Tritium labelling and degradation studies of Dmt1-endomorphin 2

Article abstract of DOI:10.1002/jlcr.1402

Two tritiated derivatives of Dmt¹-endomorphin 2 (Dmt ¹-EM2) were prepared by the catalytic tritiodehalogenation of 3′,5′-diiodo-Dmt¹-EM2 and the saturation of ^3,4ΔPro²-Dmt¹-EM2, resulting i

Full text of DOI:10.1002/jlcr.1402

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 1148–1152.
Published online 31 August 2007 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1402
JLCR
Research Article
Tritium labelling and degradation studies of
Dmt1-endomorphin 2
´
´
´
ERZSEBET SZEMENYEI and GEZA TOTH*
Isotope Laboratory, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged,
Hungary
Received 17 April 2007; Revised 14 May 2007; Accepted 14 May 2007
Abstract: Two tritiated derivatives of Dmt1-endomorphin 2 (Dmt1-EM2) were prepared by the catalytic tritiodeha-
logenation of 30,50-diiodo-Dmt1-EM2 and the saturation of 3,4DPro2-Dmt1-EM2, resulting in [30,50-3H2]Dmt1-EM2
and [3H2]Pro2-Dmt1-EM2 with specific activities of 2.88 TBq/mmol (77.8 Ci/mmol) and 1.95 TBq/mmol (52.8 Ci/
mmol), respectively. 30,50-Diiodo-Dmt1-EM2 was synthesized by the chloramine T method from Dmt1-EM2. 3,4DPro2-
Dmt1-EM2 was synthesized by using the Merrifield solid-phase method. The distributions of the tritium in the
labelled peptides were investigated by reversed-phase high-performance liquid chromatography after acidic
hydrolysis. The stability of Dmt1-EM2 in a rat brain membrane homogenate was also determined. Copyright #
2007 John Wiley & Sons, Ltd.
Keywords: opioid peptides; catalytic tritiation; Dmt1-endomorphin 2; degradation
Introduction
phenol ring may lead to an improved ligand–receptor
interaction. The appropriately oriented phenol ring
arising from the dimethylation can probably play a role
in the formation of hydrophobic forces with the
aliphatic or aromatic residues of the receptors.7
The present article relates to the synthesis of two
novel, labelled opioid ligands: [30,50-3H2]Dmt1-EM2
(H-[30,50-3H2]Dmt-Pro-Phe-Phe-NH2) and [3H2]Pro2-
Dmt1-EM2 (H-Dmt-[3H2]Pro-Phe-Phe-NH2). The aim of
the work was to acquire suitable tools for use in in vitro
and in vivo biological assays.
Since the discovery 10 years ago of the endomorphins1
(endomorphin 1, H-Tyr-Pro-Trp-Phe-NH2, EM1, and
endomorphin 2, H-Tyr-Pro-Phe-Phe-NH2, EM2), which
are endogenous neuropeptides with high affinity for the
m-opioid receptor, several new analogues have been
developed, mainly with the aim of increasing the opioid
receptor affinity, the bioactivity and the enzyme
resistance. The substitution of Dmt (20,60-dimethyltyr-
osine) for Tyr in EM2 (Dmt1-EM2) resulted in a 5-fold
increase in the m-opioid receptor affinity.2,3 Simulta-
neously, the d-opioid receptor affinity was increased 2–
Results and Discussion
3-fold, Dmt1-EM2 thereby being
a bivalent EM2
derivative.2,4 Through this substitution, the action
was shifted more spinally than supraspinally.5 The
pharmacological potency of most Dmt analogues can
presumably be attributed to the enzyme resistance at
the N-terminal.6 The increase in hydrophobicity of Dmt
opioid peptides resulting from dimethylation of the
Dmt1-EM2 and Dmt1-3,4DPro2-EM2 were synthesized
by the SPPS method on 4-methylbenzhydrylamine
resin by means of Boc chemistry. 30,50-Diiodo-Dmt1-
EM2 was synthesized by the chloramine T method.8,9
The radioligands were prepared by tritiation of the
precursor peptides containing 30,50-diiodo-Dmt and
3,4-dehydroproline. The crude tritiated products were
purified by reversed-phase high-performance liquid
chromatography (RP-HPLC). The quantitative analyses
of the chemical amounts and radioactivities of the
labelled peptides were performed by RP-HPLC via UV
and radioactivity detection using a calibration curve
produced with unlabelled Dmt1-EM2. Analytical data
*Correspondence to: Ge´za To´th, Isotope Laboratory, Institute of
Biochemistry, Biological Research Centre of the Hungarian Academy of
Sciences, P.O. Box 521, H-6701 Szeged, Hungary. E-mail: geza@brc.hu
Contract/grant sponsor: Hungarian National Bureau of Research and
Technology; contract/grant numbers: NKTH and DNT 08/2004
Contract/grant sponsor: Hungarian Research Foundation; contract/grant
numbers: OTKA 046514 and OTKA TS 09817
Copyright # 2007 John Wiley & Sons, Ltd.

TRITIUM LABELLING AND DEGRADATION STUDIES OF DMT1-ENDOMORPHIN 2 1149
on the unlabelled and radioactive peptides were
biological matrix used. In our earlier investigations,
EM1 and EM2 demonstrated long half-lives in the
presence of a rat brain membrane preparation (0.3 mg/
ml protein): 295 and 230 min, respectively, accordingly
they do not degrade during binding assays.10 In the
present study, the half-life of [3H2]Pro2-Dmt1-EM2
under the above conditions was 515 min.
The kinetics of degradation of Dmt1-EM2 was studied
in a rat brain homogenate, as compared with the earlier
published degradation kinetics of EM2.11 The protein
content of the homogenate was 5.92 mg/ml and Dmt1-
EM2 concentration was 100 mM. Figure 1 shows the
kinetics of degradation of EM2 and Dmt1-EM2 in the
rat brain homogenate. After 12 min of incubation, only
24% of the parent EM2 remained in the samples,
whereas 72% of the initial Dmt1-EM2 concentration
remained after 15 min of incubation. The logarithmic
forms of these curves were analysed by linear regres-
obtained by TLC, HPLC and MS (see Tables 1 and 2).
The distributions of the tritium labels in [3H2]Dmt1-
EM2 and [3H2]Pro2-Dmt1-EM2 were determined after
acidic hydrolysis and Fmoc derivatization by HPLC.
The Dmt and Pro contained tritium in >90% of the
theoretical level. The Phe residues in the peptides were
also partially labelled (Table 3). This phenomenon
presumably caused the overall higher specific activity
of [3H2]Dmt1-EM2 than the theoretical level. The
specific activity of [3H2]Pro2-Dmt1-EM2 was >90% of
theoretical level, and the specificity of the label was
satisfactory, resulting in an appropriate radioligand for
radioligand-binding experiments and metabolic stu-
dies.
The stability of a radioligand in radioligand-binding
studies is essential, and it is therefore necessary to
determine the biological half-life of the ligand in the
Table 1 Analytical data on Dmt-EM2 analogues
Peptides
TLCa
HPLCb
MSc
Rf (A)
Rf (B)
Rf(C)
k0
½M þ Hꢀþ
Mr
Dmt-Pro-Phe-Phe-NH2
DiiodoDmt-Pro-Phe-Phe-NH2
Dmt-DPro-Phe-Phe-NH2
0.36
0.50
0.39
0.53
0.57
0.53
0.33
0.43
0.33
3.04
5.91
3.01
600.37
852.24
598.33
599
851
597
a Rf values on silica gel 60 F254-precoated glass plates (Merck) solvent systems: (A) acetonitrile:methanol:water (4:1:1); (B)
1-butanol:acetic acid:water (4:1:1); (C) ethyl acetate:pyridine:acetic acid:water (60:20:6:11).
b Capacity factor for a Vydac 218TP54 C18 reversed-phase column (25 ꢁ 0.46 cm) with a gradient of 20–40% of organic modifier in
20 min. Flow rate 1 ml/min. All peptides were monitored at 216 nm.
c Measured and calculated molecular weights.
Table 2 Radioanalytical data on Dmt-EMs
Peptides
a (TBq/mmol)
TLCa
HPLCb
Rf (A)
Rf (B)
Rf(C)
k0
[30,50-3H2]Dmt-Pro-Phe-Phe-NH2
Dmt-[3,4-3H2]Pro-Phe-Phe-NH2
2.88
1.95
0.36
0.36
0.53
0.53
0.33
0.33
3.26
3.26
a For conditions, see Table 1.
b For conditions, see Table 1.
Table 3 Tritium distributions in Dmt-EMs
HPLCb
a
Tritiated peptides
a/amax
Fmoc-[3H]Dmt
Fmoc-[3H]Pro
Fmoc-[3H]Phe
[3H]Dmt1-EM2 (%)
Dmt1-[3H]Pro-EM2 (%)
133
92
92
1
}
95
8
4
a a/amax is the ratio of the specific to the theoretically maximum specific activity.
b Analysis on a Vydac 218TP54 C18 column (25 ꢁ 0.46 cm) with a gradient of 30–80% of organic modifier in
20 min. Flow rate 1 ml/min; l¼ 216 nm.
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1148–1152
DOI: 10.1002.jlcr

´
1150 E. SZEMENYEI AND G. TOTH
sion, which allowed calculation of the half-lives of the
EMs. As shown in Table 4, Dmt1-EM2 broke down
relatively slowly in the brain homogenate. Dmt1-EM2
had a half-life of 33.64 min, while EM2 was almost six
times less resistant than Dmt1-EM2 to the peptidases:
its half-life was 5.88 min.
mixed from 0.1% (v/v) trifluoroacetic acid (TFA) in
water and 0.08% (v/v) TFA in acetonitrile. A Bruker
Reflex III MALDI-TOF mass spectrometer was used to
identify the peptides.
Radio-thin layer chromatography (TLC) was per-
formed on silica gel 60 F254-precoated glass plates
(Merck), and the radioactive spots were detected with a
Berthold LD 2821 flow-through (9.9 methane/Ar)
Experimental
Materials
¨
Geiger–Muller counter. Radio-HPLC was performed on
Jasco instrument with Vydac 218TP54 C18
a
a
(25 ꢁ 0.46 cm, 5 mm) column and liquid scintillation
detection on a Canberra Packard Radiomatic 505TR
Flow Radiochromatography Detector with the Ultima-
Flo M scintillation cocktail. Tritiation was carried out in
Protected and unprotected amino acids and 4-methyl-
benzhydrylamine resin were purchased from Calbio-
chem-Novabiochem or Bachem.
The reagents were from Merck or Fluka. All reagents
and solvents were of analytical or reagent grade and
were used without further purification. The mobile
phases for linear gradient elutions in RP-HPLC were
a
self-designed vacuum manifold.12 3H2 gas was
purchased from Technobexport, Russia, and contained
598% tritium. The radioactivities of the tritiated
compounds were measured with a TRI-CARB 2100TR
liquid scintillation counter in a toluene–Triton X-100
cocktail.
120
Methods
100
EM2
80
Solid-phase syntheses of peptides. Peptides were
synthesized manually on 4-methylbenzhydrylamine
resin by using the Merrifield solid-phase method. Na-
t-Boc chemistry with N-hydroxybenzotriazole and N,N0-
dicyclohexylcarbodiimide as coupling agents were
employed for peptide elongation. The peptides were
cleaved from the resin with anhydrous HF (10 ml/g
resin) in the presence of anisole (1 ml/g resin) at 08C for
60 min. The peptide–resin mixtures were washed with
diethyl ether to remove the scavengers, the peptides
were then dissolved in 30% acetic acid, and the filtrate
was lyophilized. The crude peptides were purified by
Dmt1-EM2
60
40
20
2
0
0
50
100
150
200
Time (min)
Figure 1 Degradation of EM2 and Dmt1-EM2 in rat brain
homogenate. EM2 and Dmt1-EM2 were added to the brain
homogenate at a final concentration of 100 mM. HPLC analysis
was performed on supernatants of the centrifuged homoge-
nate, and 20 ml of aliquots was analysed after digestion for 0–
40 and 0–180 min. Each point represents the mean ꢂ SEM of
the EMs concentration, expressed as a percentage of the initial
EM2 and Dmt1-EM2 (n ¼ 4–6). EM2, endomorphin-2; Dmt1-
EM2, Dmt1-endomorphin-2.
RP-HPLC on
a Vydac 218TP1010 C18 (25 ꢁ 1 cm,
12 mm) column, using a linear gradient of 20–50% of
the organic modifier within 25 min at a flow rate of
4 ml/min, with UV detection at 220 nm. Peptide purity
was assessed by TLC and HPLC, and the molecular
weights of the peptides were established by MALDI-
TOF-MS (Table 1).
Syntheses of 30,50-diiodo-Dmt1-EM2. 30,50-Diiodo-Dmt1-
EM2 was synthesized by the chloramine T method.
Testing was performed to establish the optimum
reaction conditions. Accordingly, 1.4 mmol of Dmt1-
EM2 was dissolved in acetonitrile:water (1:1) and 5
equivalents of the reagents (0.067 M NaI þ 0:022 M
chloramine T in phosphate buffer, pH ¼ 7:4) was added
amount to the solution. The reaction was stopped after
30 s by adding 0.053 M Na2S2O5. The reaction mixture
was analysed and purified by RP-HPLC (a Vydac
Table 4 Half-lives of endomorphins
EM2
Dmt1-EM2
100 ꢁ k
(minꢃ1
t1/2
(min)
100 ꢁ k
(minꢃ1
t1/2
(min)
)
)
11.79 ꢂ 0.75
5.88 ꢂ 0.39
2.09 ꢂ 0.39
33.64 ꢂ 6.81
Data are arithmetic means of three to six measure-
ments ꢂ SEM. Protein content in the brain homogenate:
5.92 mg/ml. The EMs concentrations were 100 mM; k, rate
constant; t1/2, half-life.
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1148–1152
DOI: 10.1002.jlcr

TRITIUM LABELLING AND DEGRADATION STUDIES OF DMT1-ENDOMORPHIN 2 1151
218TP54 C18 RP column with a gradient of 20–50% of
microlitre aliquots were withdrawn after incubation for
5, 15, 30 or 60 min, and immediately acidified with
20 ml of 0.1 M HCl solution. Following centrifugation of
the samples (11 340 ꢁ g, 5 min, 258C), 20 ml of super-
natant was analysed by radio-HPLC.
organic modifier in 25 min, flow rate 1 ml/min).
Tritium labelling. An amount of 2.4 mg (2.88 mmol) of
30,50-diiodo-Dmt1-EM2 and 2.8 mg (3.92 mmol) of
3,4DPro2-Dmt1-EM2 were dissolved separately in 1 ml
of dimethylformamide and labelled with tritium gas.
The reaction mixture contained 1.5 ml of triethylamine
and 12 or 14 mg of PdO/BaSO4 catalyst, respectively.
Tritium gas was liberated from uranium tritide by
heating, and 555 GBq (15 Ci) of this gas was introduced
into the reaction vessel.10,12 The reaction mixture was
stirred at room temperature for 1 or 2 h and the
unreacted tritium gas was then adsorbed onto pyr-
ophoric uranium. The catalyst was filtered off on a
Whatman GF/C filter and the labile tritium was
removed by repeated vacuum evaporation of an aqu-
eous ethanolic solution of the radiolabelled product.
The crude products were purified by HPLC to give a
radioactive purity of >95%, checked by both TLC and
HPLC. The quantitative analysis of the pure, labelled
peptides was performed by HPLC with a UV detector,
using a calibration curve prepared with unlabelled
Dmt1-EM2, and the total activities of the products were
measured by liquid scintillation counting. The specific
activity of [3H]-Dmt1-EM2 was 2.88 TBq/mmol
(77.8 Ci/mmol), and that of [3H2]Pro2-Dmt1-EM2 was
1.95 TBq/mmol (52.8 Ci/mmol) (Table 2). The pure,
tritiated peptides were dissolved in ethanol and were
stored at a concentration of 37 MBq/ml under liquid
nitrogen. The stabilities of both tritiated endomorphin
analogues under these storage conditions were really
good. After 6 months, the purities were checked and
proved to be >95%.
Determination of the rates of degradation of the
peptides. About 20 ml aliquots of the 1 mM peptide
stock solutions in 50 mM Tris–HCl buffer (pH ¼ 7:4)
were added to 180 ml of rat brain homogenate, and the
mixtures were incubated at 378C. Aliquots of 20 ml were
taken from these incubation mixtures and immediately
acidified with 25 ml of 0.1 M aqueous HCl solution.
About 10 ml of each supernatant obtained after cen-
trifugation (11 340 ꢁ g, 5 min, 258C) of the samples was
analysed by HPLC. The degradation rate constants (k)
were obtained by least-square linear regression analy-
sis of the plots of logarithmic EM peak areas (ln(A/A0))
versus time, using a minimum of four points. Degrada-
tion half-lives (t1/2) were calculated from the rate
constants as ln(2/k).
Conclusions
Two Dmt1-EM2 isotopomers were labelled with tritium
in position 1 or position 2. Both radioligands exhibited
high specific radioactivity. Tritiated Dmt1-EM2 may
become a useful research tool for direct radioreceptor
binding13 (to be published elsewhere) and the isotopo-
mers may serve as important tools for degradation
studies in rat brain homogenates. Different rates of
degradation of EM2 and of Dmt1-EM2 were observed in
the rat brain homogenate. Dmt1-EM2 was six times
more resistant than EM2 to peptidases, the half-lives
being 33.64 and 5.88 min, respectively. The half-life of
[3H2]Pro2-Dmt1-EM2 proved almost 2.5 times longer
than that of EM2.
Tritium distributions in labelled peptides. An amount of
0.74 MBq of [3H2]Dmt1-EM2 or [3H2]Pro2-Dmt1-EM2
and 0.05 mg of unlabelled Dmt1-EM2 were hydrolysed
separately for 24 h in 1 ml of 6 M HCl at 1108C under
argon pressure in sealed ampoule. The solvent was
then removed by evaporation, and the sample was next
dissolved in 1 ml of 0.2 M borate buffer, pH ¼ 7:7.
The remaining radioactivity was 0.39 MBq for [3H2]
Pro2Dmt1-EM2 or 0.1 MBq for [3H2]Dmt1-EM2. To
0.2 ml of each aqueous sample, 0.2 cm3 of 9-fluorenyl-
methyl chloroformate in acetone (15 mM) was added.
After about 45 s, the mixtures were extracted with n-
pentane and the aqueous phase was analysed by HPLC
(Table 3).
Acknowledgements
This work was supported by grant NKTH, DNT 08/
2004 from the Hungarian National Bureau of Research
and Technology, and grants OTKA 046514 and OTKA
TS 09817 from the Hungarian Research Foundation.
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