Synthesis, pharmacokinetics, and metabolism of the C-terminal tripeptide of dermorphin and its diastereomer

Article abstract of DOI:10.1134/S1068162007060015

A tritium-labeled C-terminal fragment of dermorphin (H-Tyr-[3,4- ³H]Pro-Ser-NH₂) and its isomer (H-Tyr-D-[3,4- ³H]Pro-Ser-NH₂) with molar radioactivity of 35 Ci/mmol were synthesized, and their pharmacokinetics

Full text of DOI:10.1134/S1068162007060015

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2007, Vol. 33, No. 6, pp. 537–543. © Pleiades Publishing, Inc., 2007.
Original Russian Text © P.S. Gromovykh, L.S. Guzevatykh, K.V. Shevchenko, L.A. Andreeva, L.Yu. Alfeeva, V.P. Shevchenko, I.Yu. Nagaev, T.A. Voronina, N.F. Myasoedov, 2007,
published in Bioorganicheskaya Khimiya, 2007, Vol. 33, No. 6, pp. 581–587.
Synthesis, Pharmacokinetics, and Metabolism of the C-Terminal
Tripeptide of Dermorphin and Its Diastereomer
P. S. Gromovykha,1, L. S. Guzevatykhb, K. V. Shevchenkoa, L. A. Andreevaa, L. Yu. Alfeevaa,
V. P. Shevchenkoa, I. Yu. Nagaeva, T. A. Voroninab, and N. F. Myasoedova
a Institute of Molecular Genetics, Russian Academy of Sciences, pl. Akademika Kurchatova 2, Moscow, 123182 Russia
b Zakusov Institute of Pharmacology, Russian Academy of Medical Sciences, ul. Baltiiskaya 8, Moscow, 125315 Russia
Received September 21, 2006; in final form, April 25, 2007
Abstract—A tritium-labeled C-terminal fragment of dermorphin (H-Tyr-[3,4-3H]Pro-Ser-NH2) and its isomer
(H-Tyr-D-[3,4-3H]Pro-Ser-NH2) with molar radioactivity of 35 Ci/mmol were synthesized, and their pharma-
cokinetics and metabolism in rat organs were studied after their intramuscular injections. The tripeptides were
detected in the blood only for 5 min after the injection, and maximum contents of both compounds (approxi-
mately 5% of the total amount of the injected label) were registered in the kidneys after 20 min. Both stereomers
were shown to penetrate into the brain. We failed to detect any radioactive metabolite, except proline, due to
rapid proteolytic degradation of these peptides.
Key words: dermorphin, C-terminal fragment, dehydroproline analogue, synthesis, labeling with tritium,
metabolism, pharmacokinetics
DOI: 10.1134/S1068162007060015
INTRODUCTION
which leads to the formation of N-terminal tetrapeptide
and C-terminal tripeptide.
Dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-
NH2) is an endogenous opioid peptide with a high bio-
logical activity. Dermorphin and its analogue,
[Hyp6]dermorphin, were first isolated from skin of
South American frogs [1].2 Dermorphins were also
found in the central nervous system and peripheral
organs of warm-blooded animals [2]. Dermorphin
affects the thermoregulation system of warm-blooded
The N-terminal tetrapeptide is known to be a mini-
mal fragment with the analgesic activity [2]. There is no
information on biological properties of the C-terminal
tripeptide and its stereomer (H-Tyr-D-Pro-Ser-NH2) in
literature. We have here studied the pharmacokinetics
and metabolism of H-Tyr-Pro-Ser-NH2 and H-Tyr-D-
animals upon its peripheral administration and exhibit Pro-Ser-NH2 in rat organs upon their intramuscular
strong analgesic effect, which is 290 and 10–25 times
higher than the effect of morphine after the central and
peripheral administration, respectively [3]. Dermor-
phin also exhibits a wide spectrum of physiological
activities: it affects functions of cardiovascular, respira-
tory, digestive, and secretory systems and various forms
of animal behavior [4].
injections and their ability to penetrate into the rat
brain.
RESULTS AND DISCUSSION
It was necessary to synthesize the Boc-Tyr(Boc)-
DL-3,4-∆Pro-Ser-NH2 precursor (IIIc) of the isomeric
Studies of dermorphin pharmacokinetics upon its
intravenous administration in rats demonstrated that it tripeptides with the labeled Pro residue for the studies
has a short lifespan in blood (t1/2 = 1.3 min) and is accu-
mulated in liver and kidneys. It was shown that only a
third of dermorphin remains intact, two thirds are its
degradation products. The Gly–Tyr bond of the peptide
is mainly cleaved by enzymes of brain homogenate [5],
of metabolism of the C-terminal fragment of dermor-
phin in different organs of rats. We also synthesized all
the corresponding nonradioactive tri- and dipeptides:
H-Tyr-Pro-Ser-NH2 (IVa), H-Tyr-D-Pro-Ser-NH2
(IVb), H-Pro-Ser-NH2 (VIIa), H-Pro-D-Ser-NH2
(VIIb), H-Tyr-Pro-OH (VIIIa), and H-Tyr-D-Pro-OH
(VIIIb) as chromatographic standards for the metabolic
studies (scheme).
1
Corresponding author; fax: +7 (499) 196-0221; e-mail:
nagaev@img.ras.ru.
2
3
Abbreviations: 3,4-∆Pro, 3,4-dehydroproline; [3,4- H]Pro,
3,4-bistritioproline.
537

538
GROMOVYKH et al.
Boc-Tyr(Boc)-OH
HCl · H-Tyr-Xaa-OH
H+
(VIIIa,b)
H-Xaa-OH
Boc-Tyr(Boc)-Xaa-OH
H-Ser-OMe
(Ia–c)
H+
NH3
Boc-Tyr(Boc)-Xaa-Ser-NH2
Boc-Tyr(Boc)-Xaa-Ser-OMe
HCl · H-Tyr-Xaa-Ser-NH2
(IVa,b)
(IIIa–c)
(IIa–c)
H+
NH3
H-Ser-OMe
Boc-Xaa-OH
Boc-Xaa-Ser-OMe
(Va,b)
Boc-Xaa-Ser-NH2
(VIa,b)
HCl · H-Xaa-Ser-NH2
(VIIa,b)
(Ia)–(VIIIa): Xaa = L-Pro; (Ib)–(VIIIb): Xaa = D-Pro; (Ic)–(IIIc): Xaa = DL-3,4-∆Pro
tripeptide of dermorphin (IVa), its analogues, and relative dipeptides.
Scheme. Synthesis of the H-Tyr-Pro-Ser-NH C-terminal
2
Dipeptides (Ia)–(Ic), (VIa), and (VIb) were synthe- catalyst. The liquid phase hydrogenation in the atmo-
sized using the methods of mixed anhydrides and active sphere of tritium gas in dioxane for 3 h at room temper-
esters [6–8]. Tripeptides (IIIa)–(IIIc) were prepared by ature in the presence of 5% PdO/BaSO4 proved to be
the coupling of dipeptides (Ia)–(Ic) with the serine the best synthetic method with optimum balance
methyl ester and subsequent ammonolysis. The neces- between the yield and molar radioactivity of the target
sary standards of free peptides (VIa), (VIb), (VIIa), peptide. The resulting mixture of the labeled diastere-
(VIIb), (VIIIa), and (VIIIb) hydrochlorides were omers was deprotected and fractionated by HPLC, and
obtained as a result of deprotection of the correspond- labeled peptides [3,4-3H](IVa) and [3,4-3H](IVb) were
ing Boc-derivatives. They were characterized by mass obtained. Their molar radioactivities (35 Ci/mmol) was
spectrometry and HPLC (the retention times are given sufficient for the studies of pharmacokinetics and
in Table 1).
One can see from Table 1 that the isomeric [3,4-
metabolism.
The labeled peptides [3,4-3H] (IVa) and [3,4-
3H](IVa) and [3,4-3H](IVb) labeled with tritium can be
obtained by the HPLC fractionation of the diastereo-
meric mixture formed after the tritium hydrogenation
and subsequent deprotection of the derivative of race-
mic dehydroproline (IIIc). In addition, a clear fraction-
ation of the studied tripeptide and most of the potential
metabolites proceeds under the specially chosen HPLC
conditions. As expected, chromatographic mobilities of
diastereomers (VIIIa) and (VIIIb) significantly differ.
However, hydrophilic dipeptides (VIIa) and (VIIb) are
weakly retained on the reversed phase and cannot be
resolved by HPLC.
3H](IVb) were intramuscularly injected in rats for stud-
ies of penetration and cleavage rate of these peptides in
the rat organs and blood. The rats were decapitated after
the definite intervals after the administration of labeled
peptides, and the organs and blood samples were taken.
The samples of organs and blood were homogenized
at cooling in liquid nitrogen. The solution containing
the known quantities of the corresponding unlabeled
peptides was added to the samples to monitor the com-
pleteness of extraction of the peptide material. The con-
tents of the peptides were determined after the extraction,
and the corresponding corrections were entered. The pep-
tides were extracted from the samples with a 4 : 1 aceto-
nitrile–water mixture and centrifuged. The supernatant
was evaporated. The residue was dissolved in a 1 : 1
methanol–water mixture and analyzed by HPLC. An
aliquot (0.1 volume) was preliminarily taken for the
determination of radioactivity of the biological sample.
The results revealed peculiarities of pharmacokinetics
and metabolism of the tripeptides (Table 2, Figs. 1, 2).
The contents of tripeptides in the brain were calculated
by the technique given in [10, 11], according to which
the blood volume of the brain is 2.6% of the blood vol-
ume in the whole organism.
We introduced the tritium label into the dehydropro-
line precursor (IIIc) by the solid phase and liquid phase
hydrogenation [9] with tritium gas in the presence of a
Table 1. Retention times of the tripeptides and their frag-
ments at HPLC (for conditions, see the Experimental section)
Retention times, min
Substance
series a
with L-Pro
series b
with D-Pro
H-Tyr-Pro-Ser-NH2 (IV)
H-Tyr-Pro-OH (VIII)
H-Pro-Ser-NH2 (VII)
H-Pro-OH
13.56
22.28
1.49
14.21
26.43
1.49
Peptides [3,4-3H](IVa) and [3,4-3H](IVb) were
found to be detected in blood only for the first five min,
whereas the maximum contents of the tripeptides in
brain, lungs, kidneys, and liver of rats were observed
1.76
1.76
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SYNTHESIS, PHARMACOKINETICS, AND METABOLISM
539
Table 2. Change in the percent content of isomers (IVa) and (IVb) with time in the rat organs (radioactivity of the tripeptide
administered in rats is taken to be 100%)
Time, min
Substance
Organ
5
20
40
80
Blood
Kidneys
Liver
0.31 0.05
0.8 0.1
0
0
–
3.6 0.5
2.8 0.5
0.23 0.03
(IVa)
0.35 0.08
0.047 0.007
0.28 0.05
0.09 0.01
0.53 0.07
0.059 0.004
0.07 0.02
0.22 0.05
0.5 0.2
0.04 0.02
Brain
0.032 0.003
0.03 0.02
0.07 0.02
–
–
–
Heart
Lungs
Blood
Kidneys
Liver
0.42 0.09
2.4 0.4
0
0
–
5
2
4.904 0.004
0.26 0.09
0.062 0.007
0.13 0.01
0.218 0.008
0.8 0.2
0.18 0.05
0.08 0.06
0.30 0.02
0.41 0.07
0.6 0.3
0.13 0.04
0.31 0.08
0.5 0.1
1.50 0.04
(IVb)
Brain
–
–
–
Heart
Lungs
after 20 min. Content of the D-Pro-containing peptide larly, the labeled L-proline is formed from [3,4-
[3,4-3H](IVb) in the brain and lungs is about two times
higher than that of [3,4-3H](IVa) (0.13 and 0.06% in
the brain and 0.49 and 0.22% in lungs, respectively).
Similar amounts of both isomers are accumulated in
heart within the first five min, but the content of the L-
isomer quickly decreases later, whereas the content of
the D-isomer remains practically constant. This indi-
3H](IVa) faster than D-proline from [3,4-3H](IVb).
We cannot find any radioactive metabolites of the
tripeptides in the organs and blood of rats, except for
the proline isomers (Figs. 3, 4). This fact unexpectedly
indicates a high rate of the proteolytic cleavage of both
peptide bonds of such short peptides independently of
cates a higher proteolytic stability of the latter. Simi- the proline configuration.
Content, %
Content, %
4.00
3.50
3.00
0.35
0.30
0.25
0.20
(b)
(a)
5 min
5 min
20 min
40 min
2.50
2.00
1.50
1.00
0.50
0
20 min
40 min
0.15
0.10
0.05
0
Time, min
Time, min
3
3
Fig. 1. Time changes in the content of (a) H-Tyr-[3,4- H]Pro-Ser-NH [3,4- H](IVa), in lungs, brain, heart and (b) liver, kidneys,
2
and blood.
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540
GROMOVYKH et al.
Content, %
Content, %
0.60
0.50
0.40
7.00
6.00
5.00
(a)
5 min
(b)
5 min
20 min
40 min
20 min
40 min
4.00
3.00
2.00
0.30
0.20
0.10
0
1.00
0
Time, min
Time, min
3
3
Fig. 2. Time changes in the content of H-Tyr-D-[3,4- H]Pro-Ser-NH [3,4- H](IVb), in lungs, brain, (a) heart and liver, (b) kidneys
2
and blood.
Content of Pro, %
Content of Pro, %
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0
0.25
0.20
0.15
0.10
(a)
5 min
5 min
(b)
20 min
40 min
20 min
40 min
0.05
0
Time, min
Time, min
Fig. 3. Time changes in the content of Pro in lungs, brain, (a) heart and liver, (b) kidneys and blood.
Our results show that the labeled proline is more tration with equal ease and, second, the double level of
quickly formed from [3,4-3H](IVa) than from the cor-
responding D-isomer in all the rat tissues. This fact
allows assign a decreased content of [3,4-3H](IVa) in
the rat brain precisely to its faster metabolism. Note
that the total amounts of label we measured in the rat
brain are practically equal for both isomers of the trip-
eptide; i.e., they do not depend on the proline configu-
ration. One can conclude from this fact that, first, both
forms of tripeptide (IV) penetrate into the brain within
the D-proline isomer in comparison with the L-proline
isomer registered already five minutes after the admin-
istration is associated with the known enzymatic stabil-
ity of the peptides containing D-amino acid residues.
EXPERIMENTAL
Commercially available amino acids, reagents, and
the first five minutes after their intramuscular adminis- solvents were used in this study. Mps were determined
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SYNTHESIS, PHARMACOKINETICS, AND METABOLISM
541
Content of Pro, %
0.45
Content of Pro, %
0.018
0.40
0.35
0.30
0.25
0.20
0.016
0.014
0.012
0.010
0.008
0.006
0.004
(a)
(b)
5 min
5 min
20 min
40 min
20 min
40 min
0.15
0.10
0.05
0
0.002
0
Time, min
Time, min
Fig. 4. Time changes in the content of D-Pro in (a) lungs, brain, and heart and (b) liver, kidneys, and blood.
on a Boetius device (Rapido, Germany) and are given cooled solution of the mixed anhydride was added. The
without correction.
reaction mixture was stirred for 20 min at –20°ë and
1 h at –10°ë and monitored by TLC in chromato-
graphic system A. When the process was completed,
the reaction mixture was evaporated in a vacuum. The
residue was dissolved in water (20 ml), acidified with
NaHSO4 · 3ç2é (29.9 g) to pH 3, and extracted with
ethyl acetate (5 × 50 ml). The combined organic
extracts were washed with water, 10% solution of
äçSO4, water, and the saturated solution of NaCl,
dried with MgSO4, and evaporated. The residue was
reprecipitated from ethyl acetate with hexane two
times. The precipitate powder was filtered and dried in
a vacuum. The yield of (Ib) was 13.9 g (81%); Rf 0.52
(A); mp 75–77°ë.
The homogeneity of the synthesized compounds
was determined by TLC on Silufol plates (Czech
Republic) and by HPLC. The substances on TLC plates
were detected by the treatment with ninhydrin solution.
The chromatographic mobilities (Rf) in the follow-
ing chromatographic systems are given: (A) 2 : 1 : 1
acetone–benzene–acetic acid and (B) 8 : 1.75 : 0.25
chloroform–methanol–concentrated ammonia.
The labeled preparation was analyzed and purified
by HPLC on a Gilson chromatograph (France)
equipped with a Kromasil SGX C18 column (5 µm, 3 ×
150 mm) (Eka Chemicals AB, Sweden). The fraction-
ation was carried out in an isocratic regime at a flow
rate of 1 ml/min in 5% methanol in NH4H2PO4 + H3PO4
buffer (50 mM, pH 2.8).
The MultiKhrom system (OOO Ampersend, Rus-
sia) on the basis of an IBM PC/AT was used for collec-
tion and processing the data. Radioactivity was mea-
sured on a Berthold LB 506 scintillation counter (Ber-
thold, Germany) at a 30% efficiency of the tritium
registration using the dioxane scintillator. Mass spectra
were recorded on an LCQ Finnigan MAT ESI/MS spec-
trometer (United States).
Boc-Tyr(Boc)-Pro-OH (Ia) (yield of 95%; Rf 0.26,
mp 79–81°ë) and Boc-Tyr(Boc)-DL-3,4-∆Pro-OH (Ic)
(yield of 99%; Rf 0.38 (A); mp 93–95°ë) were similarly
prepared.
Boc-Tyr(Boc)-D-Pro-Ser-OCH3 (IIb). N-Hydrox-
ysuccinimide (4.07 g, 35.4 mmol) was added to a solu-
tion of (Ib) (13.02 g, 27.2 mmol) in acetonitrile (100 ml).
The reaction mixture was cooled to 0°ë, treated with
DCC (7.30 g, 35.4 mmol), and stirred for 1 h at 0°ë.
Then HCl · Ser-OCH3 (5.50 g, 35.4 mmol) and triethy-
lamine (5.0 ml, 35.4 mmol) were added, and stirring
Boc-Tyr(Boc)-D-Pro-OH (Ib). Boc-Tyr(Boc)-OH
(13.8 g, 36.2 mmol) was dissolved in acetonitrile was continued for 1 h at 0°ë and 2 days at room tem-
(125 ml), triethylamine (5.6 ml, 39.8 mmol) and piv- perature. The reaction mixture was filtered, evaporated,
aloyl chloride (4.9 ml, 39.8 mmol) were added to the diluted with ethyl acetate (250 ml), and washed with
solution at –20°ë, and the reaction mixture was kept at water (20 ml), 10% solution of KHSO4 (45 ml), water
–20°ë for 20 min. D-Proline (5 g, 43.4 mmol) was dis- (30 ml), 5% solution of NaHCO3 (30 ml), water (30 ml),
solved in water (20 ml) and diluted with acetonitrile and saturated solution of NaCl. The ethyl acetate layer
(60 ml). Triethylamine (6.08 ml, 43.8 mmol) was was dried with MgSO4, filtered, and evaporated. The
added, and the solution was cooled to –20°ë, and a oily residue was washed with ether and dried in a vac-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 6 2007

542
GROMOVYKH et al.
uum. The yield of dried residue was 10.8 g (68%); Rf 18%, respectively. Their molar radioactivities were
0.27 (A); mp 81–83°ë.
35 Ci/mmol.
Pharmacokinetics of the labeled tripeptides [3,4-
Boc-Tyr(Boc)-Pro-Ser-OCH3 (IIa) (yield 96%; Rf
0.18 (B); mp 86–88°ë), Boc-Tyr(Boc)-DL-3,4-∆Pro-
Ser-OCH3 (IIc) (yield 87%, Rf 0.24 (A) and 0.81 (B),
mp 85–87°ë), Boc-Pro-Ser-OCH3 (Va), and Boc-D-
Pro-Ser-OCH3 (Vb) were similarly prepared.
3H](IVa) and [3,4-3H](IVb) in rat organism. An aque-
ous solution (200 µl) of the mixture of labeled [3,4-
3H](IVa) or [3,4-3H](IVb) (200 µCi, 0.06 µmol) and
nonradioactive (IVa) or (IVb) (198 µg, 0.54 µmol) was
intramuscularly administered in outbred male rats
(body mass of 200 20 g). After the necessary time
interval, the rats were decapitated, and their organs and
blood samples were taken. The samples were stored at
–70°ë.
Boc-Tyr(Boc)-D-Pro-Ser-NH2 (IIIb). A solution
of tripeptide (IIb) (5.55 g, 9.6 mmol) in methanol
(200 ml) was saturated with gaseous ammonia for
60 min at 0°ë and kept for 2 days at room temperature.
The reaction mixture was coevaporated with ethyl ace-
tate (2 × 10 ml) and with anhydrous methanol (2 ×
10 ml). The product was precipitated with ether from
absolute methanol; yield of (IIIb) 5.28 g (95%); Rf 0.70
(B); mp 115–117°ë.
Isolation of the peptide fraction, preliminary purifi-
cation by the solid phase extraction, and HPLC analysis
were carried out according to the method described in
[11]. For the preparation of tissue extracts, the samples
of organs and blood were weighted, placed in a porce-
lain mortar, freezed in liquid nitrogen, crushed to a
powder, and quantitatively transferred into centrifuge
tubes. The mortar and pestle were washed with a aceto-
nitrile–water mixture adjusted with HCl to pH 2.0
(4 : 1, 10 ml). Solutions (100 µl) of the corresponding
unlabeled peptides (IVa) (11 µg), (VIIa) (41 µg),
(VIIIa) (11 µg), and Pro (15 µg) were added to the
homogenate and extracted with the same mixture (2 ×
15 ml) by centrifugation at 8000 rpm for 40 min. The
supernatants were combined, evaporated, and analyzed
for the labeled peptides. The data of three experiments
averaged for every time point are given in Table 2.
Boc-Tyr(Boc)-Pro-Ser-NH2 (IIIa) (yield 98%, Rf
0.72 (B), mp 127–129°ë), Boc-Tyr(Boc)-DL-3,4-
∆Pro-Ser-NH2 (IIIc) (yield 95%, Rf 0.69 (A), mp 123–
125°ë), Boc-Pro-Ser-NH2 (VIa) from (Va), and Boc-
D-Pro-Ser-NH2 (VIb) from (Vb) were similarly pre-
pared.
HCl · H-Tyr-D-Pro-Ser-NH2 (IVb). A solution of
(IIIb) (2.50 g, 4.4 mmol) in 1 N HCl in acetic acid
(13.5 ml) was kept for 45 min and evaporated in a vac-
uum. The residue was washed with hexane two times
and precipitated from anhydrous methanol with ether.
The precipitate was filtered and dried in a vacuum;
yield of tripeptide (IVb) was 1.8 g (97%); Rf 0.15 (B);
mp 151–155°ë; ESI MS: 365.0 [M + H]+.
ACKNOWLEDGMENTS
HCl · H-Tyr-Pro-Ser-NH2 (IVa) (yield 96%, Rf 0.36
(B), mp 157–159°ë; ESI MS: 365.0 [M + H]+), HCl ·
H-Pro-Ser-NH2 (VIIa) from (VIa), HCl · H-D-Pro-Ser-
NH2 (VIIb) from (VIb), HCl · H-Tyr-Pro-OH (VIIIa)
from (Ia), and HCl · H-Tyr-D-Pro-OH from (Ib) were
similarly prepared.
This study was supported by the Program Molecular
and Cellular Biology of Fundamental Studies of Presid-
ium of the Russian Academy of Sciences, by the Pro-
gram of Leading Scientific Schools (project nos.
NSh-2150.2003.4 and NSh-5638.2006.4), and by the
grants of the State Contract no. 2006-RN-
112.0/001/156 and no. 02.245.11.7317 09.06.2006.
HCl · H-Tyr-[3,4-3H]Pro-Ser-NH2 ([3,4-3H] (IVa))
and HCl · H-Tyr-D-[3,4-3H]Pro-Ser-NH2 ([3,4-
3H](IVb)). A solution of (IIIc) (4 mg) in dioxane (0.5 ml)
was placed in a reaction ampule and 5% PdO/BaSO4
(20 mg) was added. The ampule with the reaction mix-
ture was cooled in liquid nitrogen, evacuated, saturated
with the protium–tritium mixture (with the tritium con-
tent of 60%) to the pressure of 400 gPa, thawed, and
stirred for 3 h at room temperature. The reaction mix-
ture in an ampule was again freezed in liquid nitro-
gen, and the tritium gas was removed in a vacuum.
The catalyst was filtered off and washed with metha-
nol (3 × 1 ml), and easily exchangable tritium was
removed by evaporation with methanol (3 × 1 ml). The
labeled protected tripeptide [3,4-3H](IIIc) (Boc-
Tyr(Boc)-DL-[3,4-3H]-Pro-Ser-NH2) was isolated by
HPLC in yield of 40–45% (molar radioactivity was
35 Ci/mmol). The protecting groups were removed by
the treatment with HCl in dioxane (0.5 ml) for 1 h, sub-
sequent evaporation, and purification by HPLC. The
yields of [3,4-3H](IVa) and [3,4-3H](IVb) were 20 and
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