Synthesis of Pro-Pro-Ile, a corticoliberin fragment, labeled with tritium in proline unit

Article abstract of DOI:10.1134/S1066362210030185

Boc-δ³Pro-Pro-Ile-OBzl and Boc-Pro-δ³Pro- Ile-OBzl were synthesized. Tritium-labeled Pro-Pro-Ile was prepared by their hydrogenation in a tritium atmosphere in the presence of a catalyst. In the reduction of Boc-δ³Pro-Pro-Ile-OBzl and Boc-Pro- δ³Pro-Ile-OBzl, elimination of the Bzl protective group occurs faster than hydrogenation of the 3,4-dehydroproline residue (benzene and dioxane were used as solvents). The resulting Boc-δ³Pro-Pro-Ile-OH and Boc-Pro-δ³Pro-Ile-OH are poorly soluble in aprotic solvents, which accounts for the low yield of the desired tritium-labeled peptides. Pleiades Publishing, Inc., 2010.

Full text of DOI:10.1134/S1066362210030185

ISSN 1066-3622, Radiochemistry, 2010, Vol. 52, No. 3, pp. 325–329. © Pleiades Publishing, Inc., 2010.
Original Russian Text © V.P. Shevchenko, I.Yu. Nagaev, L.A. Andreeva, N.F. Myasoedov, 2010, published in Radiokhimiya, 2010, Vol. 52, No. 3, pp. 277–
280.
Synthesis of Pro–Pro–Ile, a Corticoliberin Fragment,
Labeled with Tritium in Proline Unit
V. P. Shevchenko, I. Yu. Nagaev*, L. A. Andreeva, and N. F. Myasoedov
Institute of Molecular Genetics, Russian Academy of Sciences,
pl. Kurchatova 2, Moscow, 123182 Russia; * e-mail: nagaev@img.ras.ru
Received July 22, 2009
Abstract—Boc–Δ3Pro–Pro–Ile–ОBzl and Boc–Pro–Δ3Pro–Ile–ОBzl were synthesized. Tritium-labeled Pro–
Pro–Ile was prepared by their hydrogenation in a tritium atmosphere in the presence of a catalyst. In the reduc-
tion of Boc–Δ3Pro–Pro–Ile–ОBzl and Boc–Pro–Δ3Pro–Ile–ОBzl, elimination of the Bzl protective group oc-
curs faster than hydrogenation of the 3,4-dehydroproline residue (benzene and dioxane were used as solvents).
The resulting Boc–Δ3Pro–Pro–Ile–ОН and Boc–Pro–Δ3Pro–Ile–ОН are poorly soluble in aprotic solvents,
which accounts for the low yield of the desired tritium-labeled peptides.
Key words: peptides, synthesis, tritium labeling
DOI: 10.1134/S1066362210030185
Pro–Pro–Ile exerts an activating effect on heart
functioning and behavior of animals. Similar activating
effect on the heart activity is known for corticoliberin.
Similarity of the vegetative and bevaior effects exerted
by cotricoliberin and its Pro–Pro–Ile fragment shows
that they interact with the same receptors [1]. For a
more detailed study of the physiological effect of Pro–
Pro–Ile using antagonists of cotricoliberin receptors, it
is necessary to use tritium-labeled Pro–Pro–Ile.
The radioactivity was measured with an LKB1215
scintillation counter (tritium recording efficiency about
30%) in a dioxane scintillator. The chromatographic
data were collected and processed with MultiChrom
1.5 system (Ampersand Private Company, Russia).
Synthesis of Pro–Pro–Ile, Δ3Pro–Pro–Ile,
and Pro–Δ3Pro–Ile Derivatives
The precursors were prepared using pivaloyl chlo-
ride as condensing agent. The Bzl protective groups
were removed in the presence of a catalyst in a hydro-
gen atmosphere, and Boc groups, by acid hydrolysis.
The peptides and their derivatives were isolated by
HPLC.
The goal of this study was the synthesis of Boc–
Δ3Pro–Pro–Ile–ОBzl and Boc–Pro–Δ3Pro–Ile–ОBzl,
whose catalytic hydrogenation in a gaseous tritium
atmosphere allows preparation of [3H]Pro–Pro–Ile and
Pro–[3H]Pro–Ile, respectively.
EXPERIMENTAL
Boc–Pro–Ile–OBzl. 0.58 g of Boc–Pro–ОН was
dissolved in 10 ml of acetonitrile. The solution was
cooled to –5°С, 460 μl of triethylamine (TEA) was
added with stirring, and the solution was cooled to
–20°С, after which 370 μl of pivaloyl chloride (PivCl)
was added with stirring over a period of 20 min at
–10°С. To the resulting mixture, cooled to –30°С, we
added a precooled solution of Ile–OBzl prepared by
dissolution of 0.55 g of HCl·Ile–OBzl in 10 ml of ace-
tonitrile, followed by addition of 420 μl of TEA and
cooling to –10°С over a period of 20 min. The mixture
was stirred for 1 h at –10°С and for 2 h at 18–20°С.
Then the mixture was evaporated, and 30 ml of ethyl
The catalysts, reagents, and solvents were commer-
cial chemicals. The initial and final products were
characterized by high-performance liquid chromatogra-
phy (HPLC) and mass spectrometry, and the yield and
purity of the products were determined by HPLC.
Mass-spectrometric data were obtained with an
LCQ Advantage MAX device (Thermo Electron,
USA) with electrospray ionization, direct inlet of a
10 μg ml–1 sample solution in 0.1% acetic acid, and
fragmentation of the molecular ion in the analyzer by
the method of ion collisions at 35 eV.
325

326
SHEVCHENKO et al.
acetate was added to the residue. The ethyl acetate so-
lution was successively washed with Н2О (3 × 3 ml),
10% KHSO4 solution (3 × 3 ml), Н2О (3 × 3 ml), 5%
NaHCO3 solution (3 × 3 ml), Н2О (3 × 3 ml), and satu-
rated NaCl solution (3 ml). The washed ethyl acetate
solution was dried over MgSO4 and filtered. The sol-
vent was removed on a rotary evaporator. To the resi-
due, ~10 ml of dry ether was added, and the product
was precipitated with hexane. In so doing, the precipi-
tate is formed as an oil. The hexane was decanted off,
and the oil was dried in a vacuum desiccator over
KOH, Р2О5, and paraffin. Yield of the peptide 60%;
thin-layer chromatography (ТСХ): acetone–benzene–
acetic acid (50 : 100 : 1) (a), Rf 0.6; benzene–ethanol
(8 : 2) (b), Rf 0.5; chloroform–methanol (9 : 1) (c),
Rf 0.8; hexane–acetone (3 : 2), Rf 0.5.
acetate solution was washed with Н2О (3 × 1 ml), 10%
KHSO4 solution (3 × 1 ml), Н2О (3 × 1 ml), 5%
NaHCO3 solution (3 × 1 ml), Н2О (3 × 1 ml), and satu-
rated NaCl solution (1 ml). The ethyl acetate solution
was dried over MgSO4. Then the solution was filtered
and evaporated. The residue was dissolved in 1 ml of
absolute ethanol, and the product was precipitated with
hexane. The hexane was decanted off, and the product
was dried in a vacuum desiccator over KОН, Р2О5, and
paraffin. Yield of the peptide 74%, Rf 0.56 (a),
0.66 (b), 0.76 (c).
Boc–Pro–Δ3Pro–Ile–OBzl. The synthesis was per-
formed as described above, starting from 14 mg of
Boc–Pro–ОН and 25 mg of TFA·Δ3Pro–Ile–OВzl (the
amounts of all the other chemicals were proportionally
decreased). Yield 65–70%.
Boc–Δ3Pro–Ile–OBzl. The synthesis was per-
formed as described above, starting from 60 mg of
Boc–Δ3Pro–ОН (the amounts of all the other chemi-
cals were proportionally decreased). Yield 45–50%.
Boc–Δ3Pro–Pro–Ile–OBzl. The synthesis was per-
formed as described above, starting from 14 mg of
Boc–Δ3Pro–ОН and 25 mg of TFA·Pro–Ile–OВzl (the
amounts of all the other chemicals were proportionally
decreased). Yield 60–65%.
TFA·Pro–Ile–OBzl. To 0.315 g of Boc–Pro–Ile–
OBzl, we added 2 ml of methylene chloride and 2 ml
of trifluoroacetic acid (TFA) and kept the mixture for
45 min at room temperature. After removing the tert-
butyloxycarbonyl protective group, the peptide solu-
tion was evaporated with absolute methanol, benzene,
and ether (two times with each solvent), the residue
was dissolved in 3 ml of benzene, and the product was
precipitated with hexane. The hexane was decanted
off, and the resulting oil was dried in a desiccator over
P2O5, KOH, and paraffin. Yield of the peptide 93%,
Rf 0.02 (a), 0.12 (b), 0.18 (c).
TFA·Δ3Pro–Ile–OBzl. The synthesis was per-
formed as described above, starting from 45 mg of
Boc–Δ3Pro–Ile–OBzl (the amounts of all the other
chemicals were proportionally decreased). Yield 90–
95%.
Boc–Pro–Pro–Ile–OН. Boc–Pro–Pro–Ile–OBzl
(10 mg) was dissolved in 0.5 ml of methanol, 5 μl of
1 M HCl and 10 mg of a catalyst (10% PdO on neutral
Al2O3) was added, and the substrate was hydrogenated
in a dry hydrogen flow at room temperature and a pres-
sure of 1 atm for 6 h. Then the catalyst was filtered off
and washed on the filter with methanol. The combined
filtrate was evaporated to dryness. The residue was
precipitated from absolute methanol with ether and
vacuum-dried, with replacing several times the desic-
cants. Yield of Boc–Pro–Pro–Ile–OН 89%. Analysis
on a Kromasil 100 C18 column, 4 × 150 mm, flow rate
1 ml min–1, methanol–water system (7 : 3), retention
time of the peptide 3.42 min.
Under similar conditions, from 1 mg of Boc–Pro–
Δ3Pro–Ile–OBzl or Boc–Δ3Pro–Pro–Ile–OBzl, we pre-
pared Boc–Pro–Pro–Ile–OН. Because the compounds
prepared from these precursors are identical, their mass
spectra were also identical.
Boc–Pro–Pro–Ile–OBzl. 0.06 g of Boc–Pro–ОН
was dissolved in 2 ml of acetonitrile. The solution was
cooled to –5°С, 44 μl of TEA was added with stirring,
the solution was cooled to –20°С, and 40 μl of PivCl
was added with stirring over a period of 20 min at
–10°С, after which the solution was cooled to –30°С.
Then a solution of 0.11 g of TFA·Pro–Ile–OВzl in
1 ml of a mixture of acetonitrile with TEA (25 : 2),
cooled to –10°C, was added over a period of 20 min.
The mixture was stirred for 1 h at –10°С and for 2 h at
18–20°С. Then the mixture was evaporated, and 6 ml
of ethyl acetate was added to the residue. The ethyl
TFA·Pro–Pro–Ile–OН. To 0.1 g of Boc–Pro–Pro–
Gly–OBzl we added 0.5 ml of methylene chloride and
0.5 ml of TFA. The mixture was kept for 45 min at
room temperature. After the deprotection, the solution
was evaporated with absolute methanol, benzene, and
ether (two times with each solvent). The residue was
dissolved in 0.5 ml of benzene and precipitated with
hexane. The hexane was decanted off, and the resulting
oil was dried in a vacuum desiccator over P2O5, KOH,
RADIOCHEMISTRY Vol. 52 No. 3 2010

SYNTHESIS OF Pro–Pro–Ile
327
and paraffin. Yield of the peptide 97%. HPLC analysis
was performed on a Reprosil C18aq column, 4 ×
150 mm, flow rate 1 ml min–1, system methanol–50
mM ammonium phosphate buffer (pH 2.8) (3 : 7), re-
tention time of Pro–Δ3Pro–Ile–OН 7.05 min.
Analysis of the Synthesized Peptides
The precursors and references required for the syn-
thesis of labeled peptides were characterized addition-
ally.
Fig. 1. Analysis of Boc–[3H]Pro–Pro–Ile or Boc–Pro–[3H]
Preparative purification of Boc–Pro–Δ3Pro–Ile–
OBzl and Boc–Δ3Pro–Pro–Ile–OBzl was performed by
HPLC on a Reprosil pur C18aq column, 20 × 150 mm,
flow rate 15 ml min–1, system methanol–water (4 : 1),
pH 3 (HCl). HPLC analysis was performed on a
Reprosil pur C18aq column, 4 × 150 mm, flow rate
1 ml min–1, system methanol–water (3 : 1); retention
times, min: Boc–Pro–Δ3Pro–Ile–OBzl 6.07, Boc–
Δ3Pro–Pro–Ile–OBzl 6.56.
Pro–Ile on a Kromasil 100 C18 column, 4 × 150 mm, flow
rate 1 ml min–1, system methanol–water (7 : 3). (6) Target
component; the other figures refer to impurities.
nol (3 × 1 ml). Boc–3HPro–Pro–Ile or Boc–Pro–
3HPro–Ile was purified by HPLC (Fig. 1).
Then the solvent was evaporated and the Boc pro-
tective group was removed as described above by
keeping the labeled compounds in a mixture of methyl-
ene chloride and TFA for 45 min at room temperature.
The yields and molar radioactivities of the labeled
products are given in the table.
The compounds used as references were character-
ized by mass spectrometry. The mass spectra of Boc–
Pro–Δ3Pro–Ile–OBzl and Boc–Δ3Pro–Pro–Ile–OBzl
contained a molecular peak at m/z 514.07 and 514.09,
and also fragmentation peaks with m/z 458 and 414,
i.e., the molecular ion lost the СН2=С(СН3)2 fragment
or Вос protective group.
RESULTS AND DISCUSSION
In this study, as precursors for preparing [3H]Pro–
Pro–Ile and Pro–[3H]Pro–Ile we used Boc–Δ3Pro–Pro–
Ile–ОBzl and Boc–Pro–Δ3Pro–Ile–ОBzl, respectively
(Scheme 1, Fig. 2).
The mass spectrum of Boc–Pro–Pro–Ile–OН con-
tained a molecular peak at m/z 426.00 and fragmenta-
tion peaks at m/z 370 and 326, i.e., the molecular ion
lost the СН2=С(СН3)2 fragment or Вос protective
group.
Yields and molar radioactivities of labeled peptides
Yield, Molar radioactiv-
Compound
Precursor
%
ity, Ci mmol–1
35.2
Boc–Δ3Pro–Pro–
[3H]Pro–Pro–Ile
24
The mass spectrum of Pro–Pro–Ile–OН contained a
molecular peak at m/z 326.07 and fragmentation peaks
at m/z 229 and 195, i.e., the molecular ion lost Pro
or Ile.
Ile–Bzl
Boc–Pro–Δ3Pro–
Pro–[3H]Pro–Ile
5
40.1
Ile–Bzl
Scheme 1. Synthesis of [3H]Pro–Pro–Ile and Pro–[3H]Pro–
Synthesis of Tritium-Labeled.Pro–Pro–Ile–OН
Ile and their precursors
Boc–ProОН + Ile–ОBzl → Boc–Pro–Ile–ОBzl
→ Pro–Ile–ОBzl,
A reaction ampule was charged with a solution of 4
mg of Boc–Δ3Pro–Pro–Ile–OBzl (or Boc–Pro–Δ3Pro–
Ile–OBzl) in 0.5 ml of dioxane and with 20 mg of 5%
Pd/BaSO4. The ampule contents were frozen with liq-
uid nitrogen and evacuated, after which tritium was
admitted to a pressure of 400 hPa, and the contents
were defrosted and stirred for 3 h at room temperature.
Then the contents were again frozen with liquid nitro-
gen, evacuated, and defrosted. The catalyst was filtered
off and washed with methanol (3 × 1 ml), after which
labile tritium was removed by evaporation with metha-
Boc–ProОН + Pro–Ile–ОBzl → Boc–Pro–Pro–Ile–ОBzl
→ Boc–Pro–Pro–IleОН → Pro–Pro–IleОН,
Boc–Δ3ProОН + Ile–ОBzl → Boc–Δ3Pro–Ile–ОBzl
→ Δ3Pro–Ile–ОBzl → Boc–ProОН + Δ3Pro–Ile–ОBzl
→Boc–Pro–Δ3Pro–Ile–ОBzl → Boc–Pro–[3H]Pro–IleОН →
→Pro–[3H]Pro–Ile,
Boc–Δ3ProОН + Pro–Ile–ОBzl → Boc–Δ3Pro–Pro–Ile–ОBzl
→ Boc– [3H]Pro–Pro–IleОН → [3H]Pro–Pro–Ile.
RADIOCHEMISTRY Vol. 52 No. 3 2010

328
SHEVCHENKO et al.
(a)
(b)
Fig. 2. Structural formulas and molecular models of (a) Boc–Δ3Pro–Pro–Ile–Bzl and (b) Boc–Pro–Δ3Pro–Ile–Bzl.
beled product did not exceed 3 and 15%, respectively.
HPLC analysis of the composition of the reaction mix-
tures showed that, along with Boc–[3H]Pro–Pro–
IleОН, they contained Boc–Δ3Pro–Pro–IleОН (77%)
and the initial Boc–Δ3Pro–Pro–Ile–ОBzl (8%). This
fact indicates that the double bond in unsaturated
proline incorporated in these peptides is poorly ad-
sorbed on active centers of the catalyst because of
steric factors. Therefore, the rate of elimination of the
benzyl protective group appears to be higher than the
hydrogenation rate. As a result, the major products are
Boc–Δ3Pro–Pro–IleОН and Boc–Pro–Δ3Pro–IleОН,
which are poorly soluble in benzene and do not further
participate in the hydrogenation. The yield of both la-
beled products was approximately two times higher in
dioxane (see table) in which these peprides are more
soluble.
To prepare labeled products with a high molar ra-
dioactivity, hydrogenation of unsaturated peptides
should be performed in aprotic solvents using their
protected precursors. Because it is known [2] that the
reaction in protic solvents is accompanied by notice-
3
3
3
able isotope dilution (Н+ + Н2 ҙ Н+ + НН), hydro-
genation of the unsaturated peptides in methanol al-
lows preparation of labeled products with the molar
radioactivity no higher than 10–15 Ci mmol–1.
Reduction of Boc–Pro–Δ3Pro–Ile–ОBzl and Boc–
Δ3Pro–Pro–Ile–ОBzl in aprotic solvents is difficult.
With benzene used as solvent in tritium labeling of
these peptides, strong dependence of the labeled prod-
uct yield on the kind of the precursor was observed. In
hydrogenation of Boc–Pro–Δ3Pro–Ile–ОBzl and Boc–
Δ3Pro–Pro–Ile–ОBzl in benzene, the yield of the la-
RADIOCHEMISTRY Vol. 52 No. 3 2010

SYNTHESIS OF Pro–Pro–Ile
329
Thus, proper choice of the reaction conditions al-
lowed us to synthesize [3H]Pro–Pro–IleОН and Pro–
[3H]Pro–IleОН with a high molar radioactivity (35–
40 Ci mmol–1).
Scientific Schools grant no. NSh-3626.2008.4 under
the guidance of Acad. N.F. Myasoedov.
REFERENCES
1. Makarenko, E.Yu., Andreeva, L.A., Borovik, A.S., and
Mart’yanov, A.A., Ross. Fiziol. Zh. im. I.M. Sechenova,
2003, vol. 89, no. 8, pp. 943–950.
2. Shevchenko, V.P., Nagaev, I.Yu., and Myasoedov, N.F.,
Mechennye tritiem lipofil’nye soedineniya (Tritium-
Labeled Lipophilic Compounds), Moscow: Nauka,
2003.
ACKNOWLEDGMENTS
The study was financially supported in part by the
Program for Basic Research of the Presidium of the
Russian Academy of Sciences “Molecular and Cellular
Biology,” Russian Foundation for Basic Research, and
RADIOCHEMISTRY Vol. 52 No. 3 2010