The Study of Stability of Proline-Containing Derivatives of Dopamine and Serotonin in the Biological Media in Vitro Experiments

Article abstract of DOI:10.1134/S1990750820020110

Abstract—: The peptides Boc-Gly-Pro-DP, Z-Gly-Pro-DP, LA-Gly-Pro-DP, Boc-Gly-Pro-Srt, Z-Gly-Pro-Srt have been synthesized for the first time. The study of their stability in the presence of leucine aminopeptidase, carboxypeptidase Y, carboxypeptidase B, and proline endopeptidase (PEP) has shown that the synthesized peptides are stable in the presence of aminopeptidases and carboxypeptidases. In the presence of PEP, dopamine (DP) and serotonin (Srt) have been cleaved from these substances. Thus, the originally synthesized proline derivatives of Srt and DP may be considered as the resources, from which Srt and DP can be gradually released. This creates the possibility of a prolonged action of these biologically active compounds on cells and, consequently, on the whole body.

Full text of DOI:10.1134/S1990750820020110

ISSN 1990-7508, Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry, 2020, Vol. 14, No. 2, pp. 150–158. © Pleiades Publishing, Ltd., 2020.
Russian Text © The Author(s), 2020, published in Biomeditsinskaya Khimiya.
The Study of Stability of Proline-Containing Derivatives of Dopamine
and Serotonin in the Biological Media in vitro Experiments
K. V. Shevchenkoa, *, L. A. Andreevaa, I. Yu. Nagaeva, V. P. Shevchenkoa, and N. F. Myasoedova
aInstitute of Molecular Genetics of the Russian Academy of Sciences, pl. Kurchatova 2, Moscow, 123182 Russia
*e-mail: ATCarma@mail.ru
Received June 18, 2019; revised July 8, 2019; accepted July 10, 2019
Abstract—The peptides Boc-Gly-Pro-DP, Z-Gly-Pro-DP, LA-Gly-Pro-DP, Boc-Gly-Pro-Srt, Z-Gly-
Pro-Srt have been synthesized for the first time. The study of their stability in the presence of leucine amino-
peptidase, carboxypeptidase Y, carboxypeptidase B, and proline endopeptidase (PEP) has shown that the
synthesized peptides are stable in the presence of aminopeptidases and carboxypeptidases. In the presence of
PEP, dopamine (DP) and serotonin (Srt) have been cleaved from these substances. Thus, the originally syn-
thesized proline derivatives of Srt and DP may be considered as the resources, from which Srt and DP can be
gradually released. This creates the possibility of a prolonged action of these biologically active compounds
on cells and, consequently, on the whole body.
Keywords: proline-containing dopamine and serotonin derivatives, peptide degradation, leucine aminopep-
tidase, carboxypeptidase, proline endopeptidase (PEP), blood plasma enzymes
DOI: 10.1134/S1990750820020110
INTRODUCTION
dase (EC 3.4.13.9; peptidase D, iminopeptidase)] [8]
and are involved in the cleavage of proline-containing
peptides [dipeptidyl peptidase IV, (EC 3.4.14.5;
DPP-IV)] [9], as well as in other processes [8, 10].
Their action on biologically active fragments linked to
a proline residue results in a gradual cleavage of such
prodrug and release of the biologically active frag-
ment, which will cause a certain cell response. In other
words binding a biologically active compound to a
proline-containing peptide can result in formation of a
drug substance characterized by prolonged life-time in
the body; such approach will reduce the dosage of the
drug at the unchanged positive effect and, therefore,
will reduce negative side-effects.
In this study, we have investigated stability of pro-
line derivatives of DP and Srt under the action of
leucine aminopeptidase (EC 3.4.11.2), carboxypepti-
dase Y (EC 3.4.16.1), carboxypeptidase B (EC
3.4.17.2) and proline endopeptidase (EC 3.4.21.26).
The aim of this work was to synthesize Boc-Gly-
Pro-DP, Z-Gly-Pro-DP, LA-Gly-Pro-DP, Boc-
Gly-Pro-Srt, Z-Gly-Pro-Srt and to study their enzy-
matic hydrolysis in experiments in vitro. Semax (Met-
Glu-His-Phe-Pro-Gly-Pro) was used as a reference
compound.
Biologically active peptide compounds can be
administered in the body by using various methods
(intravenously, intranasally, etc.). Therefore, it is very
important to determine their stability in the presence
of aminopeptidases and carboxypeptidases. In addi-
tion, it is interesting to elucidate, which metabolites
can be formed during enzymatic transformation (as
they can exhibit their own biological activity). For
example, the use of different routes of Pro-Gly-Pro-
Leu administration can result in almost 3-fold differ-
ence in the content of the resultant Gly-Pro dipeptide
[1]. This cannot be ignored due to the fact that Gly-
Pro has a pronounced pharmacological effect: it pre-
vents an increase in anxiety and a decrease in the level
of research activity [2].
Synthetic peptides are known to be stabilized by
their modification with proline residues [3]. Such pro-
line-containing compounds are highly resistant to the
proteolytic action of the most common proteases [4–
6]. Living organisms also contain prolidases.
These enzymes are present in the brain [dipeptidyl
peptidase II (EC 3.4.14.2; DPP-II)] [7], blood [proli-
Abbreviations used: AcOH—acetic acid; Boc—tert-butoxycar-
bonyl group; Bzl—benzyl group; DCCD—N, N-dicyclohexyl-
carbodiimide; DMF—dimethylformamide; DMSO—dimethyl
sulfoxide; DP—dopamine; Et N—triethylamine; HPLC—high
3
MATERIALS AND METHODS
performance liquid chromatography; LA—lauric acid; MeOH—
methanol; PEP—proline endopeptidase; Srt—serotonin; Su—
N-oxysuccinimide; TFA—trifluoroacetic acid; Z—benzyloxy-
carbonyl group.
Microsomal leucine aminopeptidase (Type VI)
from pig kidney (9.2 U/mg), carboxypeptidase Y from
150

STABILITY OF PROLINE-CONTAINING DERIVATIVES OF DOPAMINE AND SEROTONIN
151
Table 1. Chromatography-mass spectrometry analysis of
baker’s yeast (17 U/mg), carboxypeptidase B from pig
pancreas (70 U/mg), and proline endopeptidase from
flavobacteria (55 U/mg), as well as catalysts and
reagents were obtained from Sigma-Aldrich (USA).
Solvents, acids, etc. are obtained from the company
“Khimmed” (Russia).
Boc-Gly-Pro was synthesized at the Institute of
Molecular Genetics of the Russian Academy of Sci-
ences by condensation of Boc-Gly and Pro-OBzl fol-
lowed by removal of the benzyl group by hydrogena-
compounds
[M + 1]+
Compound
τ, min
Z-Gly-Pro-DP
Boc-Gly-Pro-DP
Z-Gly-Pro-Srt
Boc-Gly-Pro-Srt
LA-Gly-Pro-DP
8.13*
7.80*
8.30*
7.96*
8.80**
7.62*
7.08*
2.25*
4.07*
441
407
464
430
489
305
271
153
176
tion. Blood plasma (31 mg protein/mL) was obtained Z-Gly-Pro
from male Wistar rats (200 g). Blood (5 mL) collected
in test tube containing 20 μL of heparin (5000 U/mL)
was centrifuged at 1500 g for 10 min. Protein concen-
tration in blood plasma samples was determined by
the Hartree-Lowry method. In vitro studies were car-
ried out according to the previously described meth-
ods [11, 12].
Boc-Gly-Pro
Dopamine
Serotonin
* System 1; ** system 2; τ—retention time on the chromato-
+
graphic column; [M + 1] —molecular mass.
In the case of experiments with commercially avail-
able enzymes, reaction mixtures were analyzed with-
out preliminary purification and the range of values
during parallel measurements did not exceed 5%. In
the case of experiments with plasma samples it was
necessary to perform preliminary purification by
solid-phase extraction. Therefore, measurements were
usually repeated at least 5 times. The range of values
obtained in this case did not exceed 10–15%.
were added under stirring to a solution of 15 mg Boc-
Gly-Pro (0.055 mmol) in 1 mL of dichloromethane.
After 10 min, 20 μL of Et3N and a DP solution (9.9 mg
(0.064 mmol) in 0.5 mL of DMF) were added and
stirring was continued for 4 h. DMF was removed by
lyophilization. Solid phase extraction of Boc-Gly-
Pro-DP, applied onto Diapak C16, was carried out by
five solvent systems. Initially, 5 mL 5% methanol was
passed, then 10 mL of 20% methanol, 6 mL of 30%
methanol, 6 mL of 95% methanol, and 5 mL of 100%
methanol were passed. The ratio of the content of the
desired product in the fractions was 0 : 1 : 5 : 20 : 0.01.
The purity of the resultant preparation increased
threefold. Preparative purification of Boc-Gly-Pro-
DP was carried out using a Reprosil-Pur C18aq col-
umn (Maisch Gmbh, Germany, 20 × 150 mm, parti-
cle size 10 μm) in the system MeOH−AcOH−TFA
(45 : 0.1 : 0.01). The flow rate of the eluent was
20 mL/min. Retention time was 3.95 min. The yield of
Boc-Gly-Pro-DP was 58% and the chemical purity
was 98%.
Analysis of Reaction Mixtures
Reaction mixtures were analyzed at 210 nm,
220 nm, 230 nm, 240 nm, 254 nm on a Milichrom-
A02 chromatograph (Russia) using a ProntoSIL-120-
5-C18 AQ DB-2003 column (Bishcoff Chromatogra-
phy, Germany, 2 × 75 mm, particle size 5 μm), in a
gradient of methanol and buffer (0.2 M LiClO4 +
0.005 M HClO4, pH 2.24) for 12 min at 35°C. Analysis
of Z-Gly-Pro-DP, Boc-Gly-Pro-DP, Z-Gly-Pro-Srt
and Boc-Gly-Pro-Srt was carried out by increasing
the methanol concentration from 5% to 100%
(system 1). The analysis of LA-Gly-Pro-DP was car-
ried out by increasing the concentration of methanol
from 5% to 45% in 5 min and from 85% to 100% in
6.0–12.0 min (system 2). The flow rate of the eluent
was 0.2 mL/min. The retention times of the test com-
pounds are shown in Table 1.
Compounds were analyzed using mass spectromet-
ric data obtained on a LCQ Advantage MAX instru-
ment (Thermo Electron Corp., USA) with electro-
spray ionization by direct injection of a sample solu-
tion (10 μg/mL in 0.1% acetic acid) and further
fragmentation of the molecular peak in the analyzer by
the ion collision method at 35 eV (Table 1).
Synthesis of Boc-Gly-Pro-Srt
A solution of 24 mg Boc-Gly-Pro (0.088 mmol),
7 mg 1-hydroxybenzotriazole (0.05 mmol), and 29 mg
DCCD (0.141 mmol) in 1 mL of DMF was stirred for
20 min at room temperature. After addition of 20 μL
Et3N (0.14 mmol) and stirring for 10 min and then
addition of 15 mg Srt (0.085 mmol) it was stirred for
4 h. DMF was removed by lyophilization and prepar-
ative purification was performed as above. Retention
time was 5.02 min. The yield of Boc-Gly-Pro-Srt
was 64%.
Synthesis of Z-Gly-Pro-DP
Synthesis of Boc-Gly-Pro-DP
At room temperature 10 mg 1-hydroxybenzotri-
At room temperature 1.4 mg 1-hydroxybenzotri- azole (0.071 mmol) and 130 mg DCCD (0.63 mmol)
azole (0.01 mmol) and 20.8 mg DCCD (0.1 mmol) were added under stirring to a solution of 170 mg of
BIOCHEMISTRY (MOSCOW), SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol. 14 No. 2 2020

152
SHEVCHENKO et al.
Z-Gly-Pro (0.56 mmol) in 0.3 mL DMF. After 0.2 mL/min. The retention time of Boc-Gly-ProOMe
10 min, 0.2 mL Et3N and a solution of 100 mg DP and Boc-Gly-ProOBzl was 6.53 min and 7.11 min,
respectively.
Preparative purification of Boc-Gly-ProOMe and
(0.65 mmol) in 0.3 mL DMF were added. Stirring was
continued for 4 h. DMF was removed by lyophiliza-
tion and preparative purification was performed as
described above. Retention time was 5.53 min. The
yield of Z-Gly-Pro-DP was 66%.
Boc-Gly-ProOBzl was performed using a Reprosil-
Pur C18aq column (20 × 150 mm, particle size 10 μm)
in the system MeOH−H2O−AcOH (50 : 50 : 0.1). The
retention times of Boc-Gly-ProOMe and Boc-Gly-
ProOBzl were 6.53 min and 7.11 min, respectively. The
yield of Boc-Gly-ProOMe and Boc-Gly-ProOBzl
was about 70%.
Synthesis of Z-Gly-Pro-Srt
A solution of 28.5 mg Z-Gly-Pro (0.093 mmol),
7 mg 1-hydroxybenzotriazole (0.05 mmol), and 21 mg
DCCD (0.102 mmol) of 0.5 mL of DMF was stirred
for 20 min at room temperature. After addition of
20 mg Srt (0.113 mmol) in 0.5 mL DMF and 25 μL
Et3N (0.179 mmol) the resultant solutions was stirred
for 4 h. Preparative purification was carried out as
described above. Retention time was 5.95 min. The
yield of Z-Gly-Pro-Srt was 65–70%.
Synthesis of LA-Gly-ProOMe and LA-Gly-ProOBzl
(a) A solution of 200 mg LA (1 mmol) in 3 mL of
absolute benzene was mixed with 321 mg thionyl chlo-
ride (2.70 mmol). The solution was boiled for 2.5 h.
Benzene and the excess thionyl chloride were evapo-
rated and the solution evaporated twice more after
addition of 5 mL absolute benzene. LA chloroanhy-
dride was used without additional purification.
Boc-protection with Boc-Gly-ProOMe and Boc-
Gly-ProOBzl was removed by treating a solution of
100 mg of the protected peptide with a mixture of 1 mL
chloroform and 0.5 mL of TFA for 1 h at room tem-
perature. Then the solution was evaporated and after
addition of 5 mL methanol was evaporated twice
more. The residue was lyophilized and used without
additional purification.
A solution of Gly-ProOMe in 1 mL chloroform
was treated under stirring with 0.4 mL Et3N and
0.35 mmol LA chloroanhydride. After 2 h, the reac-
tion mixture was treated with methanol and lyo-
philized. A similar reaction was carried out with Gly-
ProOBzl. The analysis was performed on a Mili-
chrome A-02 chromatograph in a gradient of 0.1%
acetic acid-methanol from 10% to 100% in 10 min,
then methanol for 2.5 min. The flow rate of the eluent
was 0.2 mL/min. The retention time of LA-Gly-
ProOMe and LA-Gly-ProOBzl was 10.30 min and
10.96 min, respectively.
Preparative purification of LA-Gly-ProOBzl and
LA-Gly-ProOMe was performed using a Reprosil-
Pur C18aq column (20 × 150 mm, particle size 10 μm)
in the system MeOH−H2O−AcOH−TFA (90 : 10 : 0.1 :
0.01). The flow rate of the eluent was 20 mL/min. The
retention times of the compounds were 2.94 min and
2.71 min, respectively. The yields of substances were
87% and 79%, respectively.
Synthesis of DP and Srt Derivatives Based on the Use
of Boc-Gly-Pro-Su and Z-Gly-Pro-Su
After addition of 214 mg DCCD (1.039 mmol) to
the solution of 1 mmol Boc- or Z-amino acid and
115 mg N-oxysuccinimide (1 mmol) in 5 mL dry diox-
ane, the mixture was stirred for 2 h at 15°C and then
left under stirring overnight at room temperature. The
urea was filtered off, the filtrate was washed with 2 mL
dioxane, and the solvent was evaporated. The precipi-
tate was dissolved in 6 mL ethanol. 1 mmol DP or Srt
was dissolved in 2 mL ethanol with 0.15 mL Et3N.
Then, 0.5 mmol of Boc-Pro-Su or Z-Gly-Pro-Su in
3 mL of ethanol was mixed with 0.5 mmol of DP or Srt
solution in 1 mL ethanol. The solutions were stirred
overnight at room temperature. Preparative purifica-
tion was performed as described above. The yield of
Boc-Gly-Pro-DP, Z-Gly-Pro-DP, Z-Gly-Pro-Srt,
and Boc-Gly-Pro-Srt was 82%, 86%, 81%, and 79%,
respectively.
Synthesis of Boc-Gly-ProOMe and Boc-Gly-ProOBzl
A solution of 875 mg Boc-Gly (5.57 mmol) in 5 mL
chloroform was treated with 1444 mg DCCD
(7 mmol) for 20 min at room temperature and after
addition of 0.7 mL Et3N (5.04 mmol) it was stirred for
10 min. Then a solution of 778 mg ProOCH3
(5.37 mmol) in 4 mL of chloroform was added and the
mixture was stirred for 3 h at room temperature and
then 72 h at 5°C. The precipitate was filtered off and
(b) After addition of 245.3 mg DCCD (1.19 μmol)
chloroform was evaporated. Boc-Gly-ProOMe was to a solution of 216 mg LA (1.08 mmol) and 124.2 mg
dissolved in 10 mL of ethyl acetate, washed with 5 mL N-oxysuccinimide (1.08 mmol) in 5 mL of dry diox-
1 M HCl and 5 mL water. Ethyl acetate was evapo- ane, the solution was stirred for 2 h at 15°C. The urea
rated, the residue was lyophilized. Boc-Gly-ProOBzl was filtered off, the filtrate was washed with dioxane
was synthesized in a similar manner. The analysis was (2 mL × 2), and the solvent was evaporated. The pre-
performed on a Milichrome A-02 chromatograph in cipitate was dissolved in 1 mL dioxane and treated
the gradient of 0.1% CH3COOH−methanol from 30% with a solution of 250.5 mg Gly-Pro-OBzl in 2 mL
ethanol with 0.2 mL of Et3N. After 21 h, the solution
to 100% in 12.5 min. The flow rate of the eluent was
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STABILITY OF PROLINE-CONTAINING DERIVATIVES OF DOPAMINE AND SEROTONIN
153
Synthesis of LA-Gly-Pro-DP
100
90
80
70
60
50
40
30
20
10
60 mg DCCD (0.29 mmol) was added, to a solu-
tion of 90 mg (0.26 mmol) of LA-Gly-Pro in 0.15 mL
of DMF under stirring. After 10 min, 150 μL Et3N and
then a solution of 50 mg DP (0.327 mmol) in 0.3 mL
DMF were sequentially added. Stirring was continued
for another 4 h. After addition of 4 mL chloroform
stirring was continued overnight. DMF was removed
by lyophilization. The residue was dissolved in ethyl
acetate (6 mL), and then 2 mL water and 0.1 mL of
acetic acid were added. After centrifugation, water and
urea were separated. The aqueous fraction was
extracted again with ethyl acetate (4 mL) and centri-
fuged. Preparative purification of LA-Gly-Pro-DP
was performed using a Reprosil-Pur C18aq column
(20 × 150 mm, particle size 10 μm) in the system
MeOH−H2O−AcOH−TFA (85 : 15 : 0.1 : 0.01). Reten-
tion time was 2.86 min. The yield of LA-Gly-Pro-DP
was 46%.
0
100
200
300
400
Time, min
Analysis of the Effect of the Presence of Ethanol
or DMSO in the Incubation Medium on the of Leucine
Aminopeptidase and Carboxypeptidase Y Functioning
Fig. 1. Kinetics of alkaline hydrolysis of LA-Gly-
ProОСН .
3
The effect of the presence of ethanol or DMSO on
the enzyme activity in the incubation medium was
analyzed using the following procedure. The effect of
ethanol or DMSO was investigated using 100 μL of
phosphate-buffered saline (PBS; 27.4 mM NaCl,
0.4 mM KCl, 2 mM Na3PO4 in 100 mL H2O, pH 7.4)
containing 0.068 μmol of Semax and 0.0039 U of
leucine aminopeptidases or 0.935 U of carboxypepti-
dase Y. The ratio PBS to ethanol or DMSO ranged
from 100 : 0 to 50 : 50 (Table 2). The incubation mix-
ture was stirred and thermostated at 30°C; aliquots
(15 μL) were taken at certain time intervals. Enzy-
matic hydrolysis was stopped by adding an equal vol-
ume of methanol to the samples taken.
was evaporated, dissolved in 5 mL ethyl acetate,
washed twice with 1 mL water. The aqueous layers
were combined and extracted with 1 mL ethyl acetate.
Ethyl acetate was evaporated. The yield of LA-Gly-
ProOBzl was 52%.
Synthesis of LA-Gly-Pro
After addition of 0.5 mL of 2 M NaOH to a solution
of 110 mg LA-Gly-ProOCH3 in 4 mL methanol under
stirring and incubation for 7 h (Fig. 1) the solution was
acidified with 1 M HCl and evaporated. The residue
was dissolved in 3 mL ethyl acetate and washed with
water until neutral pH. The solution was evaporated,
lyophilized and used without additional purification.
The yield of LA-Gly-Pro was 90%.
Evaluation of Stability of Peptide Derivatives
in the Presence of Leucine Aminopeptidases,
Carboxypeptidases Y and B, and PEP
In the case of leucine aminopeptidases, carboxy-
peptidases, and PEP, conditions of enzymatic hydro-
lysis were optimized by studying degradation of the
investigated peptides at different substrate : enzyme
Benzyl protection of LA-Gly-ProOBzl was
removed in methanol by hydrogenation in the pres-
ence of 5% PdO/BaSO4 (30 mg) for 90 min. The cat-
alyst was filtered off, the solution was evaporated, and ratios. For subsequent work, the ratio of 1 μmol/U was
used for leucine aminopeptidases, 0.092 μmol/U for
carboxypeptidase Y, 0.022 μmol/U for carboxypepti-
dase B, 10, 1 and 0.10 μmol/U for PEP.
the residue was lyophilized. The resultant substance
was used without additional purification. The yield of
LA-Gly-Pro was 98%.
Enzymatic hydrolysis of proline derivatives of DP
and Srt was carried out according to the following pro-
cedure. A solution of 0.5 μmol of a proline-containing
derivative in 260 μL PBS (27.4 mM NaCl, 0.4 mM
KCl, 2 mM Na3PO4 in 100 mL H2O, pH 7.4) was
treated with 0.47 U of leucine aminopeptidases or
5.45 U of carboxypeptidase Y or 22.46 U of carboxy-
The analysis was performed on a Milichrome A-02
chromatograph in a gradient of 0.1% acetic acid−
methanol from 10% to 100% in 10 min, then in meth-
anol for 2.5 min. The flow rate of the eluent was
0.2 mL/min. The retention time of the LA-Gly-Pro
was 10.10 min.
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SHEVCHENKO et al.
Table 2. The effect of ethanol and DMSO on the enzymatic hydrolysis of Semax (the reaction was carried out for 180 min)
Percent of ethanol and DMSO
Enzyme
Organic solvent
Ethanol
0.0
1.0
1.5
5.0
20.0
50.0
Leucine aminopeptidase
Carboxypeptidase Y
Leucine aminopeptidase
Carboxypeptidase Y
1.2*
14.6
1.2
1.8
19.2
2.1
3.0
21.3
9.2
5.0
27.5
17.1
54.5
88.0
76.9
96.3
97.5
97.7
98.1
97.9
DMSO
14.6
23.4
29.3
44.0
* Residual Semax content in the incubation solution, %.
peptidases B in the same buffer (PBS). For different experiments we used DCCD, thionyl chloride, various
proline derivatives of DP and Srt, 0.05 U, 0.5 U, or protective groups. The Boc group was removed under
5.0 U of PEP were added. The incubation mixture was acidic conditions, benzyl group was removed using
stirred and thermostated at 30°C and aliquots (20 μL) hydrogen gas in the presence of a catalyst, while
were taken at certain time intervals. Enzymatic hydro- methyl group was removed under alkaline conditions.
lysis was stopped by adding an equal volume of meth- Saponification of methyl ester occurred rather slowly:
anol to the samples taken.
90% of the reaction was completed only after 6 h
(Fig. 1).
Two main methods were used to obtain the above
substances. The first method involved the use of
DCCD for the condensation of compounds; the sec-
ond method used DCCD and N-oxysuccinimide to
obtain the corresponding esters, which were subse-
quently used to obtain desired substances. The first
method was more promising for synthesis of com-
pounds containing LA, while the second method was
preferable for reactions with DP and Srt.
Evaluation of Stability of Peptide Derivatives
in the Presence of Blood Plasma
A solution of 0.74 μmol of a peptide derivative in
820 μL of PBS (27.4 mM NaCl, 0.4 mM KCl, 2 mM
Na3PO4 in 100 mL H2O, pH 7.4) was mixed with
380 μL of blood plasma (31 mg protein/mL). The
incubation mixture was stirred and thermostated at
30°C; aliquots (100 μL) were taken at certain time
intervals. The obtained samples were purified by
reverse phase solid phase extraction: the peptide frac-
tion was applied onto a cartridge packed with a reverse
phase Lichroprep RP-18, followed by elution of the
peptides with methanol with 0.1% TFA. The mixture
was then evaporated and dissolved in 150 μL of a mix-
ture of methanol : water (5 : 95). The mixture was ana-
lyzed by HPLC.
All substances were characterized by chromato-
graphic methods (HPLC) and using mass spectros-
copy.
The difficulty of in vitro experiments with Boc-
Gly-Pro-DP, Z-Gly-Pro-DP, LA-Gly-Pro-DP,
Boc-Gly-Pro-Srt, Z-Gly-Pro-Srt consisted in their
insufficient solubility in water. Therefore, we first
evaluated the effect of ethanol or DMSO, which were
added to increase solubility of the DP and Srt deriva-
tives, on the enzymatic hydrolysis of peptides. In these
experiments Semax was used as a reference com-
pound.
RESULTS AND DISCUSSION
The N-terminal residue is especially vulnerable
peptide fragment in the compounds Boc-Gly-Pro-
DP, Z-Gly-Pro-DP, LA-Gly-Pro-DP, Boc-Gly-
Pro-Srt, Z-Gly-Pro-Srt and therefore, in the com-
pounds used, its amino group was protected (Boc, Z,
fatty acid). The idea of amino group protection with
fatty acid was inspired by the results obtained in [13].
These experiments have shown that the ethanol or
DMSO content less than 5% did not exhibit any
noticeable decrease in the efficiency of the enzymatic
hydrolysis of Semax (Table 2).
The study of the resistance of Pro-Gly-Pro-Leu
and its acylated analogues to leucine aminopeptidase
has shown that the degradation of the original peptide
was much higher than that of its N-acyl derivatives
and, therefore, the physiological effect of the latter can
be longer [13]. Consequently, the use of N-protected
peptides usually leads to an increase in their resistance
to degradation in biological media [13, 14].
Results demonstrate preferential use of ethanol.
Solutions of DP and Srt derivatives were prepared by
dissolving Boc-Gly-Pro-DP, Z-Gly-Pro-DP, Boc-
Gly-Pro-Srt, Z-Gly-Pro-Srt in 25% ethanol. After
addition of the necessary aliquot to the incubation
medium, the ethanol concentration did not exceed
1.5%. In the case of LA-Gly-Pro-DP, the concentra-
tion of ethanol or DMSO in the incubation medium
Synthesis of Boc-Gly-Pro-DP, Z-Gly-Pro-DP, had to be increased to 3%. Therefore, under such con-
LA-Gly-Pro-DP, Boc-Gly-Pro-Srt, Z-Gly-Pro-Srt ditions, the effect of ethanol on the efficiency of
was carried out using known methods [15, 16]. In the enzymes was minimal.
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STABILITY OF PROLINE-CONTAINING DERIVATIVES OF DOPAMINE AND SEROTONIN
155
Table 3. Stability of Boc-Gly-Pro-DP, Z-Gly-Pro-DP, LA-Gly-Pro-DP, Boc-Gly-Pro-Srt, Z-Gly-Pro-Srt in the pres-
ence of amino- and carboxypeptidases
Time, min
Substrate
Enzyme
2
30
60
180
1440
4320
Boc-Gly-Pro-DP
Z-Gly-Pro-DP
LА-Gly-Pro-DP
Boc-Gly-Pro-Srt
Z-Gly-Pro-Srt
Boc-Gly-Pro-DP
Z-Gly-Pro-DP
LА-Gly-Pro-DP
Boc-Gly-Pro-Srt
Z-Gly-Pro-Srt
Boc-Gly-Pro-DP
Z-Gly-Pro-DP
LА-Gly-Pro-DP
Boc-Gly-Pro-Srt
Z-Gly-Pro-Srt
Leucine aminopepti-
dase
100*
100
100
100
100
100
100
100
100
100
100
100
100
100
100
89.3
77.3
83.5
99.3
94.7
96.6
85.0
86.7
100
100
92.5
98.4
92.5
79.6
73.2
67.1
99.0
94.7
88.2
76.7
73.4
100
100
89.7
93.3
85.0
99.6
67.4
53.6
40.1
98.3
89.5
79.8
61.6
55.6
55.1
48.1
33.7
93.8
79.0
65.7
58.8
34.4
33.7
33.2
27.2
92.4
73.7
36.5
30.0
29.5
Carboxypeptidase B
Carboxypeptidase Y
100
100
100
94.4
88.7
91.7
73.3
98.4
88.9
80.3
83.2
73.0
97.2
94.4
83.3
54.0
62.6
40.5
96.8
88.9
100
100
100
100
* The initial content of the substance in the incubation solution, %.
Besides commercially available enzymes listed in idue and the presence of protective groups on the N-
Table 2, experiments with of DP and Srt derivatives terminal amino acid.
were performed using carboxypeptidase B and PEP
Different rates of decrease in the content of DP and
(Tables 3, 4).
Srt derivatives observed in the presence of the pepti-
As can be seen from the presented data, the stability
of Srt derivatives in the presence of leucinaminopepti-
dase was higher than that of the corresponding DP
derivatives. The same tendency was observed in the
presence of carboxypeptidases B and Y. Compounds
containing Boc protection in the presence of leucine
dases listed above indicate that this process is deter-
mined not by hydrolysis of the proline-containing
fragment of these derivatives, but by the degradation of
the dopamine or serotonin moieties of these com-
pounds. This is also indicated by the fact that during
mass spectrometric analysis of the reaction mixtures,
molecular peaks that could have formed from a pro-
line-containing fragment were not found.
aminopeptidases and carboxypeptidases
B were
more stable than compounds with Z protection or
containing lauric residue. In the presence of carboxy-
peptidase Y, compounds with Z-protection appeared
to be more stable than compounds with Boc-protec-
tion and containing lauric residue. In general, it can be
concluded that all synthesized compounds are highly
resistant to both amino and carboxypeptidases. In
contrast to Semax, the initial compounds remain in
the incubation mixture for many hours; such stability
is obviously explained by the presence of a proline res-
Experiments with PEP required optimization of its
concentration. Initially, the substrate-enzyme ratio
was used, as was the case with other enzymes. How-
ever, under these conditions degradation of DP and
Srt derivatives in the presence of PEP occurred very
quickly. Thus, we had to decrease PEP concentration
during the kinetic study of the enzymatic hydrolysis of
the synthesized compounds (Table 4).
As can be seen from the above data, stability of
compounds with Z-protection in the presence of PEP
was lower than that of compounds with Boc-protec-
tion. At the substrate-enzyme ratio of 5.7 μmol/U, the
remaining content of Z-Gly-Pro-DP after 60-min
incubation was 30%, while the content of Boc-Gly-
Pro-DP was about 90%. At the substrate-enzyme ratio
of 0.49 μmol/U Z-Gly-Pro-DP underwent complete
degradation; in the case of Boc-Gly-Pro-DP about
17% remained. Therefore, the study on time-depen-
dence of stability of Boc-Gly-Pro-DP, Z-Gly-Pro-
Table 4. Optimization of the substrate-PEP ratio (the reac-
tion was carried out for 60 min)
Substrate-enzyme ratio, μmol/U
Substrate
0.05
0.49
0.0 28.0
17.5 92.4 100
* The initial content of the substance in the incubation solution, %.
5.7
58
94.1 100
100
597
Z-Gly-Pro-DP
0.0*
Boc-Gly-Pro-DP 0.0
BIOCHEMISTRY (MOSCOW), SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol. 14 No. 2 2020

156
SHEVCHENKO et al.
(b)
(a)
(c)
100
90
80
70
60
50
40
30
20
10
100
90
80
70
60
50
40
30
20
10
100
90
80
70
60
50
40
30
20
10
0
40 80 120 160 200 240
Time, min
0
40 80 120 160 200 240
Time, min
0
40 80 120 160 200 240
Time, min
Fig. 2. Stability of Z-Gly-Pro-DP (j), Z-Gly-Pro-Srt (m), Boc-Gly-Pro-DP (r), Boc-Gly-Pro-Srt (d), LA-Gly-Pro-DP (×)
used at various concentrations in the presence of PEP: (a) 10 μmol/U; (b) 1 μmol/U; (c) 0.1 μmol/U.
DP, LA-Gly-Pro-DP, Boc-Gly-Pro-Srt, Z-Gly-Pro-
Srt in the presence of PEP was carried out for com-
pounds with Z- and Boc-protection at different sub-
strate-enzyme ratios (Fig. 2).
The kinetic study of the reaction product formation
under the PEP action was performed both for com-
pounds with Z-protection and for compounds with
Boc-protection. It was shown that enzymatic hydroly-
sis occurred at the bond between DP / Srt and proline
residue (Fig. 3, Table 6).
Thus, under the PEP action, a gradual release of
DP and Srt occurs. Maximum values (about 90%)
were obtained in a few hours. Consequently, it is rea-
sonable to expect a longer exposure to DP and Srt,
formed from these compounds, on processes occur-
ring both in cells and in the whole body.
As can be seen from the presented results, the shape
of the kinetic curves for all compounds was identical.
Enzymatic hydrolysis of derivatives with Z-protection
occurred much faster than compounds with Boc-pro-
tection (Fig. 2a). The kinetics of LA-Gly-Pro-DP
hydrolysis could be determined only after 10-fold
increase in the enzyme concentration (Fig. 2c). The
DP and Srt residues had a minor contribution to the
stability of compounds at the low enzyme content. A
decrease in the substrate-enzyme ratio to 1 μmol/U
was accompanied by a higher stability of the Srt deriv-
atives (Fig. 2b). The experiment performed for several
days has shown that at the lowest substrate-enzyme
ratio (0.1 μmol/U), only LA derivatives (LA-Gly-Pro-
A comparison of the data obtained using commer-
cially obtained enzymes and the enzyme system exist-
ing in rat blood plasma showed that persistence of the
main patterns (Table 7).
As can be seen from the data obtained (Table 7),
DP) can be detected (Table 5). At the ratio 10 μmol/U among the DP derivatives the compound containing
significant amounts of compounds with Boc protec- LA was the most stable as compared with the deriva-
tion were detected, while compounds with Z protec- tives with Boc protection and, especially with deriva-
tion were basically absent in the incubation medium or tives containing Z protection. As already emphasized
a few percents of the initial value could be detected above the overall stability of compounds with the same
(Table 5). At the substrate enzyme ratio of 1 μmol/U protective group was determined by the stability of
compounds with Z-protection and Boc-protection dopamine or serotonin fragments. It can be concluded
were completely hydrolyzed in one day (Table 5).
that in the presence of rat blood enzymes in experi-
Table 5. PEP-based enzymatic hydrolysis of substances during reaction time for more than one day
Time
72 h
Time
72 h
CPEP
μmol/U
,
CPEP,
μmol/U
Substrate
24 h
120 h
24 h
120 h
Z-Gly-Pro-DP
Boc-Gly-Pro-DP
Z-Gly-Pro-Srt
Boc-Gly-Pro-Srt
LА-Gly-Pro-DP
10
0.0*
72.46
5.23
84.54
–
0.0
34.30
2.83
68.42
–
0.0
23.70
2.40
56.40
–
1
0.0
0.0
0.0
0.5
49.1
0.0
0.0
0.0
0.0
44.3
0.0
0.0
0.0
0.0
43.1
0.1
* The initial content of the substance in the incubation solution, %.
BIOCHEMISTRY (MOSCOW), SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol. 14 No. 2 2020

STABILITY OF PROLINE-CONTAINING DERIVATIVES OF DOPAMINE AND SEROTONIN
157
(a)
(b)
100
90
80
70
60
50
40
30
20
10
100
90
80
70
60
50
40
30
20
10
DP from Z-Gly-Pro-DP
Srt from Z-Gly-Pro-Srt
DP from Boc-Gly-Pro-DP
Srt from Boc-Gly-Pro-Srt
0
40
80
120 160 200 240
Time, min
0
40
80
120 160 200 240
Time, min
Fig. 3. Formation of DP and Srt from their peptide derivatives at various substrate-enzyme (PEP) ratios: (a) 10 μmol/U;
(b) 1 μmol/U.
Sciences “Innovative Developments in Biomedicine” and
“Postgenomic Technologies and Promising Solutions in
Biomedicine.”
ments in vitro, derivatives of DP and Srt are stable.
Therefore, the synthesized DP and Srt derivatives can
be the sources of DP and Srt for several hours. It is rea-
sonable to suggest that they may be drugs that can be
used for a long-term effect on pathological processes
with the aim of their localization and treatment.
COMPLIANCE WITH ETHICAL STANDARDS
All animal experiments were carried out in accordance
with the International Recommendations of the “European
Convention for the Protection of Vertebrate Animals Used
FUNDING
The work was partially supported by the Program of the for Experiments or for Other Scientific Purposes” (The
Basic Research of the Presidium of the Russian Academy of European Convention, 1986).
Table 6. Formation of DP and Srt from their peptide derivatives during their 5-day with PEP (the substrate-enzyme ratio
for Z-derivatives was 10 μmol/U, for Boc-derivatives—1 μmol/U)
Time, days
3
Time, days
3
Source of DP
Source of Srt
1
5
1
5
Z-Gly-Pro-DP
Boc-Gly-Pro-DP
66.8*
75.7
16.2
54.9
12.9 Z-Gly-Pro-Srt
19.8 Boc-Gly-Pro-Srt
88.5
56.7
74.9
50.1
61.3
26.3
* The content of the enzymatic hydrolysis product in the incubation solution, %.
Table 7. Stability of Boc-Gly-Pro-DP, Z-Gly-Pro-DP, LA-Gly-Pro-DP, Boc-Gly-Pro-Srt, Z-Gly-Pro-Srt in the pres-
ence of rat plasma
Time, min
180 300
Z-Gly-Pro-DP 99.0 0.7* 87.1 4.9 79.1 4.8 48.8 6.9 30.8 4.3 20.0 2.6 10.3 1.5
Boc-Gly-Pro-DP 100
Compound
10
30
60
120
420
1440
0
2880
0
4320
0
0
97.4 1.5 96.0 1.4 56.6 3.3 50.0 4.1 29.0 4.2 14.5 2.0 1.6 0.2 1.1 0.2
LA-Gly-Pro-DP 99.0 0.5 95.9 2.1 88.8 3.9 81.6 4.7 70.4 4.4 67.4 2.2 66.3 0.9 60.2 2.9 46.3 4.8 23.5 3.3
Z-Gly-Pro-Srt
Boc-Gly-Pro-Srt
100
100
100 80.0 8.5 60.0 9.2 40.0 6.6 24.9 3.5 13.4 1.9
100 98.7 0.7 82.9 8.6 65.8 5.8 65.8 4.2 64.5 4.6 63.2 4.7 61.8 5.1 59.2 4.5
0
0
0
* The initial content of the substance in the incubation solution, %.
BIOCHEMISTRY (MOSCOW), SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol. 14 No. 2 2020

158
SHEVCHENKO et al.
CONFLICT OF INTEREST
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Acta, 1988, vol. 954, no. 2, pp. 161−169.
The authors declare that they have no conflict of interest
10. Hooper, N.M., Htyszko, J., and Turner, A.J., Biochem.
J., 1990, vol. 267, no. 2, pp. 509–515.
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Translated by A. Medvedev
BIOCHEMISTRY (MOSCOW), SUPPLEMENT SERIES B: BIOMEDICAL CHEMISTRY Vol. 14 No. 2 2020