Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 3, pp. 193-196, 2005

Labeling of Bifunctional Chelating Agent, MAG3, with Carrier-free 188Re
Kazuyuki Hashimoto,*, a W. H. B. B. Wan Kamal,b and Hiromitsu MatsuokaaaDepartment of Research Reactor, Japan Atomic Energy Research Institute, Tokai-mura, Ibaraki-ken 319-1195,
Japanb
Malaysian Institute for Nuclear Technology Research, Bangi, 43000 Kajang, Selangor, Malaysia
Received: July 31, 2005; In Final Form: September 30, 2005
The labeling of MAG3 with carrier-free 188Re from the 188W/188Re generator was investigated by the direct Sn
reduction (solid-phase synthesis) and the indirect labeling method using citrate or gluconate as a transfer ligand.
Optimum pH range were 12.1–12.6 for the direct Sn reduction, 2 –5 for the use of citrate and 2.6 –3 for the use of
gluconate. Although the differences of the optimum conditions such as pH and reaction time at room temperature
were observed by using a different transfer ligand, the labeling yield of 188Re-MAG3 synthesized by the all methods was over 90% under the optimum conditions. The solid-phase synthesis requires the operation under a streamof nitrogen gas and the evaporation of solvent. On the other hand, the method using a transfer ligand is one-pot
preparation by just heating a reaction mixture. Thus, judging from the ease of operations, the method using atransfer ligand is more convenient.

1. Introduction

2. Experimental

The radioisotopes of rhenium (186Re and 188Re) are attractive
radionuclides for radiotherapy because of their energetic beta
particles and gamma rays suitable for imaging.1−3 Especially,rhenium-188 has an advantage of easy availability, because
188
Re can be obtained in a carrier-free level from a 188W/188Re
generator. Oxorhenium(V) and oxotechnetium(V) complexes
with quadridentate ligands such as N3S and N2S2 ligands areoften used as radiopharmaceuticals.4, 5 Among them, mercaptoacetyltriglycine, MAG3 (N3S ligand, Figure 1) labeled with99m
Tc is widely used as a renal imaging agent. Furthermore,
MAG3 is a useful bifunctional ligand in labeling biomolecules(such as monoclonal antibodies6, 7 and peptides8) with 99mTc forradioimmunoscintigraphy of cancer and with 186,188Re for radioimmunotherapy of cancer.
In this work, the labeling of MAG3 with carrier-free 188Re
from a 188W/188Re generator was investigated in detail. The
188
Re-MAG3 complex was synthesized by the direct Sn reduction (solid-phase synthesis) 6 and by the indirect labelingmethod using a transfer ligand (citrate 8 or gluconate). The
dependence of the labeling yield upon the reaction conditions
such as the concentrations of tin(II) chloride dihydrate as a
reducing agent, S-benzoyl MAG3 and the transfer ligand, pH,temperature, reaction time and the addition of a carrier (final
concentration: 20 µg Re/mL) was examined.

2.1. Production of 188W/188Re generator. Typically 25 –35
mg of enriched 186W as WO3 (99.79% enrichment: Euriso-top,France) was irradiated for 26 –52 days in JAERI JMTR (a thermal neutron flux of 2.7 × 1014 n cm−2 s−1).9 The irradiated target
was allowed to stand for more than 4 weeks for 187 W decay.
The 188W/188Re generator was prepared by the alumina column
system.10 The irradiated WO3 was dissolved in 2 M NaOH.
The pH of the 188W solution was adjusted to about 2 using HCl.
This solution was passed through the alumina column (10 × 60mm; BIO-RAD, AG-4, 100 – 200 mesh), which was conditioned with 0.01 M HCl. The column was then washed withnormal saline. Rhenium-188 was eluted with normal saline
after the equilibrium between 188W and 188 Re had almost
reached. Rhenium-188 solution (0.2 –3 MBq/mL) was obtained
from the generator in a saline solution and was used for labeling purposes without further purification.
2.2. Synthesis of 188Re-MAG3 complex. S-Benzoyl-MAG3(S-Bz-MAG3) was supplied from Center for Development ofRadioisotopes and Radiopharmaceuticals, BATAN (the National
Nuclear Energy Agency, Indonesia). S-Triphenylmethyl-MAG3(S-Tr-MAG3) was a gift from Prof. Arano, Chiba University.
All other chemicals used were of guaranteed reagent grade.
(a) Direct Sn reduction (solid-phase synthesis) 6
To 150 µL of 1 M Na2CO3, 500 µL of a 188Re solution fromthe generator, 150 µL of a freshly prepared Na2SO3 solution (100mg/mL), 75 µL of HCl or NaOH solution for pH adjustment, 25µL of S-Bz-MAG3 (2 mg/mL acetonitrile/H 2O, 9:1) and 100µL of a freshly prepared 2 mg/mL tin(II) chloride dihydrateaqueous solution were added. The reaction mixture in a closed
vial was heated in boiling water for 10 min and evaporated todryness under a stream of N2 gas. The mixture was further
heated for another 15 min. After cooling on ice, the mixture
was reconstituted with 500 µL of water. The pH was broughtin 5.7 to 6.3 with 490 µL of 1 N H2SO4 and 25 –70 µL of 1 M
Na2CO3, and the solution was filtered through a 0.22 µm filter.
Radiochemical yields of 188Re-MAG3 were determined by
HPLC. Free MAG3 was also used instead of S-Bz-MAG3.
Free MAG3 was prepared from S-Tr-MAG3 by treating withtrifluoroacetic acid and triethylsilane just before radiolabeling.

O
O

NH

HN

SH

HN

O
COOH

Figure 1. Chemical structure of MAG3.
*Corresponding author. E-mail: hashimoto.kazuyuki@jaea.go.jp.
FAX: +81-29-282-5932.

© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 12/28/2005

194 J. Nucl. Radiochem. Sci., Vol. 6, No. 3, 2005

Hashimoto(b) Citrate as a transfer ligand
To 0.55 mg of S-Bz-MAG3, 450 µL of solution for pH adjustment (0.1~0.6 M HCl, 0.2 M NaOH, 0.2 M acetic aid and/or 0.2
M sodium acetate) and 450 µL of a freshly prepared SnCl2·2H2O
in 0.1 M citrate-buffer (pH = 5) were added. The reaction mixture was vigorously stirred by ultrasonic waves and 300 µL of
a 188Re solution from the generator was added. The final concentration of Re varied from carrier-free levels to 20 µg Re/mL(1.07 × 10 –4 M) by adding NH4ReO4 to the 188Re solution. After
stirring the solution by vortex, the mixture in a closed vial wasallowed to react in boiling water or at room temperature for 1 h.
The mixture was cooled on ice for 5 –10 min. After the solution was filtered through a 0.22 µm filter, radiochemical yieldsof 188Re-MAG3 were determined by HPLC.
The labeling yield of 188Re-citrate was determined by silica
gel TLC (Merck No. 5735/acetone) and silica gel ITLC
(Gelman Sciences/0.9% NaCl) as well as 99mTc-gluconate.11
The distribution of 188Re in TLC and ITLC was measured witha radioanalytic imaging system (AMBIS-100).
(c) Gluconate as a transfer ligand
To 0.55 mg of S-Bz-MAG3, 450 µL of solution for pH
adjustment (0.0~10.1 M HCl, 0.01– 0.2 M NaOH and/or 0.2 M
sodium acetate), 225 µL of sodium gluconate aqueous solution
and 225 µL of a freshly prepared SnCl 2 ·2H 2O in 0.1 M HCl
were added. The reaction mixture was vigorously stirred byultrasonic waves and 300 µL of a 188Re solution from the generator was added. The final concentration of Re varied fromcarrier-free levels to 20 µg Re/mL by adding NH4ReO4 to the188
Re solution. The mixture in a closed vial was allowed toreact in boiling water or at room temperature for 1 h. The
white precipitation was occurred at pH < 11. After the pH of
the solution was brought to about 12 by adding NaOH, the precipitation was dissolved. Therefore, the same volume of 0.1 M
NaOH was added to the reaction mixture just before the HPLC
analysis when the precipitation had occurred. Radiochemicalyields of 188Re-MAG3 were determined by HPLC.
2.3. HPLC analysis. The liquid chromatograph used was a
Waters 2690 separations module equipped with a Waters 996
photodiode array detector and a radio-HPLC detector (Packard
Radiomatic 515TR). The column was kept at 25 ˚C. Radiochemical yield of 188Re-MAG3 was determined by reversed
phase HPLC (Hypersil BDS-5C18, 4.6 × 150 mm, Chemco Science Co., Japan) using 4% EtOH - 0.01 M phosphate buffer (pH= 7). The flow rate was 1.0 mL/min. Typical chromatograms
are shown in Figure 2. Retention times of 188ReO4 − and 188ReMAG3 were 2.4 min and 3.7–3.9 min, respectively.
2.4. Stability studies of 188Re-MAG3 complex. The pH of
188
Re-MAG3 solution (100 µL) was changed to higher values(pH 2 –14) by adding 400 µL of HCl, NaOH and/or sodium
188

pH 2.7

Radioactivity / arbit. unit

Re-MAG3

188

pH 5.1

Re-MAG3

188

-

ReO4

acetate solution for pH adjustment. At regular intervals (1 hourto 70 hours), the radiochemical yield of 188Re-MAG3 was determined by HPLC as mentioned earlier.
3. Results and Discussion
3.1. Direct Sn reduction (solid-phase synthesis). By justheating the reaction mixture, no formation of 188Re-MAG3 wasobserved. When the solution was evaporated at 100 ˚C under astream of N2 until dry and heating continued, 188Re-MAG3 formation occurred. This phenomenon would be due to increase ofconcentration of the reagents (SnCl2, MAG3) during the evaporation process.
The yield of 188Re-MAG3 increased with the concentrationof tin chloride and reached a constant value at 2 mg/mL oftin(II) chloride dehydrate in the initial solution. The concentration of tin chloride was fixed at 2 mg/mL in this work (0.2mg/mL in the initial reaction mixture), however the concentration in the literature6 using 186Re was 1 mg/mL of tin(II) chloride dehydrate solution.
The yields of 188Re-MAG3 were 83% and 89% at pH12.5,
when 1 mg/mL and 2 mg/mL of S-Bz-MAG3 solution wereused respectively. The concentration of S-Bz-MAG3 was fixedat 2 mg/mL in this work (0.05 mg/mL in the initial reaction
mixture), however the concentration in the literature 6 was 1
mg/mL of S-Bz-MAG3 solution.
The dependence of the labeling yield of 188Re-MAG3 on pH
in the initial reaction mixture was shown in Figure 3. The
yield was more than 90% in the pH range 12.1 – 12.6. Our
results are somewhat different from the results in the literature6
that the optimum pH was 11.7. The formation of 188Re-MAG3
by the direct Sn reduction consists of some reactions such as
the deprotection of benzoly-group from S-Bz-MAG3, thereduction of Re(VII) to Re(V) by tin chloride and the reactionof MAG3 and Re(V). The pH dependence of the yield of 188ReMAG3 may reflect individual pH dependences in these reactions. The yield of 188Re-MAG3 has the maximum value in thealkaline pH region, as shown in Figure 3. Generally, the deprotection of benzoly-group from S-Bz-MAG3 is carried out in
NaOH solution. If the deprotection process is important for
determination of the pH dependence of the 188Re-MAG3 yield,
the pH dependence will be changed by using free MAG3 insteadof S-Bz-MAG3. As shown in Figure 3, the pH dependence using
free MAG3 produced from S-Tr-MAG3 was almost the sameas that using S-Bz-MAG3. Thus, the deprotection of benzoyl
group is less important for determination of the pH dependenceof the 188Re-MAG3 yield.
100

Labeling yield of cf-188Re-MAG3 / %

8

80

60

40

: Using S-Benzoyl MAG3
: Using MAG3 converted from Tr-MAG3

20

0
0

2

4

6

8

10

12

14

Retention time / minFigure 2. Typical chromatograms of carrier-free 188Re-MAG3 synthesized by using citrate at different pH.

10

11

12

13

14

pH
Figure 3. Influence of pH on the labeling yield of carrier-free 188ReMAG3 prepared by the solid-phase synthesis.

J. Nucl. Radiochem. Sci., Vol. 6, No. 3, 2005 195

Labeling of Bifunctional Chelating Agent, MAG3

yield decreased sharply above pH 3. In the alkaline region (pH
10 –13), the labeling yield using gluconate was higher than thatusing citrate. Furthermore, the labeling yield using gluconate
increased with the concentrations of tin chloride (as describedabove) and gluconate (as described later).
The pH dependence of the yield of 188Re-MAG3 by the indirect labeling method may reflect some factors such as the
deprotection of benzoly-group from S-Bz-MAG3, the reductionof Re(VII) to Re(V) by tin chloride, the reaction of MAG3 andRe(V), the formation of Re(V)-X and the reaction of MAG3and Re(V)-X (X = transfer ligand). The pH dependence of the
188
Re-MAG3 labeling yield was influenced by a transfer ligand
as mentioned above. And, it was reported that the optimum pH
was 5 – 6 for 188Re-MAG3 prepared by using sodium potassium
tartrate as a transfer ligand.12 It was different from our results
using citrate or gluconate. Thus, the reactions in which a transfer ligand participates such as the formation of Re(V)-X andthe reaction of MAG3 and Re(V)-X would be important for
determination of the pH dependence. Furthermore, it would
support the above induction that the pH dependence (such as
the optimum pH range) of the labeling yield of 186,188Re-citrate13
as shown in Figure 6 was similar to that of 188Re-MAG3 prepared by using citrate (Figure 4).
100

Labeling yield of 188Re-MAG3 / %3.2. Indirect labeling method using a transfer ligand.
3.2.1. Effect of the concentration of S-Bz-MAG3. Citrate: The
influence of S-Bz-MAG3 concentration (0.45 –1.67 mg/mL) in
the reaction mixture was studied at pH 1.7–1.8, [SnCl2 ·2H2O]= 2.25 mg/mL. The labeling yield of 188Re-MAG3 was almost
constant (about 90%) for the carrier-free 188Re. However, theyield increased with the concentration of S-Bz-MAG3 andreached a constant value (about 90%) at 1.3 mg/mL for the carrier-added 188Re (20 µg Re/mL). Higher concentration of SBz-MAG3 was required to obtain more than 90% of the yieldfor the carrier-added 188Re.
Gluconate: The influence of S-Bz-MAG3 concentration(0.11–1.66 mg/mL) in the reaction mixture was studied at pH 3,[SnCl2·2H2O] = 2.25 mg/mL. The labeling yield of 188Re-MAG3
was almost constant (about 90%) for the carrier-free 188Re.
However, the yield increased with the concentration of S-BzMAG3 and reached a constant value (about 90%) at 0.8 mg/mL
for the carrier-added 188Re.
The results indicated almost the same tendency for citrate
and gluconate.
3.2.2. Effect of the concentration of tin chloride. Citrate:
The influence of SnCl2 ·2H2O concentration (0.38 – 4.5 mg/mL)in the reaction mixture was studied at pH 1.6 – 1.9, [S-BzMAG3] = 0.45 mg/mL. The labeling yield of 188Re-MAG3 wasabout 90% for the carrier-free 188Re and about 80% for the carrier-added 188Re not less than 1.1 mg/mL (4.9 × 10−3 M). The
yield decreased below 0.38 mg/mL (1.7 × 10−3 M).
Gluconate: The influence of SnCl 2 ·2H 2O concentration(0.59 – 9.0 mg/mL) in the reaction mixture was studied at pH
2.8 –3.1, [S-Bz-MAG3] = 0.46 mg/mL. The labeling yield of
188
Re-MAG3 was about 90% in the range 1–3 mg/mL for boththe carrier-free and the carrier-added 188Re. A precipitate wasformed and the yield decreased more than 3 mg/mL. At pH
12, the yield increased (36 –38% at 1.1 mg/mL of SnCl2, 70 –72% at 9.0 mg/mL of SnCl2) with the concentration of tin chloride for both the carrier-free and the carrier-added 188Re.
3.2.3. Effect of pH. Citrate: The influence of pH on the labeling yield was investigated at [SnCl2 ·2H2O] = 2 mg/mL, [S-BzMAG3] = 0.45 mg/mL for the carrier-free 188Re, 1.67 mg/mL for
the carrier-added 188Re, as shown in Figure 4. The maximum
labeling yield (more than 90%) was obtained in the pH range
2 –5 and the yield decreased sharply above pH 5.
Gluconate: The influence of pH on the labeling yield wasinvestigated at [SnCl2 ·2H2O] = 2 mg/mL, [S-Bz-MAG3] = 0.46
mg/mL, as shown in Figure 5. The maximum labeling yield(more than 90%) was obtained in the pH range 2.6 –3 and the4020

carrier-freecarrier-added
0

2

4

6

8

10

12

14

pH
Figure 5. Influence of pH on the labeling yield of 188Re-MAG3 prepared by using gluconate as a transfer ligand ([SnCl2 ·2H2O]= 2 mg/
mL, [S-Bz-MAG3]=0.46 mg/mL).
100

Labeling yield of 186,188Re-citrate / %Labeling yield of 188Re-MAG3 / %

60

0

100

80

60

40

20

: carrier-free: carrier-added
0

80

0

2

4

6

pH

8

10

12
188

14

Figure 4. Influence of pH on the labeling yield of Re-MAG3 prepared by using citrate as a transfer ligand ([SnCl2 ·2H2O]= 2 mg/mL,[S-Bz-MAG3]= 0.45 mg/mL for the carrier-free 188Re, 1.67 mg/mL
for the carrier-added 188Re).

80

60

40

20

0

0

2

4

6

8

10

12

14

pH
Figure 6. Influence of pH on the labeling yield of 186,188Re-citrate (○:This work (cf- 188Re, [SnCl2 ·2H2O] = 2.25 mg/mL, [citrate] = 0.038
M), ●: R. Konířová et al. (186Re)13).

196 J. Nucl. Radiochem. Sci., Vol. 6, No. 3, 2005

Hashimoto

0.046

0.092

0.138

0.183

0.229

Labeling yield of 188Re-MAG3 / %

100

80

60

40
188

: carrier-free Re, pH 3188
: carrier-free Re, pH 12188: carrier-added Re, pH 3188
: carrier-added Re, pH 12

20

0
0

10

20

30

40

50

Concentration of sodium gluconate / (mg/mL)
Figure 7. Influence of the concentration of gluconate on the labelingyield of 188Re-MAG3 prepared by using gluconate as a transfer ligand([SnCl2 ·2H2O] = 2.25 mg/mL, [S-Bz-MAG3] = 0.46 mg/mL).

3.2.4. Effect of the concentration of a transfer ligand. Citrate:
The influence of citrate concentration (7.9 and 39 mg/mL,
0.038 and 0.19 M) was studied at pH 5.6, 7.4, 13 –14, [SnCl2 ·2H2O]= 2.25 mg/mL, [S-Bz-MAG3] = 0.45 mg/mL. The labelingyield using 0.19 M citrate was a little higher (5 –10 points) thanthat using 0.038 M citrate at all pH.
Gluconate: The influence of gluconate concentration (2.0 –49.1 mg/mL, 0.0094 – 0.225 M) was studied at pH 3 and 12,[SnCl2 ·2H2O] = 2.25 mg/mL, [S-Bz-MAG3] = 0.46 mg/mL, asshown in Figure 7. The labeling yield at pH 3 indicated a maximum at 8.2 mg/mL of gluconate. The labeling yield at pH 12
increased up to 85% with the concentration of gluconate forboth the carrier-free and the carrier-added 188Re.
3.2.5. Effect of reaction time and temperature. At the optimum
pH (2.4 –2.5 for citrate and 2.8 –3.0 for gulconate), the labelingyield was more than 90% for 15 min in boiling water for the
carrier-free 188Re. For the carrier-added 188Re, the reaction timein boiling water was required 60 min for citrate and 30 min forgluconate to obtain more than 90% of the yield. The labelingyield prepared at room temperature was lower than that in boiling water for both citrate and gluconate. The labeling yield using
gluconate at pH 3 for 30 min at room temperature was more than
88% for the carrier-free 188Re, however the labeling yield using
citrate was lower than 15% under the same conditions.
3.3. Stability of 188Re-MAG3. The influence of pH on thestability of the 188Re-MAG3 prepared by using citrate or gluconate under the optimum conditions was investigated. The pHof 188Re-MAG3 was changed to a higher value (pH 2 –14). The
decomposition of 188Re-MAG3 prepared by using citrate wasobserved only when the pH was over 12, as shown in Figure 8.
The survival { = (the amount after the pH change/the initialamount) × 100} of carrier-free 188Re-MAG3 was over 97% at pH
6.7 and 94% at pH 11.3 even after 70 hours. The same results
were obtained for 188Re-MAG3 prepared by using gluconate.
Furthermore, there was no difference in stability between carrier-free 188Re-MAG3 and carrier-added one.
4. Conclusion
The labeling of MAG3 with carrier-free 188Re from theW/188Re generator was investigated. The labeling yield of
188
Re-MAG3 synthesized by the direct Sn reduction (solid-phase
synthesis) and the indirect labeling method using citrate or gluconate as a transfer ligand was over 90% under the optimum
188

Survival of cf 188Re-MAG3 / %Concentration of gluconate / M
0.000

100
80

: pH= 2.3
: pH= 6.7
: pH =11.3: pH =12.7

60
40
20
0
0

10

20

30

40

50

60

70

80

Time / hour
Figure 8. Stability of carrier-free 188Re-MAG3 prepared by using citrate.

conditions. The solid-phase synthesis requires the operation
under a stream of nitrogen gas and the evaporation of solvent.
On the other hand, the method using a transfer ligand is one-pot
preparation by just heating a reaction mixture. Thus, judging
from the ease of operations, the method using a transfer ligand
is more convenient. However, the solid-phase synthesis required
the smaller amounts of the reagents (S-Bz-MAG3: 0.05 mg per
1 mL of the reaction mixture, SnCl2 ·2H 2O: 0.2 mg) than themethod using a transfer ligand (S-Bz-MAG3: 0.5 mg per 1 mLof the reaction mixture, SnCl2 ·2H2O: 1–2 mg) because the concentrations of the reagents in the solid-phase synthesis increased
gradually during the evaporation process. The effect of pH on
the labeling yield of 188Re-MAG3 was influenced by the difference of transfer ligands. Citrate works effectively in the acidpH region and gluconate dose in the alkaline pH region.
Acknowledgements. The authors are grateful to Prof. Yasushi
Arano, Faculty of Pharmaceutical Sciences, Chiba University forproviding S-Tr-MAG3 and to the staff of Center for Developmentof Radioisotopes and Radiopharmaceuticals, National Nuclear
Energy Agency, Indonesia for providing S-Bz-MAG3. The authors
would like to thank Mr. S. Uchida for his technical assistance.
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