PEPTIDE DERIVATIVES
175
Table 2. Distribution of proline isotopomers for different con-
The reaction was carried out with stirring for 12–17 h
at room temperature. The reaction mixture was
worked-up by the procedure described in [6]. The
Boc-Gly-[2H]Pro dipeptide was condensed with Dox,
DOPA, or serotonin, as described in Table 1. The
yields reached values of about 30–40%. Apparently,
this is due to the necessity of preliminary preparation
of glycine N-hydroxysuccinimide ester and conduc-
tion of condensation in the presence of water.
A similar procedure was used to prepare Boc-Gly-
ΔPro. Hydrogenation of Boc-Gly-ΔPro was con-
ducted in ethyl acetate in the presence of 5%
PdO/BaSO4 in a deuterium atmosphere (400 hPa).
Hydrogenation in aprotic solvent made it possible to
introduce, on average, 1.91 deuterium atoms into the
peptide. The contents of isotopomers are summarized
in Table 2. Furthermore, isotope labeling at a later
stage minimizes the overall loss of the label during the
synthesis of isotope-modified compounds. High-per-
formance liquid chromatography of Boc-Gly-[2H]Pro
and Boc-Gly-ΔPro was conducted on a ProntoSIL-
120-5-C18 AQ DB-2003 column (2.0×75 mm; parti-
cle diameter of 5 µm) in the system of A, 0.1%
CH3COOH, and B, acetonitrile, with 0 to 100% gradi-
ent of B in 12.5 min; the eluent flow rate was
0.2 mL/min. The retention time was 5.76 min.
ditions of deuteration (deuterium gas pressure of 400 hPa)
Content of isotopomers in hydrogenated ΔPro, %
2H
atoms
2H2O*
H2O*
DMF** 140°C*** EtAc****
0
1
2
3
4
5
6
7
13.2
17.2
69.6
4.0
1.1
–
7.9
14.9
64.2
10.6
2.5
–
5.4
24.5
63.6
4.8
1.7
–
29.8
18.9
23.4
12.4
8.1
4.4
2.2
0.8
3.5
20.9
60.2
13.5
2.2
–
–
–
–
–
–
–
–
–
2
* ΔPro (80 mg), H O or H O (0.4 mL), 5% PdO/BaSO (20 mg),
2
2
4
2 h; ** ΔPro (2 mg), DMF (0.4 mL ), 5% PdO/BaSO (4 mg), 2 h;
4
*** 5% PdO/BaSO (117 mg) was impregnated with ΔPro (11.7 mg)
4
in water (40 µL) and freeze-dried, the reaction was carried out for
15 min at 140°C; **** Boc-Gly-ΔPro (5 mg), ethyl acetate (0.4 mL),
5% PdO/BaSO (11 mg ), 2 h.
4
Table 3. Calculated distributions of Dox, DOPA, and sero-
tonin between blood and brain tissues
AUCbrain/AUCblood
Compound
Boc-Gly-Pro-DOPA
Boc-Gly-Pro-Dox
Boc-Gly-Pro-Srt
Z-Gly-Pro-DOPA
Z-Gly-Pro-Dox
Z-Gly-Pro-Srt
0.085
0.019
0.079
0.059
0.013
0.054
Thus, we obtained and identified Boc-Gly-Pro-
Dox, Boc-Gly-Pro-DOPA, and Boc-Gly-Pro-Srt
and their deuterated analogues.
With a large number of compounds at hand, their
comprehensive biological testing presents a certain
problem. This takes a lot of time and is experimentally
sophisticated, for example, when it is necessary to find
out how efficiently these compounds overcome the
blood–brain barrier (BBB). The time is required for
the preparation of rats, injection of the agent, isolation
of the biological material, analysis of the obtained
specimens, etc. This work can be simplified by using
recently developed approaches [7, 8]. These
approaches provide a tentative idea of the brain con-
tent of the test compound (AUCbrain) if the blood level
of the compound is known (AUCblood) (Table 3).
due to isotope exchange under these conditions. This
is also indicated by the formation of a large amount of
Pro isotopomers containing less than two deuterium
atoms per amino acid molecule. It is evident that some
double bonds are reduced by protons activated via iso-
tope exchange. For the above reasons, preparative
amounts of hydrogen isotope-labeled proline should
better be obtained using an aqueous solution, because,
as indicated above, the isotope dilution is minimized
As can be seen from the presented data, this value
and the labeling procedure is markedly more facile and is affected by not only the molecular groups responsi-
does not depend on many factors that should be borne ble for the biological activity of the compound, but
in mind to conduct the solid-phase hydrogenation.
also groups attached to free amine of the amino acid.
The Boc-protection is somewhat more advantageous
than Z-protection for penetration of the same agent
through the BBB. Among the groups determining the
biological activity, the best results were found for
DOPA, which was followed by Srt; the Dox-contain-
ing compounds were 4 to 5 times inferior.
For the synthesis of Boc-Gly-[2H]Pro, equimolar
amounts of Boc-Gly, N-hydroxysuccinimide, and
DCHC (a chloroform solution) were stirred overnight
to give Boc-GlyOSu. The solvent was evaporated. The
residue was extracted with ethyl acetate and centri-
fuged. The condensation of Boc-GlyOSu (without
additional purification) with [2H]Pro was carried out
by a reported procedure [4]. For this purpose, [2H]Pro
hydrochloride (40 mg, 0.26 mmol) in water (0.2 mL)
containing Et3N (0.1 mL) was treated with an ethanol
solution of Boc-GlyOSu (1 mL) (1 : 2 molar ratio).
FUNDING
This work was partially supported by the Basic Research
Program of the Presidium of the RAS “Basic Research for
Biomedical Technologies.”
DOKLADY CHEMISTRY Vol. 487 Part 1 2019
Shevchenko
