Role of protecting groups in the preparation of thiolate complexes of Technetium-99 m using cysteine as a model

Article abstract of DOI:10.1002/jlcr.1084

An attempt has been made to develop a suitable protecting group for the thiolate function for ^99mTc binding ligands having such function and which could be deprotected automatically during ^99mTc-chelation without the use of any additional reagents. As a model ligand a simple molecule like L-cysteine was selected. Seven S-protected derivatives of this amino acid were synthesized, radiolabelled with technetium-99 m under a variety of experimental conditions and the yield of the desired chelate was compared to that of ^99mTc-L-cysteine, the authentic standard chelate, by HPLC. The corresponding ⁹⁹Tc chelate of cysteine from L-cystine and S-thiomethyl L- cysteine was also prepared. It was found that the ⁹⁹Tc chelates exhibited similar retention profiles to those of the corresponding ^99mTc chelates in reverse phase HPLC. The results of the biodistribution studies after ^99mTc chelation were likewise compared to those of ^99mTc-L-cysteine. The effect of probenecid on renal excretion was studied only on the ^99mTc chelate of S-thiomethyl-L- cysteine to determine whether tubular excretion was involved. The results suggest that the S-thiomethyl group could be used as an ideal protective group to mask the high reactivity of thiolate functions attached to different ^99mTc binding ligands. Copyright

Full text of DOI:10.1002/jlcr.1084

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 835–847.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1084
Research Article
Role of protecting groups in the preparation of thiolate
complexes of Technetium-99 m using cysteine as a
model
1
U. Roy , M. Chatterjee Debnath *, Kasturi Sanyal , M. K. Das and
1,
1
2
3
S. Banerjee
1
Nuclear Medicine Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick
Road, Kolkata 700032, India
2
Radiopharmaceuticals Lab of BRIT, Variable Energy Cyclotrone Centre, Kolkata
7
3
00064, India
Saltlake, AE-413, Kolkata 700064, India
Summary
An attempt has been made to develop a suitable protecting group for the thiolate
9
9m
function for
Tc binding ligands having such function and which could be
9
9m
deprotected automatically during
Tc-chelation without the use of any additional
reagents. As a model ligand a simple molecule like l-cysteine was selected. Seven
S-protected derivatives of this amino acid were synthesized, radiolabelled with
technetium-99 m under a variety of experimental conditions and the yield of the
9
9m
desired chelate was compared to that of
9
Tc–l-cysteine, the authentic standard
9
chelate, by HPLC. The corresponding Tc chelate of cysteine from l-cystine and
S-thiomethyl l- cysteine was also prepared. It was found that the Tc chelates
9
9
9
9m
exhibited similar retention profiles to those of the corresponding
Tc chelates in
9m
Tc chelation
9
reverse phase HPLC. The results of the biodistribution studies after
9
9m
were likewise compared to those of
excretion was studied only on the
Tc-l-cysteine. The effect of probenecid on renal
9
9m
Tc chelate of S-thiomethyl-l- cysteine to
determine whether tubular excretion was involved. The results suggest that the
S-thiomethyl group could be used as an ideal protective group to mask the high
9
9m
reactivity of thiolate functions attached to different
2006 John Wiley & Sons, Ltd.
Tc binding ligands. Copyright
#
Received 8 January 2006; Revised 10 April 2006; Accepted 11 April 2006
*Correspondence to: M. Chatterjee Debnath, Nuclear Medicine Division, Indian Institute of Chemical
Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, India. E-mail: mita chdebnath@yahoo.com
Contract/grant sponsor: Indian Council of Medical Research; contract/grant number: 5/4/1/Exm/2002-
NCD-II
Copyright # 2006 John Wiley & Sons, Ltd.

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U. ROY ET AL.
Key Words: L-cysteine; protecting group; thiolate function; technetium-99&99m;
S-thiomethyl-l-cysteine
Introduction
Protecting groups are used in different branches of organic chemistry to
conduct a chemical reaction of a multifunctional compound selectively at one
reactive site and to avoid undesirable by-product formation. In the
preparation of inorganic chelate complexes, masking of heteroatoms of the
ligands with easily removable protecting groups to produce specific chelates
has been a standard practice
Technetium-99 m is the most widely used radioisotope in diagnostic nuclear
medicine. However, in many cases of technetium chelation the ligands used are
mostly unprotected and with such compounds very often more than one
technetium chelate is formed under slightly different experimental conditions.
Most frequently this problem is encountered with ligands that contain one or
1
,2
99m
99m
more thiolate function. In
Tc-MAG , the most important
3
Tc chelate
for renal function studies benzoyl protection on the sulphur atom of the ligand
3
during chelation was used. Whether the benzoyl group is the ideal protecting
group in this experiment could be questioned since reproducible yields of the
chelate could not be obtained in many cases and different methods, each
claiming its superiority over that of others, have appeared in the literature over
4–6
a long period of time.
To provide information relating to the formation of undesired technetium
chelates with thiolate containing ligands, one may look into the chemistry of
9
9
chelation with the long-lived isotopes of technetium ( Tc). In some cases the
desired chelate can be produced in good yield only when the oxotechnetium
derivative and the thiolate ligand are allowed to react in stoichiometric
99
proportion, and a relatively pure Tc-chelate can be obtained only when the
thiolate group is derivatized with acetyl amino methyl, benzyl, benzamido-
7À9
methyl, trityl etc.
We observed that cysteine, a common and simple thiolate ligand, also shows
this abnormal behaviour during chelation with technetium. It produced the
99
desired oxotechnetium Tc bis cysteinate in exellent yield only when the
99
ligand and TcO
À
is allowed to react in stoichiometric ratio, but the presence
4
of any excess of cysteine in the reaction mixture furnished only polymeric
products. However, this problem could be avoided by using the corresponding
disulphide (cystine) as monosulphide precursor; use of large excess of the
1
0
former produced the above chelate in excellent yield. Therefore it may be
concluded that the protection of monosulphide by disulphide formation,
which is a useful tool in peptide chemistry, may be successfully applied in
thiolate chelation with technetium.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49:835–847
DOI: 10.1002/jlcr

ROLE OF PROTECTING GROUPS IN PREPARATION OF THIOLATE COMPLEXES 837
The objective of the present paper is to show how the aforementioned
9
methods of chelation of thiolate compound with Tc can be transferred to the
9
9
9m
preparation of
Tc-radiopharmaceuticals where technetium is used at tracer
level. Since, for clinical application, the required radiopharmaceutical needs to
be available as a kit the chelation is best carried out, at near, neutral pH.
Under such conditions it is inconvenient to use symmetrical disulphides like
cystine because of poor aqueous solubilities of such compounds. We have
observed that thiolated compounds, when coupled with thiomethyl chloride,
produce the mixed disulphides which have a better solubility in aqueous
conditions.
9
9m
Preparation of the desired
Tc-cysteine complex from cystine is a well-
1
0À12
known procedure.
Using this chelate preparation as standard we
undertook the comparative HPLC studies of different technetium–l-cysteine
preparations using several S-protected l-cysteine derivatives which include
S-thiomethyl and S-thiophenyl cysteine as disulphide precursor as well as
S-benzyl, S-acetamidomethyl cysteine etc. as proclaimed by different
1
3,14
groups.
From such a study we expect the derivative best suitable for
9
9m
efficient use in
Tc radiopharmaceutical preparation to emerge. The results
are discussed in the following sections.
Experimental
General
S-t butyl l-cysteine (III) was purchased from Aldrich Chemical Company,
USA. Proton NMR spectra were recorded on a 300 MHz Bruker DPX 300
spectrometer. Elemental analysis was performed in the microanalytical
laboratory of the Indian Association for Cultivation of Science, Kolkata.
9
9MoO4 was purchased from the Bhabha Atomic Research Centre and
9m
9
TcO4 was obtained by 2-butanone extraction from a 5(N) NaOH solution
À
99
of MoO . Ammonium Tc-pertechnetate was purchased as an aqueous
4
solution (0.38 mmol ml) from Amersham. g-Counting was performed using a
well type counter from Electronic Corporation of India Model LV4755. HPLC
analyses were performed on a reverse phase C-18 column (4.6Â 250mm particle
size 5 mm) fitted to Water Associates (USA) solvent delivery system consisting of
two M515 pumps driven by a M680A automatic gradient controller and
Rheodyne 77251 injector. Radioactivity in the eluate was monitored using a
Beckman Model 170 detector and integrated by a Water M746 data Module.
9
9
For Tc chelates the UV detector (Waters Model 481) was set at 425nm.
Synthesis
In most cases the thiol protection of l-cysteine was performed by literature
1
3
methods leading to the preparation of S-benzyl-l-cysteine(I), S-p-methoxy
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49:835–847
DOI: 10.1002/jlcr

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U. ROY ET AL.
1
5
14
benzyl-l-cysteine (II), S-acetamido methyl-l-cysteine (IV), l-thiazolidine-
4
16
17
-carboxylic acid (V) and S-thiophenyl-l-cysteine (VI). The synthesized
compounds (Figure 1) had comparable melting points with those reported in
1
the literature and were further characterized by TLC analysis and HNMR
spectroscopy. Methyl sulphenyl chloride was prepared by the reported
1
8
method
with some modifications. S-thiomethyl l-cysteine (VII) was
prepared by the following two step synthesis:
(a) Preparation of methyl sulphenyl chloride: A solution of chlorine (15 g) in
dry carbon tetrachloride (70 ml) was added to a solution of dimethyl
disulphide (6.76 g) in carbon tetrachloride (15 ml) at À208C. A white solid
(5 g) appeared which was filtered and was added to dimethyl disulphide
(2.9 ml) pre-cooled at –208C. A deep orange liquid was formed which was
stored at À208C.
b) Preparation S-thiomethyl-L-cysteine (VII): An aliquot of the above
solution having the theoretical amount of methyl sulphenyl chloride (1.07 g,
.013 mol) was added dropwise to a suspension of l-cysteine hydrochloride
(
0
monohydrate (1.89 g, 0.010 mol) and sodium bicarbonate (1.09 g, 0.13 mol) in
ethanol (34 ml) under nitrogen. The reaction mixture was allowed to stand at
room temperature until cysteine was no longer detectable by nitroprusside.
Pyridine (1.45 ml) was added when the reaction mixture showed a neutral pH
and the solution was concentrated to half of the original volume; the
precipitate was then filtered and washed with ethanol. The crude material was
purified from its ammoniacal aqueous solution by adding acetic acid to pH-5
to furnish the pure material (1.56 g, 69%). TLC single spot R , 0.65 EtOH:H O
f
2
(
7:2); and R , 0.70 (BuOH:Py:H O:AcOH;5:3:4:1), cellulose plate; m.p. 1808C;
f
2
1
HNMR (D O) 2.44 (s, 3 H, CH ), 3.12–3.64(t, 2 H, S-CH -C), 4.40–4.64
2
3
2
(t, 1 H, -CH-). Calculated for C H NS O C, 28.71; H, 5.42; N, 8.37; found C,
4 9 2 2
2
8.61; H, 5.09; N, 8.94.
Radiolabelling of ligands
99m
Tc-chelation of S-benzyl-l-cysteine (I), S-p-methoxy benzyl-l cysteine (II)
and S-thiophenyl-l-cysteine (VI). The appropriate amount of either (I) or (II)
was suspended in nitrogen purged water (0.2 ml) and sodium hydroxide
solution was added to just dissolve the ligands. In the case of S-thiophenyl-l-
cysteine (VI) was dissolved in EtOH (0.2 ml), water (0.1 ml) was added and the
pH adjusted to 12 with alkali. To each of the ligand solutions 99mTcO4
À
solution (0.1 ml, 74–185 MBq) was added followed by the addition of freshly
prepared stannous chloride solution (50 ml; 50 mg). The pH of the solution was
then adjusted to 7 and after 30 min the chelate solution was centrifuged. The
supernate, which contained most of the radioactivity, was used in subsequent
experiments.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49:835–847
DOI: 10.1002/jlcr

ROLE OF PROTECTING GROUPS IN PREPARATION OF THIOLATE COMPLEXES 839
9
9m
Tc Chelation of S-t-butyl-l-cysteine (III), s-acetamido-methyl-l-cysteine
(
IV) and l-thiazolidine-4-carboxylic acid (V). To a solution of the appro-
priate amount of the ligand in water (0.4 ml), acetate buffer (pH 4, 0.2M,
0
7
.4 ml) was added followed by the addition of aqueous 99mTcO4
À
(0.1 ml,
4–185 MBq) and stannous chloride solution (50 ml containing 20 mg of SnCl2).
The chelate solution was allowed to stand for 30 min at room temperature
before using for subsequent experiment.
99m
Tc Chelation of S-thiomethyl l-cysteine (VII). The desired amount of
ligand was dissolved in water (0.4 ml) and pH of the solution was adjusted to
; to this solution was added 99mTcO4
(0.1 ml, 74–185 MBq), followed by the
8
À
addition of stannous chloride solution (50 mg in 50 mL) prepared as described
above, and allowed to stand at room temperature before use.
9
9m
11
Preparation of
Tc-l-cysteine chelate. The chelate was prepared as reported
99m
method from l-cystine and
TcO4.
99m
Preparation of
Tc-l-cysteine kits from S-thiomethyl-l-cysteine. A kit
preparation of l-cysteine was chosen using a 5 ml vial. To the solution
(
0.5 ml, pH 8.0) of ligand (VII, 5 mg), Na HPO solution (6.75 mg; 300 ml),
2
4
1
00 ml of a solution containing ascorbic acid (1.5 mg) and NaCl (5.5 mg) was
added followed by the addition of freshly prepared stannous chloride
dihydrate solution (50 ml, 0.225 mg). The vial was lyophilized and stored for
a specified period. The kit was reconstituted by the addition of 2.0 ml of
sodium pertechnetate containing the required activity to the lyophilized vial,
and mixed well and heated in a boiling water bath for 2–3 min.
9
9
Tc-chelation of S-thiomethyl-l-cysteine. The chelation was carried out as
9
9
reported in our method for the preparation of oxotechnetium ( Tc) –l-
cysteinate. To an aqueous solution (1 ml, pH 8.5) of S-thiomethyl-l-cysteine
(
135 mg, 1 mmol), NH TcO solution (0.22 ml, 0.085 mmol) and a freshly
4 4
prepared solution (0.5 ml) of Na S O (33 mg, 0.185 mmol) was added and
2
2
4
incubated at room temperature for 4 h. The pH was adjusted to 7.0. The
reaction mixture was then passed through celluloseCM and sephadex-LH
columns to furnish pure chelate. UVlmax:425 nm.(4255), 307(1147), IR (KBr)
À1
n; 942 cm (Tc=O).
Aeration of the above chelates
3
An aliquot of each chelate (0.5 ml) was aerated by bubbling air (20 cm ) and
subsequently heating (958C) for 3 min.
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

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U. ROY ET AL.
Radioanalytical purification studies
HPLC studies of the radiochemicals were performed on a C-18 m bondapak
column. The column was eluted isocratically with tetrabutylammonium
chloride (0.005 M): MeOH in 1:1 ratio (volume) at a flow rate of 1 ml/min.
In each case the retention time and per cent purity were recorded.
Biodistribution studies
Experiments using mice were carried out in compliance with the relevant
national laws relating to the conduct of animal experimentation. Male swiss
albino mice (20–25 g) were hydrated by intraperitoneal administration of
9
9m
saline (0.9%, 3 ml) for 1 h. After another 1 h, the
Tc-chelate of ligands
1
31
I–VII or l-cysteine (185–259 kBq) premixed with I-OIH (74–111 kBq) in a
total volume of 0.03 ml was injected via a tail vein. The mice were sacrificed
either at 30 or 10 min (for probenecid study) post-injection and the desired
organs were collected. The radioactivity in each organ is expressed as the
percentage of injected dose. Blood was assumed to be 7% of the body weight.
In the probenecid experiment mice were pre-treated with probenecid
À1
(50 mg Kg ), 10 min prior to radiopharmaceutical injection.
Results
S-benzyl –l-cysteine (I), S-p-methoxy benzyl-l-cysteine (II) and l-thiazoli-
dine-4-carboxylic acid (V) were prepared using reported methods. S-
Acetamido methyl l-cysteine (IV) was prepared by reacting N-hydroxy
methylacetamide with l-cysteine under strongly acidic conditions. The two
NH2
CH
CO H
2
H C
CH
NH
2
RS
CH2
COOH
S
CH 2
L - Cysteine ; R = H
S - Benzyl - L - cysteine ; R =
CH2
( I )
L - Thiazolidine - 4 - carboxylic acid ( V )
S - p - Methoxy benzyl - L - cysteine ; R =
CH3
CH2
OCH3 ( II )
S - t - Butyl - L - cysteine ; R =
C
CH3
( III )
CH3
S - Acetamido methyl - L - cysteine ; R = -CH2-NH-CO-CH3 ( IV )
S - Thiophenyl - L - cysteine ; R =
S
(
VI )
S - Thiomethyl - L - cysteine ; R =
S
CH3
( VII )
Figure 1. Chemical structures of thiol protected l-cysteine derivatives (I–VII)
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49:835–847
DOI: 10.1002/jlcr

ROLE OF PROTECTING GROUPS IN PREPARATION OF THIOLATE COMPLEXES 841
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49:835–847
DOI: 10.1002/jlcr

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U. ROY ET AL.
disulphides S-thiophenyl-l-cysteine (VI) and S-thiomethyl-l-cysteine(VII)
were prepared by condensing l-cysteine with either benzene sulphenyl chloride
or with methane sulphenyl chloride, respectively. Benzene sulphenyl chloride
was prepared by chlorinating thiophenol and similar reaction using dimethyl
disulphide under careful reaction condition produced the methane analogue.
1
All these ligands (I–VII) were characterized by TLC analysis and HNMR
spectroscopy.
99m
Tc radiolabelling of the ligands I–VII was carried out using the standard
stannous chloride reduction procedure under different experimental conditions
involving the amount of ligand, pH of the solution and aeration of the chelate
solution with subsequent heating in boiling water bath for 2 min to maximize
the yield. The analysis was done by HPLC, which revealed that all the chelates
consisted of a single component. The results of complexation studies (n=3) at
various ligand concentrations are shown in Table 1.
S-benzyl-l-cysteine(I), S-p-methoxy benzyl-l-cysteine(II) and S-thiophenyl
–l-cysteine(VI) exhibited a maximum complexation yield at pH-12. For
S-t-butyl–l-cysteine (III), S-acetamido methyl–l-cysteine(IV) and l-thiazoli-
dine-4-carboxylic acid (V) the optimum pH for maximum yield was between
pH 4–4.8. The labelling yield of S-thiomethyl–l-cysteine(VII) reached an
optimum at pH 8. S-thiophenyl-l-cysteine (VI) is not soluble in water so it was
complexed in an alcoholic medium.
It was evident from the HPLC result (Table 1) that in most of the cases the
labelling yield was higher with 5 mg of ligand concentration than that for 1 or
2
mg of ligand concentration. In most cases the aeration step increased the
stability of the desired chelate.
Of all the chelates studied it is evident from Table 1 that l-thiazolidine-4-
carboxylic acid (V), S-thiophenyl-l-cysteine (VI) and S-thiomethyl-l-cysteine
9
9m
99m
(
VII) after
Tc labelling produced the corresponding
Tc-complexes in the
9m
Tc-chelates from
9
best yields (93–98%). The yield of the corresponding
other ligands (I–IV) was comparatively low (70–88%) (Table 1) and the
product was contaminated with other impurities. The radiochemical yield of
9
9m
the
Tc–l-cysteine chelate prepared from VII was 96% when it was
formulated as single vial lyophilized kit.
Biodistribution studies in mice at 30 min post-injection were undertaken
only with those preparations where the yield of the desired chelate (HPLC;
9
9m
99m
Table 1) was maximum. The biodistribution results of
9
Tc-I to
I-OIH is depicted in Table 2 as per cent dose per
Tc-VII,
9m
131
Tc-l-cysteine and
organ. From the table it is evident that the chelates
9
9m
99m
Tc-I to
Tc-IV which
were contaminated with radioacative artefacts, exhibited elevated hepatobiliary
uptake (10.76–14.72%) and decreased urinary excretion (51–55%) whereas
9
9m
99m
Tc-VI and
Tc-VII exhibited comparatively high urinary excretion of
5
8.58, and 60.38%, respectively with decreased hepatobiliary uptakes of 8.01
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49:835–847
DOI: 10.1002/jlcr

ROLE OF PROTECTING GROUPS IN PREPARATION OF THIOLATE COMPLEXES 843
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49:835–847
DOI: 10.1002/jlcr

8
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U. ROY ET AL.
9
9m
Table 3. Effect of probenecid on the biodistribution of
Tc-S-thiomethyl-l-cysteine
I-orthoiodohippurate (results in parenthesis) in
mice at 10 min time point expressed as per cent dose/organ
9
9m
131
and
Tc-cysteine coinjected with
9
9m
99m
Organs
Tc-S-thiomethyl-l-cysteine
Tc-l-cysteine
Control
Probenecid
Control
Probenecid
Blood
Urine
3.87 Æ 0.76
3.91 Æ 1.50)
33.94 Æ 2.38
48.31 Æ 4.94)
10.64 Æ 1.63
11.55 Æ 1.63)
3.82 Æ 0.73
8.49 Æ 3.17
(4.53 Æ 1.15)
21.35 Æ 2.38
(42.81 Æ 3.75)
2.97 Æ 1.00
(7.64 Æ 1.04)
4.24 Æ 1.00
4.41 Æ 0.08
(3.81 Æ 0.37)
32.64 Æ 2.10
(52.41 Æ l.78)
13.85 Æ 0.17
(5.12 Æ 1.20)
3.34 Æ 1.17
7.65 Æ 1.42
(5.20 Æ 0.25)
16.33 Æ 5.46
(42.39 Æ 6.07)
4.86 Æ 0.12
(3.64 Æ 0.61)
5.44 Æ 0.70
(
(
(
Kidney
Liver
(
3.81 Æ 0.14)
4.03 Æ 0.79
5.00 Æ 0.19)
(2.98 Æ 0.75)
3.64 Æ 0.49
(4.25 Æ 1.36)
(2.29 Æ 0.03)
4.72 Æ 0.62
(2.66 Æ 0.55)
(2.81 Æ 0.42)
7.45 Æ 1.13
(5.11 Æ 0.50)
Intestine
(
and 7.64%, respectively. In this respect these two chelates exhibited more
9
9m
similarity to
9m
Tc-l-cysteine, the authentic standard, than others. Although
Tc-VI exhibited pronounced renal excretion and less hepatic uptake due to
9
its water insolubility further study with this complex was discontinued.
However, S-thiomethyl-l-cysteine is soluble in water at pH-8 and this could be
used for easily reconstitutable kit formulation consisting of a lyophilized
mixture of S-thiomethyl-l-cysteine and SnCl2, 2H O at pH 8. The HPLC
2
elution pattern of the above radiolabelled kit was also comparable to that of
9
9m
the
To determine the exact pathway of elimination
probenecid depression studies and the results (Table 3) were compared with
Tc–l-cysteine standard.
9
9m
Tc-VII was subjected to
9
9m
131
Tc-l-cysteine and
and kidney retention of
I-OIH. It was observed that both urinary excretion
99m
Tc-VII at 10 min post-injection in mice pre-treated
with probenecid was significantly reduced. This significant change in the
biodistribution pattern proved that both compounds were eliminated via the
renal tubular excretory pathway.
99
We prepared the Tc chelate of VII from the S-thiomethyl-l-cysteine ligand
following exactly the same method adopted earlier in this laboratory for the
9
9
10 99
preparation of Tc-l-cysteinate.
Tc-chelate of VII exhibited UV -max at
À1
4
25 nm and IR stretching bands at 942 cm which were comparable to those
À1
99
10
of Tc-l-cysteinate (lmax 426 nm and IR band 940 cm ) reported earlier.
9
The reverse phase HPLC retention time of Tc and
9
99m
Tc-l-cysteinate
prepared from l-cystine as starting ligands were 5.03 and 5.15 respectively,
9
whereas the Tc and
9
99m
Tc chelates prepared from S-thiomethyl-l-cysteine
VII) exhibited retention times of 5.08 and 5.18, respectively on the same
(
reverse phase HPLC system (Figure 2).
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49:835–847
DOI: 10.1002/jlcr

ROLE OF PROTECTING GROUPS IN PREPARATION OF THIOLATE COMPLEXES 845
Discussion
Among the different protecting groups that are used in peptide chemistry, and
also for thiol protection of different SH containing ligands in technetium
chelation, we selected benzyl, p-methoxy benzyl, acetamidomethyl and t-butyl.
They could mask the high reactivity of the thiolate function and could be
9
9m
removed instantaneously and quantitatively during
Tc-chelation. The
Tc-chelates of these ligands were
99m
radiochemical yield of the corresponding
between 70 and 88% (Table 1). This variation in yield of the desired chelate is
dependent on various factors like the amounts of ligand, pH of the ligand
solution and oxidation of the chelate solution by aeration and subsequent
heating. These variations in HPLC profile are also reflected in the
biodistribution results. Many of these chelates exhibited appreciable amount
9
9m
of hepatobiliary uptake with less urinary excretion than that of
cysteine under comparable experimental conditions.
Tc-l-
1
0
After the establishment of the molecular structure of technetium cysteine
it was apparent that disulphide could be the appropriate precursor of
monosulphide towards the preparation of desired technetium chelates. On this
basis we further selected S-thiophenyl and S-thiomethyl as two other
protective groups. It is expected that both S-thiophenyl-l- cysteine(VI) and
S-thiomethyl-l-cysteine (VII) being mixed disulphide, could be more appro-
priate for use as a precursor for the preparation of desired technetium
cysteinate chelate in quantitative yield because they may readily undergo metal
induced disulphide cleavage.
9
9m
99m
It was observed that both
chelate in almost quantitative yield. Their biodistributive properties were
Tc-VI and
Tc-VII produced the desired
9
9m
comparable to
that in the presence of probenecid the renal excretion of
inhibited to a greater extent and this behaviour was also comparable to
Tc-l-cysteine in every respect. It was further observed
9
9m
Tc-VII was
9
9m
Tc-
l-cysteine. However, VI is insoluble in water and its chelation could be done
only in alcoholic medium. This may result in inconvenience during kit
formulation and kit reconstitution so further study with this ligand was
discontinued.
Therefore, it appears that amongst the series of S-protected derivatives
of cysteine studied S-thiomethyl-l-cysteine (VII) produced the desired
Tc-chelate in quantitative yield, which excreted exactly in the same way
9
9m
9
9m
as
Tc-l-cysteine through the renal pathway. Moreover, S-thiomethyl-l-
cysteine is soluble in water at pH 8 so it is easily adaptable for kit formulation
and kit reconstitution. The 99-technetium chelate obtained from S-thiomethyl
l-cysteine exhibited a virtually identical retention time in HPLC to that of
99
oxotechnetium ( Tc)-l-cysteinate (Figure 2) whose structure was already
established from this laboratory. The thiomethylated precursor of l-cysteine,
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49:835–847
DOI: 10.1002/jlcr

8
46
U. ROY ET AL.
99m
99
Tc and (b) Tc chelate of l-cysteine
Figure 2. HPLC profiles for (a)
prepared from l-cystine (1) and S-thiomethyl-l-cysteine (2)
after technetium (99@99 m) labelling, produced the desired technetium
(99@99 m)-l-cysteine chelate in quantitative yield.
Conclusion
It can be concluded that S-thiomethyl (S-CH ) is an ideal protective group
3
that can be used to mask the high reactivity of thiolate function attached to
9
9m
different
quantitatively during
for masking thiolate function of other clinically important
Tc-binding ligands and can be removed instantaneously and
99m
Tc-chelation. An attempt to use this protective group
9
9m
Tc-radio-
Tc-MAG3 and
Tc-DMSA is also underway. This type of modification of the ligand
99m
99m
99m
pharmaceuticals like
Tc-L,L’EC,
Tc-L,L’-ECD,
9
9m
molecules may lead to the development of a single vial kit for the
aforementioned radiopharmaceuticals and therefore improve the diagnostic
capability of various nuclear medicine centres.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49:835–847
DOI: 10.1002/jlcr

ROLE OF PROTECTING GROUPS IN PREPARATION OF THIOLATE COMPLEXES 847
Acknowledgement
We acknowledge the financial assistance provided by the Indian Council of
Medical Research (5/4/1/Exm/2002-NCD-II) to carry out this work.
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Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49:835–847
DOI: 10.1002/jlcr