Myelopeptide MP-5 and fluorescent derivatives: Synthesis and biological activity

Article abstract of DOI:10.1134/S106816200804002X

The Val-Val-Tyr-Pro-Asp bone marrow peptide (MP-5) and its analogue (MP-5-Lys) were synthesized. Fluorescent derivatives, Ftc-MP-5 and MP-5-Lys(Ftc), were prepared. The iological activity of MP-5 and MP-5-Lys was studied in vitro and in vivo. The MP-5 peptide caused 60-84% inhibition of growth of the following mouse cancers: lymphatic leukemia P388, melanoma B-16, and cervical carcinoma CUC-5. These peptides also restored functional activity of T-lymphocytes that was inhibited by metabolic products of the HL-60 leukemic cell line. MP-5-Lys(Ftc) was shown to preserve the functional properties of MP-5 towards T-lymphocytes, but Ftc-MP-5 was practically inactive.

Full text of DOI:10.1134/S106816200804002X

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2008, Vol. 34, No. 4, pp. 404–408. © Pleiades Publishing, Ltd., 2008.
Original Russian Text © L.A. Fonina, A.A. Az’muko, V.N. Kalikhevich, M.L. Lewit, Z.A. Ardemasova, S.A. Gur’yanov, R.G. Belevskaya, M.A. Efremov, E.M. Treshchalina,
A.A. Mikhailova, 2008, published in Bioorganicheskaya Khimiya, 2008, Vol. 34, No. 4, pp. 451–456.
Myelopeptide MP-5 and Fluorescent Derivatives:
Synthesis and Biological Activity
L. A. Foninaa,1, A. A. Az’mukob, V. N. Kalikhevichc, M. L. Lewitc, Z. A. Ardemasovac,
S. A. Gur’yanova, R. G. Belevskayaa, M. A. Efremova, E. M. Treshchalinad, and A. A. Mikhailovaa
a Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia
b Research Institute of Experimental Cardiology, Russian Cardiological Research and Production Complex, Russian Agency
of Public Health and Social Development, Moscow, Russia
c Research Institute of Chemistry, St. Petersburg State University, St. Petersburg, Russia
d Research Institute of Experimental Therapy and Diagnostics of Cancers, Cancer Research Center,
Russian Academy of Medical Sciences, Moscow, Russia
Received May 14, 2007; in final form, June 1, 2007
Abstract—The Val-Val-Tyr-Pro-Asp bone marrow peptide (MP-5) and its analogue (MP-5-Lys) were synthe-
sized. Fluorescent derivatives, Ftc-MP-5 and MP-5-Lys(Ftc), were prepared. The iological activity of MP-5 and
MP-5-Lys was studied in vitro and in vivo. The MP-5 peptide caused 60–84% inhibition of growth of the fol-
lowing mouse cancers: lymphatic leukemia P388, melanoma B-16, and cervical carcinoma CUC-5. These pep-
tides also restored functional activity of T-lymphocytes that was inhibited by metabolic products of the HL-60
leukemic cell line. MP-5-Lys(Ftc) was shown to preserve the functional properties of MP-5 towards T-lympho-
cytes, but Ftc-MP-5 was practically inactive.
Key words: myelopeptide MP-5, VVYPD, fluorescent analogues, synthesis, T-lymphocytes, proliferative
response, antitumor activity
DOI: 10.1134/S106816200804002X
INTRODUCTION
sarcoma S-180, Lewis lung carcinoma, and melanoma
B-16 [4]. We proposed on the basis of high homology
of the MP-2 and MP-5 structures that MP-5 would
exhibit the biological activity similar to that of MP-2.
The goal of this study is synthesis and investigation
of biological activity of the MP-5 myelopeptide. Two
fluorescent analogues of MP-5 were prepared for pro-
found studies of the mechanism of its action and for
detection of its target cell.
The Val-Val-Tyr-Pro-Asp pentapeptide (MP-5)
belongs to the group of endogenous regulatory pep-
tides, myelopeptides, which are produced by cells of
the bone marrow [1].2 This peptide is homologous to
the Leu-Val-Val-Tyr-Pro-Trp myelopeptide (MP-2) for
which the mechanism of action and functional activity
were studied in detail. MP-2 was shown to restore the
proliferative response of T-lymphocytes to PHA that
was suppressed by the products of the HL-60 tumor
cells of human myeloleukemia [2]. This effect is
accompanied by recovery of the CD3+ and CD4+ phe-
RESULTS AND DISCUSSION
MP-5 was synthesized by the solid phase method on
notypes of T-cells that were damaged by the cancer [3]. the co-polymer of styrene and 1% divinyl benzene with
the HMP acid-labile anchoring group according to the
standard Fmoc-technique. The crude product of solid
phase synthesis was purified by a preparative HPLC
MP-2 were also found to inhibit the growth of a number
of transplantable tumors of mice: lymphatic leukemia
P-388, adenocarcinoma of the mammary gland Ca 755,
1
and characterized by H NMR spectroscopy and mass
1
Corresponding author; phone: +7 (495) 330-7256; fax: +7 (495)
spectrometry.
330-7210; e-mail: stas@ibch.ru
MP-5 peptide with the additional Lys residue on its
C-terminus was prepared for the subsequent introduc-
tion of the fluorescent label. This peptide was synthe-
sized by methods of conventional peptide synthesis in
solution using Boc and Z protective groups and a com-
bination of fragment condensation with a stepwise
elongation of the amino acid chain by the method of
2
Abbreviations: CM, conditioned medium; DIC, diisopropylcar-
bodiimide; F-complex, adduct of pentafluorophenol with dicyclo-
hexylcarbodiimide; FITC, fluorescein isothiocyanate; Ftc, fluo-
resceinthiocarbamoyl; HMP, hydroxymethylphenoxy; MP,
myelopeptides, MP-5, Val-Val-Tyr-Pro-Asp pentapeptide of the
MP family; NMP, N-methylpyrrolidone; HOBt, hydroxybenzotri-
azolyl; Pfp, pentafluorophenyl; PHA, phytohemagglutinin; TFA,
trifluoroacetic acid.
404

MYELOPEPTIDE MP-5 AND FLUORESCENT DERIVATIVES
405
Table 1. Effects of MP-5, Ftc-MP-5, and MP-5-Lys(Ftc)
on restoration of the PHA-induced proliferation of human
T-lymphocytes that was decreased by the action of HL-60 CM
pentafluorophenyl active esters. The C-terminal tet-
rapeptide was prepared by the condensation of the two
dipeptides, and two valine residues were attached step-
wise to this tetrapeptide. Benzyloxycarbonyl and tert-
butyloxycarbonyl protections were used for blocking of
the α-amino group and the ε-amino group of lysine res-
idue, respectively. The β-carboxyl group of aspartic
acid residue was temporary protected by benzyl ester
on the stage of preparation of the dipeptide. The
α amino group of the labeled hexapeptide was removed
by the treatment with hydrogen bromide in trifluoro-
acetic acid.
The fluorescent label was introduced in MP-5 and
MP-5-lys using fluorescein isothiocyanate (see Experi-
mental). The labeled peptides were purified by the
reversed phase HPLC.
Biological activity of the prepared peptides were
determined according to a degree of restoration of the
PHA-induced proliferation of human T-lymphocytes
(in vitro) and according to the ability of peptides to
inhibit growth of the mouse transplanted cancers (in
vivo).
The cancer cells are known to release substances
that inhibit functional activity of T-lymphocytes [5, 6].
Functional state of the lymphocytes subjected to the
action of cancer toxins was determined according to
their ability to blasttransformation in the presence of
PHA. T-lymphocytes of peripheral blood of healthy
donors were used in this study. Their functional activity
was inhibited in CM of the cells of the HL-60 line of
leukemic cells. Proliferative response of T-lymphocytes
to PHA was evaluated according to [3H]-thymidine
incorporation in DNA. The proliferation level of the
PHA-stimulated cells was taken to be 100%. The
experimental results are presented in Table 1.
As one can see from Table 1, decrease in the prolif-
eration level (to 46%) of the PHA-induced response of
T-lymphocytes is observed in the presence of the CM of
HL-60 cells. In this model, MP-5 and MP-5-Lys(Ftc)
exhibit the equally high activity in a wide range of con-
centrations (from 1 × 10–6 to 1 × 10–9 g/ml) and restore
the level of proliferation of T-lymphocytes to 76–93%.
However, the MP-5 analogue with the N-terminal
amino group labeled with fluorescein proved to be inac-
tive. Preservation of the high functional activity of MP-
5-Lys(Ftc) allows an application of this peptide as an
adequate instrument for a search for a target cell for
MP-5 peptide and studies of the mechanism of its
action.
Peptide con-
Proliferation
Added components
centration,
level, %
µg/ml
PHA (control)
PHA + LH-60 CM
–
–
100
46
4
1
89 10*
93 7*
86 9*
82 8*
0.1
MP-5
0.01
0.001
1
86 6*
84 11*
90 8*
89 16*
0.1
PHA
+ HL-60 CM
MP-5-Lys(Ftc)
Ftc-MP-5
0.01
0.001
1
44 18
48 11
48 19
47 14
0.1
0.01
0.001
* p < 0.01.
Table 2. Effect of MP-5 (1 mg/kg, five times with 24-h in-
tervals) on growth of the transplanted mouse tumors
Days after the
tumor trans-
plantation
Mouse
line
ITG*,
%
Type of the tumor
Cervical carcinoma CUC-5 CBA
7
84
83
81
74
66
59
60
63
61
14
21
7
Melanoma B-16
C57BLb
14
21
7
Lymphatic leukemia P-388 DBA2
14
21
* ITG was calculated as a ratio of difference in the averaged vol-
umes of tumors in the control and treated groups to the tumor vol-
ume in the control group.
Cytotoxic activity of T-lymphocytes is known to be
one of factors of the antitumor protection of an organ-
ism. Ability of MP-5 to restore the functional activity of
T-lymphocytes impaired by the products of leukemic
cells is a prerequisite for studies of the antitumor activ-
ity of this peptide on the in vivo models.
melanoma B-16. Experiments demonstrated that
administration of MP-5 to the animals with the trans-
planted tumors resulted in significant inhibition of
growth of all the examined cancers (Table 2). The pep-
tide was found to have no direct action on a tumor cell,
Three types of the transplanted mouse tumors were but it supported active antitumor immunity in an organ-
chosen for examination of the antitumor effect: cervical ism of a tumor-bearer by the inhibition of action of
carcinoma CUC-5, lymphatic leukemia P-388, and tumor toxins on T-lymphocytes.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

406
FONINA et al.
Thus, the biological activity of MP-5 in the used
The peptides were characterized by mass spectrom-
tests in vitro and in vivo is practically the same as that etry on a Thermo BioanalysisVision 2000 spectrometer
of MP-2 described by us previously [7]. In the case of (England).
MP-2, the attachment of fluorescent label to its N-ter-
Z-Asp(OBzl)-Lys(Boc)-OH·DCHA (I). The solu-
minal amino group resulted in a complete loss of its
tion of Z-Asp(OBzl)-OPfp (3.14 g, 6 mmol) in dioxane
functional properties. In this study, we demonstrated
(20 ml) was added to the solution of H-Lys(Boc)-OH
that elongation of MP-5 by an additional ë-terminal
(1.48 g, 6 mmol) in 1 N NaOH (6 ml) on stirring. The
Lys residue and the attachment of the fluorescent label
reaction mixture was kept for 24 h at 18°ë, and dioxane
to its ε-amino group resulted in the fluorescent ana-
was evaporated. The obtained aqueous solution was
logue that preserved the activity in vitro. We can con-
acidified with 0.5 N H2SO4 to pH 3. The oily precipitate
clude on the basis of these facts that the common Val-
was extracted with ethyl acetate (3 × 20 ml). The ethyl
Val-Tyr-Pro fragment of these peptides with unmodi-
acetate solution was washed with water (2 × 20 ml) and
fied N-terminal amino group was responsible for their
saturated solution of NaCl (2 × 20 mm), dried over the
functional activity, whereas the nature of ë-terminal
anhydrous Na2SO4, filtered, and evaporated. The resi-
residue was unimportant. Therefore, various analogues
due was dissolved in ether (20 ml), and dicyclohexy-
with the specific functional activity could be prepared.
lamine (1.2 ml, 6 mmol) was added. The precipitated
It is interesting that the direction of a peptide chain is
crystals were filtered, washed with ether, and dried in
also unimportant, at least in the case of MP-2. For
vacuum. Recrystallization from ethyl acetate yielded
example, the Trp-Pro-Tyr-Val-Val retro-analogue,
3.45 g (75%) of Z-Asp(OBzl)-Lys(Boc)-OH · DCHA;
mp 144-145.5°C; Rf 0.88(A), 0.75 (B).
which was described by us previously, exhibits the
activity similar to that of MP-2 in vitro and in vivo [7].
H-Asp-Lys(Boc)-OH (II). 0.5 N H2SO4 was added
We propose on the basis of these results that the
to the suspension of compound (I) (1.31 g, 1.71 mmol)
MP-5 endogenous immunoregulatory peptide, likely to
to pH 3 on stirring. The ethyl acetate layer was washed
MP-2, can participate in the processes of anti-carcino-
with water (3 × 15 ml) and saturated solution of NaCl
genesis that occur in a healthy organism and prevent
(2 × 15 ml), dried over Na2SO4, filtered, and evaporated.
development of cancers resulting from mutations, car-
The oily residue was dissolved in methanol (30 ml),
cinogenic actions and other factors.
and 10% Pd/C (0.1 g) was added to the solution. The
reaction mixture was subjected to the catalytic hydro-
genolysis in the H2 atmosphere to the disappearance of
EXPERIMENTAL
the starting compound according to TLC. The peptide
Commercially available amino acids and their deriv-
atives (Merck, Germany and Fluka, Switzerland) were
used for the peptide synthesis. The 10% palladium on
activated charcoal (Merck, Germany) served as catalyst
for hydrogenolysis. The fluorescent label was intro-
duced by the treatment with FITC (AppliChem, Ger-
many). Homogeneity of the compounds on the interme-
diate stages of synthesis was determined by TLC on a
Kieselgel 60 plates (Merck, Germany) in the following
chromatographic systems: (A) n-butanol–acetic acid–
water (3 : 1 : 1), (B) chloroform–methanol–acetic acid
(9 : 1 : 0.5), (C) chloroform–hexane–acetic acid (9 : 1 : 1),
and (D) chloroform–methanol–acetic acid–water (60 :
45 : 6.4 : 13.6). The substances were detected with the
use of the chlorine-benzidine reagent.
Analytical HPLC was performed on a LC-10ADvp
chromatographic system (Shimadzu, Japan) equipped
with an Ultrasphere C18 column (4.6 × 250 mm, Beck-
man, United States) in a concentration gradient of ace-
tonitrile in 0.1% trifluoroacetic acid (from 0 to 80%
within 32 min) at a flow rate of 1.6 ml/min with the
detection at 214 and 280 nm.
precipitated in the course of hydrogenolysis was dis-
solved by the addition of 10% solution of aqueous
ammonia. The catalyst was filtered off, and the filtrate
was evaporated. The residue was dissolved in isopro-
panol (20 ml) and evaporated again. Acetone (50 ml)
was added to the residue, and the formed precipitate
was filtered, washed with petroleum-ether, and dried in
vacuum. The yield of H-Asp-Lys(Boc)-OH was 0.61 g
(98%); mp 165–166°ë; Rf 0.61 (A).
Z-Tyr(Bzl)-Pro-OH (III). The solution of
Z-Tyr(Bzl)-OPfp (4.61 g, 8.06 mmol) in dioxane
(30 ml) was added to the solution of H-Pro-OH (1.11 g,
9.67 mmol) in 1 N NaOH (9.67 ml) on stirring. The
reaction mixture was kept for 24 h at 18°ë and evapo-
rated. The residue was diluted with water (30 ml). The
aqueous solution was washed with ether (2 × 15 ml) and
acidified with 0.5 N H2SO4 to pH 3. The oily precipitate
was extracted with ethyl acetate (3 × 15 ml). The ethyl
acetate layer was washed with water (3 × 15 ml) and
with saturated solution of NaCl (2 × 15 ml), dried over
anhydrous Na2SO4, filtered, and evaporated. The resi-
due was crystallized in petroleum-ether, filtered, and
dried in vacuum. The yield of compound (III) was
3.85 g (95%); mp 54–56°ë. Rf 0.81 (A), 0.51 (C).
Preparative HPLC was performed on a System
Gold chromatographic system (Beckman, United
States).
Z-Tyr(Bzl)-Pro-OPfp (IV). The solution of F-com-
1
The H NMR spectra were recorded on a Bruker plex [8] (3.77 g, 4.97 mmol) in ethyl acetate (25 ml)
WM500 spectrometer (Germany) with a working fre- was cooled to –5°ë and added to the solution of com-
quency of 500 MHz at 37°ë in DMSO-d6.
pound (III) (2.5 g, 4.97 mmol) in ethyl acetate (20 ml).
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MYELOPEPTIDE MP-5 AND FLUORESCENT DERIVATIVES
407
The reaction mixture was kept at 0°ë for 24 h. The pre- (2 × 20 ml), dried over anhydrous Na2SO4, filtered, and
cipitated crystalline dicyclohexylurea was filtered off evaporated. The residue was triturated with ether
and washed with ethyl acetate (2 × 20 ml). The filtrate (30 ml). The formed precipitate was filtered, washed
was evaporated. The oily residue was dried in vacuum. with the mixture of ether and petroleum-ether (1 : 1)
They yield of compound (IV) was 3.2 g (96%); Rf 0.78 (C). (2 × 20 ml), and dried in vacuum. The yield of peptide
(VII) was 1.36 g (80%); mp 157–160°ë. Rf 0.86 (A),
0.24 (B).
Z-Tyr(Bzl)-Pro-Asp-Lys(Boc)-OH (V). The solu-
tion of compound (IV) (3.6 g, 5.38 mmol) in dioxane
Z-Val-Val-Tyr-Pro-Asp-Lys-OH (VIII). Com-
pound (VII) (0.22 g, 0.24 mmol) was treated with trif-
luoroacetic acid (10 ml) for 40 min at 18°ë, evaporated,
and mixed with ether. The precipitate was filtered,
washed with ether (3 × 20 ml), and dried in vacuum.
The product was purified by the preparative HPLC on a
Diasorb-130-C16T column (25 × 250 ml) (BioKhim-
Mak, Russian Federation) in a concentration gradient
of isopropanol in 0.1 M acetic acid (from 20 to 50%
within 30 min) at a flow rate of 40 ml/min. The yield of
product (VIII) was 0.11 g (49%); mp 183–185°ë.
(20 ml) was added to the solution of compound (II)
(1.77 g, 4.89 mmol) in 1 N NaOH (9.78 ml, 9.78 mmol)
on stirring. The reaction mixture was kept for 24 h at
18°ë. Dioxane was evaporated. The residue was diluted
with water (20 ml), washed with ether (2 × 15 ml), and
acidified with 0.5 N H2SO4 to pH 3. The oily precipitate
was extracted with ethyl acetate (3 × 20 ml), washed
with water (2 × 20 ml) and with saturated solution of
NaCl (2 × 20 ml), dried over anhydrous Na2SO4, fil-
tered, and evaporated. The residue was triturated with
petroleum-ether (40 ml). The formed crystalline precip-
itate was filtered and dried in vacuum. Peptide (V) was
Amino acid analysis: Asp 1.04 (1), Val 1.77 (2), Pro
prepared with the yield of 2.49 g (60%) as amorphous 0.95 (1), Lys 1.05 (1), Tyr 1.00 (1).
powder with Rf 0.81 (B).
Z-Val-Val-Tyr-Pro-Asp-Lys(Ftc)-OH (IX). The
solution of FITC (42.05 mg, 0.108 mmol) in dimethyl-
formamide (2.27 ml) was added to the solution of com-
pound (VIII) (73 mg, 0.086 mmol) in 0.1 M Na2CO3
(11.3 ml). The reaction mixture was kept for 2 h at 18°ë
in the dark and for 24 h at 0°ë and evaporated. Isopro-
panol (10 ml) was added to the residue, and the solution
was evaporated again. The residue was dissolved in
water (5 ml) and acidified with 1 N HCl to pH 3. The
oily precipitate was extracted with n-butanol (2 × 10 ml),
washed with water (2 × 5 ml), and evaporated. The res-
idue was dissolved in isopropanol (10 ml) and evapo-
rated again. Triturating with ether yielded 92 mg of
peptide (IX); Rt 20.1 (the analytic HPLC).
Z-Val-Tyr-Pro-Asp-Lys(Boc)-OH (VI). The 10%
Pd/C (0.15 g) was added to the solution of compound
(V) (2.49 g, 2.94 mmol) in methanol (30 ml). The reac-
tion mixture was subjected to the catalytic hydrogenol-
ysis in current of hydrogen gas until the disappearance
of the starting compound according to TLC. The cata-
lyst was filtered off, and the filtrate was evaporated. The
residue was dissolved in 1 N NaOH (5.88 ml,
5.88 mmol), and the solution of Z-Val-OPfp (1.23 g,
2.94 mmol) in dioxane (30 ml) was added on stirring.
The reaction mixture was kept for 24 h at 18°ë. Diox-
ane was evaporated, and the residue was acidified with
0.5 N H2SO4 to pH 2.5. The oily precipitate was
extracted with ethyl acetate (3 × 20 ml). The ethyl ace-
HBr·H-Val-Val-Tyr-Pro-Asp-Lys(Ftc)-OH (X).
tate extract was washed with water (2 × 20 ml) and with The solution of compound (IX) (60 mg) in trifluoroace-
the saturated solution of NaCl (2 × 20 ml), dried over tic acid (5 ml) was completely saturated with dry HBr
anhydrous Na2SO4, filtered, and evaporated. The resi- gas. The reaction mixture was evaporated. The residue
due was triturated with petroleum-ether (30 ml), and was dissolved in isopropanol (5 ml) and evaporated
the formed crystals were filtered and dried in vacuum. again. The residue was triturated with ether (10 ml).
The recrystallization from ethyl acetate yielded 1.52 g The formed precipitate was filtered washed with ether
(61%) of peptide (VI); mp 124–126°ë; Rf 0.9 (A), (10 ml), and dried in vacuum. The product was purified
0.42 (B).
by HPLC on an Ultrasphere ODS C18 column (10 ×
250 mm, 5 µm, Beckman, United States) in a concen-
tration gradient of acetonitrile in 0.1% TFA (from 0 to
100% within 32 min) at a flow rate of 8 ml/min with the
detection at 214 and 280 nm. The yield of peptide (X)
was 25 mg (44%); Rt 14.6 min (the analytical HPLC);
MALDI-MS (m/z): 1191.3 [M + H]+ (calculated value
was 1190.27).
TFA·H-Val-Val-Tyr-Pro-Asp-OH, MP-5, (XI)
was synthesized on an Applied Biosystems 431A auto-
matic peptide synthesizer (United States) starting from
375 mg (0.25 mmol) of the Wang polymer with the pre-
liminary attached Fmoc-Asp(OBut) (0.67 mmol/g) pur-
chased from Bachem (Switzerland).
Z-Val-Val-Tyr-Pro-Asp-Lys(Boc)-OH (VII). The
solution of compound (VI) (1.52 g, 1.78 mmol) in
methanol (20 ml) was subjected to catalytic hydro-
genolysis in the presence of 10% Pd/C (0.1 g) in a cur-
rent of hydrogen gas until the disappearance of the
starting compound according to TLC. The catalyst was
filtered off, and the filtrated was evaporated. The resi-
due was dissolved in 1 N NaOH (3.56 ml, 3.56 mmol),
and the solution of Z-Val-OPfp (0.74 g, 1.78 mmol) in
dioxane (20 ml) was added on stirring. The reaction
mixture was kept for 24 h at 18°ë. Dioxane was evap-
orated, and the residue was acidified with 0.5 N H2SO4
to pH 3. The oily precipitate was extracted with ethyl
acetate (3 × 20 ml). The extract was washed with water
The peptide was synthesized according to the stan-
(2 × 20 ml) and with the saturated solution of NaCl dard protocol with a one-step condensation of Fmoc-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

408
FONINA et al.
amino acids. The synthetic cycle involved deprotection body weight) purchased from the Stolbovaya breeding
nursery of the Russian Academy of Medical Sciences.
Groups of 10–12 animals were formed. The cancers
were subcutaneously transplanted in the amount of
1 × 106 cells per one mouse in 0.3 ml of the culture
medium 199 [1]. MP-5 was dissolved ex tempore in
0.9% solution of NaCl and administered to the mice
48 h after the transplantation of carcinoma CUC-5 and
melanoma B-16 or 24 h after the transplantation of lym-
phatic leukemia P-388. MP-5 was daily subcutaneously
administered for 5 days at a dose of 1.0 µg/g. The 0.9%
sterile solution of NaCl (0.1 ml) was administered to
the control groups. The mice were observed until their
death, and volumes of their cancers were measured
three times. The antitumor effect of MP-5 was evalu-
ated according to the dynamics of inhibition of growth
of a tumor node (ITG) on the 7th, 14th, and 24th day
after the tumor transplantation. ITG was calculated as a
ratio of difference in the averaged volumes of tumors in
the control and treated groups to the tumor volume in
the control group.
of α-amino group by the treatment with 20% piperidine
solution in NMP for 20 min, activation of 1 mmol of the
attached amino acid in the presence of equivalent
amounts of DIC and HOBt in NMP for 20 min, conden-
sation with 1 mmol (fourfold excess) of the acylating
agent in NMP for 37 min, and all the necessary inter-
mediate washings of the peptidylpolymer.
For the final deprotection and cleavage of the pep-
tide from the polymer, the peptidylpolymer was sus-
pended in the deprotecting mixture of TFA and H2O
(10 : 1, 10 ml) and stirred for 2 h at 20°ë. The resin was
filtered off and washed with TFA (2 × 1 ml). The filtrate
was evaporated to the volume of 3–5 ml and mixed with
anhydrous ether (100 ml). The precipitate was filtered,
washed with ether and ethyl acetate, and dried. The
obtained precipitate was dissolved in 1% TFA (5 ml)
and applied onto a Diasorb C-16T column (25 ×
250 mm). The column was eluted with a gradient of
acetonitrile in 0.1% TFA (0.5% per one min) at a flow
rate of 12 ml/min with the detection at 226 nm. The
fraction containing the target product (XI) were joined
and evaporated. The residue was dissolved in water and
lyophilized. The yield of MP-5 was 80%; the purity was
ACKNOWLEDGMENTS
This study was supported by the grant of the Mos-
98% according to HPLC; Rt 9 min on the analytical cow government “Elaboration and Practical Applica-
HPLC column; MALDI-MS (m/z): 592.7 [M + H]+ (the
calculated molecular mass was 591.68).
tion of New Methods and Remedies of Prophylaxis,
Diagnostics, and Treatment of Cancers, Infections, and
Other Dangerous Diseases”.
Ftc-MP-5 (XII). The reaction was performed in the
mixture of dimethylformamide and 0.1 M sodium car-
bonate (volume ratio of 1 : 3, pH 9.0) with 1.5-fold
molar excess of FITC. The obtained Ftc-MP-5 was
purified on a Diasorb C18 column (10 × 250 mm) in a
concentration gradient of acetonitrile in 0.1% TFA
(from 0 to 80% within 32 min) at a flow rate of
9 ml/min with the detection at 280 nm. Rt of Ftc-MP-5
was 15.9 min (the analytical HPLC); MALDI-MS
(m/z): 981 [M + H]+ (the calculated molecular mass was
980.05).
REFERENCES
1. Petrov, R.V., Mikhailova, A.A., Fonina, L.A., and
Stepanenko, R.N., in Mielopeptidy (Myelopeptides),
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