imidazole and diethylamine derivatives
were mostly dissolved in the reaction solvent and the other
amide-derivatives were better sorbed on silochrom. In case of
arginine-amides all the reaction products are mostly distributed
on the solid carrier. We believe that such a distribution patterns
are related to the hydrophobicity of the whole target molecule. In
most cases the work-up of only one phase was sufficient for the
isolation of target peptamide in pretty good yield.
Run
Inhibitor
Bleeding-stop time, s
1
2
3
4
5
6
For-Ala-Phe-Lys-al
For-Ala-Phe-Lys-Pip
For-Ala-Phe-Lys-Mrf
␧-Aminocaproic acida
Tranexamic acida
Control
83
60
70
5
1
7
7
140
98 15
237 13
a
Commercial positive control, common drug for bleeding termination.
also shown good Ki values against subtilisin. With these in mind, we
made the in vitro and in vivo experiments to establish the activity
of newly synthesized compounds in bleeding termination.
For the in vitro experiment we used the euglobulin factor XIIa-
dependent lysis model (Table 5). The results show that some of
synthesized amides with plasmin-specific sequence Ala-Phe-Lys
are capable of significant prolongation of euglobulin clot in compar-
ison with previously described aldehyde and control experiment.
Piperidinamide and morpholinamide were found to be the best
agents in this model and were evaluated for in vivo activity in
rabbits.
The experiments with animals were made with acute bleeding
model (Table 6). This model simulates the general surgical prob-
lem of termination of strong bleeding of inner organs and includes
the acute capillary-parenchymatous bleeding of liver-wound. Our
investigation has shown that compounds For-Ala-Phe-Lys-Pip and
For-Ala-Phe-Lys-Mrf have the bleeding-terminating activity almost
twice higher than standard commercial agents tranexamic and ␧-
aminocaproic acids. Previously described For-Ala-Phe-Lys-al was
evaluated too and has shown the activity similar to amides, but
its instability in aqueous media restricts the application of this
aldehyde in surgical area.
It was of great interest to us to find out why subtilisin was capa-
ble of catalyzing the synthesis of an inhibitor for itself and all the
while it did not become inactivated when the concentration of
target inhibitor in reaction mixture increased. Schechter and Ziv
reported that competitive inhibitors can activate proteases to cat-
alyze the synthesis of peptide bonds [51]. They also showed that
acyl–enzyme complex formed in reaction medium served as initia-
tor of tripartite reaction and the process became cyclic. In addition
to already reported data we demonstrated that, contrary to the
common process of enzyme inhibition in solution, the inhibition of
subtilisin distributed on silochrom in acetonitrile did not take place.
The comparative experiments showed that the carrier-distributed
subtilisin was slightly less active than the native enzyme, but the
addition of subtilisin inhibitor Cbz-Ala-Ala-Leu-Mrf activated the
enzyme in time-dependent manner. The results are shown in Fig. 3.
As can be seen, the activity of the enzyme distributed on
silochrom (E/C-80, dry composition) and the activity of distributed
enzyme under acetonitrile layer (E/C-80 + MeCN) decreased slowly
during the course of experiment. The activity of the enzyme dis-
tributed on the carrier and treated with inhibitor in acetonitrile
(Z/C-80 + MeCN + inhibitor) dropped dramatically at first, but over
time the activity grew strongly and eventually became almost as
high as that in above mentioned cases. This is in agreement with
the data of Ziv and Schechter and gives the answer for the posed
question.
4. Conclusions
Biological evaluation of some of synthesized compounds was
started by measuring the binding constants (Ki) against plasmin
and subtilisin (Table 4). The Ki data show that morpholinamide and
piperidinamide with the amino-acid sequence Ala-Phe-Lys (spe-
cific for plasmin) possess the similar Ki value when compared with
previously reported aldehyde [37]. Diethylamide and morpholi-
namide with the sequence Ala-Ala-Lys (specific for subtilisin) have
A convenient and high-yield approach was suggested for the
synthesis of peptidyl secondary amides. This involves the appli-
cation of subtilisin-72 distributed on macroporous silica carrier as
catalyst for the reaction of acylation of secondary amines with pep-
tidyl esters in organic medium. The applied method allows to obtain
peptide secondary amides in high yields under mild conditions.
Thus obtained peptide amide derivatives could be efficiently used

66
M.E. Sergeev et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 58–66
as inhibitors of serine and cysteine proteases and as so they could
be efficiently used in medicine.
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Acknowledgements
We thank very much Dr. V.A. Makarov, Dr. L.S. Malykhina, Dr.
S.G. Malykhina and Dr. O.E. Nevedrova, from the Laboratory for
Pathology and Pharmacology of Hemostasis, Hematology Research
Center, Russian Academy of Medical Sciences for their great sup-
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Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.molcatb.2012.04.019.
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