A Novel and Efficient Method for the Technetium-99m Labelling of Disulfide Compounds Using a Tetrahydroborate Exchange Resin

Article abstract of DOI:10.1246/bcsj.76.1977

A novel and efficient method for the technetium-99m labelling of disulfide compound 7 was established with high radiochemical purity in which tetrahydroborate exchange resin (BER) simultaneously carries out the reduction of 7, the reduction of [^99mTc]pertechnetate and chelation of 7 with technetium-99m. The labelling occurs in a one-pot three-step procedure that is amenable to the preparation of ^99mTc-radiopharmaceuticals.

Full text of DOI:10.1246/bcsj.76.1977

ꢀ 2003 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 76, 1977–1981 (2003) 1977
A Novel and Efficient Method for the Technetium-99m Labelling
of Disulfide Compounds Using a Tetrahydroborate Exchange Resin
Ã
Sang Hyun Park, Hui Jeong Gwon, Young Ho Kim,y and Kyung Bae Park
Division of Radioisotope Production and Application, HANARO center, Korea Atomic Energy Research Institute,
150 Deokjin-dong, Yuseong-gu, Daejeon 305-353, Korea
yDepartment of Fine Chemical Engineering and Chemistry, Chungnam National University,
220 Gung-dong, Daejeon 305-764, Korea
Received March 12, 2003; E-mail: parksh@kaeri.re.kr
A novel and efficient method for the technetium-99m labelling of disulfide compound 7 was established with high
radiochemical purity in which tetrahydroborate exchange resin (BER) simultaneously carries out the reduction of 7, the
reduction of [99mTc]pertechnetate and chelation of 7 with technetium-99m. The labelling occurs in a one-pot three-step
procedure that is amenable to the preparation of 99mTc-radiopharmaceuticals.
Technetium-99m (99mTc) is the most widely used radionu-
clide for diagnostic radiophamarceuticals due to its great ad-
vantages in clinical use such as low cost, ready availability,
short half-life (6.01 h), and a gamma energy (140 keV) appro-
priate for obtaining a gamma picture.1,2 99mTc generally forms
a complex with compounds having nonbonding electrons such
as isocyanates, amines, carboxyls, and thiols, and this complex
is then used as a radiopharmaceutical agent for imaging vari-
ous organs including the lung, liver, and brain, etc. Diamine
dithiol has been synthesized and served as a bifunctional che-
lating agent for the preparation of 99mTc-radiopharmaceuticals.
However, the method for the preparation of the 99mTc-labelled
thiol compound is very limited since the thiol compound is
easily oxidized into a disulfide form during the labelling reac-
tion with 99mTc.3 To avoid this, the 99mTc labelling of the thiol
compound has been carried out simultaneously by chelating
99mTc and removing the protecting group of the S-protected
precursor synthesized in advance. For example, the protection
of the 2-aminoethanethiol derivative (1) was achieved by re-
acting it with p-methoxybenzyl chloride for 5h at room tem-
perature in the presence of a basic catalyst, and the deprotec-
tion of the S-protected precursor (2) was achieved by reacting
it with mercury(b) acetate at 0 ꢀC for 15min and H 2S gas for 5
min before labelling 99mTc in order to form a 99mTc-labelled
thiol compound (3) (Scheme 1).4–6 The previous method has
the disadvantage of protection of thiol group and deprotection
of the S-protected precursor requiring laborious synthetic steps
prior to reduction of [99mTc]pertechnetate. Jeong and co-
workers have recently reported the prepartion of 99mTc-label-
led thiol compounds via reduction of disulfide compounds.7,8
The method also has the disadvantage of reduction of disulfide
group and separation of the thiol precursor requiring laborious
synthetic steps prior to labelling of 99mTc. Therefore, there has
been a continuous need to develop a one-pot conversion that
simultaneously carries out the reduction of disulfide com-
pounds and the reduction of [99mTc]pertechnetate under mild
conditions.
In this paper we report a novel, simple, and efficient method
for the 99mTc labelling of disulfide compounds using tetrahy-
droborate exchange resin (BER), which can be employed to
prepare various types of 99mTc-radiopharmaceuticals.
Results and Discussion
Preparation and Labelling Efficiency of 99mTc-
Glucoheptonate.9
99mTc-glucoheptonate (99mTc-GHA, 4)
was prepared by the mixing of lyophilized glucoheptonate
and tin(b) chloride with sodium [99mTc]pertechnetate in saline
at room temperature for 20 seconds in order to perform the
transchelation (Scheme 2). The assay for formation and struc-
ture of Na99mTcO4, 99mTcO2, and 99mTc-GHA, radiolabelled
compounds with [99mTcð=O]þ3 species, was achieved by in-
vestigating their position using an instant thin-layer chroma-
tography (ITLC). In this experiment, 98% of labelling effi-
ciency of 4 was determined by performing ITLC. The label-
ling efficiency (LE) was calculated as follows, LE = 100 À
À
%
99mTcO4 À %99mTcO2. Table 1 shows the results of
thin-layer chromatography for 4 by performing ITLC on silica
gel impregnated glass fiber sheets using acetone and saline as
developing solvents.
Preparation, Radiochemical Purity, and Labelling Effi-
ciency of 99mTc-DADS by Transchelation. The strategy fol-
lowed for the synthesis of 3,3,10,10-tetramethyl-1,2-dithia-
5,8-diazacyclodecane (diamine disulfide, DADS, 7) is shown
1
in Scheme 3. The IR and H NMR spectral data of 7 support
the structure of 7 as given in Scheme 3. 99mTc-GHA (4) was
reacted with 7 in the presence of BER for transchelation in
which the [99mTcð=O]þ3 species in 4 was transferred to 7 re-
sulting in the formation of 99mTc-DADS (3) (Scheme 4).
Radiochemical purity (RCP) and labelling efficiency of 3 were
determined by using ITLC and reverse-phase HPLC. ITLC
was performed using acetone and a mixture of methanol and
HCl (v/v ratio, 99.5:0.5) as developing solvents, and the results
are given in Table 2. In addition, reversed-phase HPLC was
carried out using a C-18 reverse-phase column as a stationary
Published on the web October 15, 2003; DOI 10.1246/bcsj.76.1977

1978 Bull. Chem. Soc. Jpn., 76, No. 10 (2003)
99mTc Labelling of Disulfide Using BER
Scheme 1.
Scheme 2.
Table 1. ITLC Analysis of 99mTc-GHA
Chromatographic system
99mTc species at
Support
Solvent
Orgin
100% of 99mTc-GHA and 99mTcO2
2% of 99mTcO2
Solvent front
0% of 99mTcO4
À
ITLC-SG
ITLC-SG
Acetone
Saline
À
98% of 99mTc-GHA and 99mTcO4
Scheme 3. Reagents and conditions: i. S2Cl2, CCl4, 50–55 ꢀC, 3 h; ii. H2NCH2CH2NH2, PTSA, toluene, 2 h, reflux; iii.
ꢀ
Na[BH3CN], CH3OH, 55–60 C, 15min.

S. H. Park et al.
Table 2. ITLC Analysis of 99mTc-DADS by Transchelation
Chromatographic system
Bull. Chem. Soc. Jpn., 76, No. 10 (2003) 1979
99mTc species at
Support
Solvent
Orgin
100% of 99mTc-GHA and 99mTcO2
4% of 99mTcO2
Solvent front
0% of 99mTcO4
À
ITLC-SG
ITLC-SG
Acetone
Methanol and HCl
(99.5:0.5, v/v)
À
96% of 99mTc-GHA and 99mTcO4
determined by using ITLC and reverse-phase HPLC. ITLC
was performed using acetone and a mixture of methanol and
HCl (99.5:0.5, v/v) as developing solvents, with the results giv-
en in Table 3. In addition, reversed-phase HPLC was carried
out using a C-18 reverse-phase column as the stationary phase,
and a mixture of tetraethylammonium phosphate buffer solu-
tion and methanol as the mobile phase, while maintaining a
flow rate of 1 mL/min. The results are as same as those of
Fig. 1. Only one peak was observed with a retention time of
19.4 min, which is the retention time of the compound of inter-
est, demonstrating the formation of 3 with a labelling efficien-
cy of over 99%. In the case of the conventional method using
tin(b) chloride as a reducing agent, the formation of 3 was not
observed (Scheme 5).
The key step in this new procedure involves the introduction
of BER followed by the simultaneous reduction of the disulfide
bond and pertechnetate. In this procedure, BER provides not
only the reduction of 99mTc, but also the cleavage of the disul-
fide bond, providing mercapto groups for chelating with 99mTc
resulting in high labelling efficiency and radiochemical purity.
Generally, the reaction was completed within 30 min depend-
ing on the ligand and conditions used. Based on the preferred
embodiment of the present study, we have recognized that the
99mTc-DADS (3) has more than 99% of RCP and 98% of label-
ling efficiency.
Scheme 4.
phase and a mixture of tetraethylammonium phosphate buffer
solution and methanol as a mobile phase, while maintaining a
flow rate of 1 mL/min. The results are given in Fig. 1. As
shown in Fig. 1, only one peak was observed with a retention
time of 19.4 min, which is the retention time of the compound
of interest, demonstrating the formation of 3 having over 99%
of RCP. The RCP was determined from relative peak areas of
HPLC chromatograms with monitoring at 254 nm.
Preparation, Radiochemical Purity, and Labelling Effi-
ciency of 99mTc-DADS by Tetrahydroborate Exchange
Resin. Tetrahydroborate exchange resin (BER) is comprised
of a tetrahydroborate ion (BH4À) bound to a quaternary ammo-
nium cation supported on a polymer. BER played an essential
role in reducing the disulfide compound to a thiol compound,
as well as the [99mTcg]pertechnetate to the [99mTce=O]þ3 spe-
cies with low oxidation state simultaneously affording a 99mTc-
labelled compound directly from the disulfide compound. The
tetrahydroborate exchange resin was used enough to reduce
both the disulfide compound and pertechnetate.
Conclusions
We have established a novel and efficient method in which
BER simultaneously carries out the reduction of disulfide com-
pounds and the reduction of pertechnetate under mild condi-
tions, so that the labelling of technetium with sulfide com-
DADS (7) was reacted with Na99mTcO4 in the presence of
BER resulting in the formation of 99mTc-DADS (3) (Scheme
5). 99% of RCP and 97% of labelling efficiency of 3 were also
Fig. 1. Elution profile of 99mTc-DADS (3) by transchelation at 30 min post labelling from a C18 RP-HPLC column.

1980 Bull. Chem. Soc. Jpn., 76, No. 10 (2003)
99mTc Labelling of Disulfide Using BER
Table 3. ITLC Analysis of 99mTc-DADS by BER
Chromatographic system
99mTc species at
Support
Solvent
Orgin
100% of 99mTc-GHA and 99mTcO2
3% of 99mTcO2
Solvent front
0% of 99mTcO4
À
ITLC-SG
ITLC-SG
Acetone
Methanol and HCl
(99.5:0.5, v/v)
97% of 99mTc-GHA and 99mTcO4
À
gamma-ray detector.
Preparation of Tetrahydroborate Exchange Resin (BER).
The tetrahydroborate Exchange Resin (BER) was prepared by
the reported method with a minor modification.11 Chloride-form
resin (Amberlite1 ion exchange resin, 12.5g) was slurry-packed
with water into a 30-mL fritted glass funnel mounted on a filter
flask. Then, aqueous sodium tetrahydroborate solution (200
mL, 0.25M) was slowly passed through the resin over a period
of 30 minutes. The resulting resins were washed thoroughly with
distilled water until free of excess, and finally with ethanol (10 mL
 3). The tetrahydroborate form anion exchange resin (BER) was
then partially air-dried by removing the ethanol on the surface of
BER. This resin was analyzed for tetrahydroborate content by hy-
drogen evolution upon acidification with 0.08 M HCl, and the
average capacity of BER was found to be 2.5meq of tetrahydro-
borate ion per gram. BER was stored under nitrogen at 4 ꢀC. The
hydride content was constant over 5weeks.
Preparation of 3,3,10,10-Tetramethyl-1,2-dithia-5,8-diaza-
cyclodecane (7, diamine disulfide; DADS). 3,3,10,10-Tetra-
methyl-1,2-dithia-5,8-diazacyclodecane (7) was prepared by the
reported method with minor modifications.12
Synthesis of 2,20-dimethyl-2,20-dithiadipropanal (5): A so-
lution of isobutyraldehyde (353 g, 4.89 mol) in carbon tetrachlo-
ride (350 mL) was heated to 50 ꢀC and disulfur dichloride was
added dropwise under nitrogen atmosphere in order to keep the
evolution of hydrogen chloride under control. Upon completing
the addition, the solvent was removed by distillation under suction
at 60–80 ꢀC. Vacuum distillation gave a 56% yield of the title
compound. 1H NMR (CDCl3) ꢂ 1.4 (s, 12H, 4 CH3), 9.1 (s,
2H, 2 CHO).
Scheme 5. Reagents and conditions: i. Na99mTcO4, BER,
r.t., 30 min; ii. Na99mTcO4, SnCl2, r.t., 30 min.
pounds with high radiochemical purity and high yield can be
carried out. This novel labelling method could be applicable
not only to the 99mTc labelling, but also to the 186=188Re
labelling. In this study, we first introduced BER as a new re-
ducing agent and established a new labelling method for the
facile preparation of 99mTc-complexes. This new labelling
method, employing simple and mild labelling conditions, is
useful for the preparation of 99mTc-radiopharmaceuticals by
99mTc labelling of biologically active molecules containing di-
sulfide bonds. Therefore, we have developed a method in
which the reduction of a disulfide compound, the reduction
of 99mTcO4À, and labelling with 99mTc occur in a one-pot
three-step procedure that is amenable to ‘kit’ formation. Cur-
rently, we are in the process of using this method for the prep-
aration of 99mTc labelling of a disulfide compound (N2S2),10 a
potential probe for the ꢀ-amyloid protein of Alzheimer’s dis-
ease, the results of which will be reported in due course.
Experimental
Synthesis of 3,3,10,10-tetramethyl-1,2-dithia-5,8-diazacyclo-
deca-4,8-diene (6): Ethylenediamine (3.6 g, 60 mmol) was add-
ed to a solution of 5 (10.3 g, 50 mmol) in ethanol (70 mL), and the
reaction mixture was heated to reflux for 1 h. The precipitate
formed at room temperature and was allowed to stand at 5 ꢀC
overnight and was then filtered and recrystallized from ethyl ace-
tate in order to give 90% yield of the title compound: mp 163–164
ꢀC; IR (cmÀ1, KBr pellet) 3420, 2949, 2842, 1672, 1448, 1373;
1H NMR (CDCl3) ꢂ 1.38 (s, 6H, 2 CH3), 1.46 (s, 6H, 2 CH3),
3.26 (d, J ¼ 6:39 Hz, 2H, 2 NCH), 4.16 (d, J ¼ 6:3 Hz, 2H, 2
NCH), 6.88 (s, 2H, 2 N=CH).
Synthesis of 3,3,10,10-tetramethyl-1,2-dithia-5,8-diazacyclo-
decane (7): To a solution of 6 in ethanol (120 mL) was added
sodium tetrahydroborate (6.1 g, 160 mmol). The reaction mixture
was heated to reflux for 1 h and allowed to stand at room tem-
perature for 18 h. The solution was evaporated to dryness and the
residue was extracted with dichloromethane. After washing with
water (50 mL) twice, the organic layer was dried over anhydrous
magnesium sulfate and evaporated. Recrystallization from etha-
nol gave 82% of the title compound (7.7 g): mp 57–59 ꢀC; IR
(cmÀ1, KBr pellet) 3450, 2964, 2964, 2735, 2734, 2456, 1560,
1468, 1388; 1H NMR (CDCl3) ꢂ 1.17 (s, 6H, 2 CH3), 1.29 (s,
Unless otherwise noted, all starting materials were obtained
from commercial suppliers and used without further purification.
Na99mTcO4 was obtained by the solvent extraction method from
99Mo produced by a 30-MW multi-purpose research nuclear reac-
tor (HANARO) in the Korea Atomic Energy Research Institute,
Daejeon, Korea. Lyophilized glucoheptonate was obtained from
the Korea Atomic Energy Research Institute, Daejeon, Korea.
Flash column chromatography was performed on silica gel 60
(230–400 mesh, Merck) and all chromatographic separations were
monitored by TLC analyses, performed using glass plates precoat-
ed with 0.25-mm, 230–400 mesh silica gel impregnated with a flu-
orescent indicator (254 nm). IR spectra were recorded on a Bo-
men MB154 FTIR (KBr pellets). 1H NMR was recorded on a
Bruker 500-MHz FTNMR spectrometer in CDCl3, and chemical
shifts were recorded in ppm units using SiMe4 as an internal
standard. Labelling efficiency and radiochemical purity were de-
termined by an instant thin-layer chromatography (ITLC) and re-
versed phase high performance liquid chromatography (RP-
HPLC). The ITLC system consists of a ITLC scanner and 1-di-
mentional analysis of Berthold chroma program. The RP-HPLC
system was equipped with a ꢁBondapak C-18 column
(3:9 Â 300 mm, 10 mm, Waters, USA), ultraviolet detector and
6H, 2 CH3), 1.75(bs, 2H, 2 NH), 2.4–3.0 (m, 8H, 4 NCH ).
2

S. H. Park et al.
Bull. Chem. Soc. Jpn., 76, No. 10 (2003) 1981
Preparation of 99mTc-Glucoheptonate (4). Na99mTcO4 in
saline (0.5mL, 25mCi) was added to a vial containing lyophilized
glucoheptonate and tin(b) chloride. The reaction mixture was stir-
red for about 20 s at room temperature under nitrogen atmosphere,
and the reaction was carried out until the glucoheptonate powder
was completely dissolved. It was filtered through a membrane fil-
ter having a pore size of 0.22 mm and gave a 98% labelling effi-
ciency of the title compound.
Preparation of 99mTc-DADS (3) by Transchelation. To a
vacuum vial containing 5.0 mg of BER were simultaneously add-
ed prepared 99mTc-glucoheptonate and a solution of 7 (1 mg, di-
amine disulfide; DADS) in 0.1 mL of distilled water. The reaction
mixture was stirred for about 20 min at room temperature under
nitrogen atmosphere. It was filtered through a membrane filter
having a pore size of 0.22 mm and gave a 96% labelling efficiency
of the title compound as a technetium-labelled compound of inter-
est.
Preparation of 99mTc-DADS (3) by Using BER. To a vial
containing tetrahydroborate exchange resin (5mg) were injected
simultaneously a solution of 7 (0.1 mg, diamine disulfide; DADS)
in distilled water (0.9 mL) and an aqueous solution of sodium per-
technetate, [Naþ][99mTcO4À] (0.1 mL, 5mCi). After stirring for
30 min, 97% of labelling efficiency of the title compound was ob-
tained by filtering through a membrane filter having a pore size of
0.22 mm.
This work was supported by the Mid- and Long-term
Nuclear R & D Program of the Korean Ministry of Science
and Technology.
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