Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 3, pp. 183-185, 2005

Mechanism of Carbonylation Reactions of Technetium-99m
G. E. Kodina,*, a A. O. Malysheva,a O. E. Klement’eva,a A. A. Inkin,a N. I. Gorshkov,bA. A. Lumpov,b and D. N. Suglobovb
a1SSC Institute of Biophysics, 123082, Moscow, Russia, Zhivopisnaya, 46
RPA Khlopin Radium Institute, 194021, St.-Petersburg, Russia, 2nd Murinskii, 28b

Received: July 31, 2005; In Final Form: September 30, 2005
The aim of this work was to study products produced in basic and acidic media upon atmospheric pressure andhigh-pressure ca rbonylation and to propose probable mechanism of reaction. It was found thattricarbonyltechnetium(I) fragment forms strong complexes with charged ligands. The polydentate anions (likecitrate anion), which occupies three coordination places in octahedral technetium arrangement forms strongest
complexes. So Tc(CO)3 moiety in aqueous solutions exhibits properties of a typical d-element and a “soft” acid.

1. Introdouction
Within 40 years technetium-99m occupies the leading position among the radionuclides used in diagnostic nuclear medicine.
One of perspective methods of synthesis of bioconjugateswith technetium is the embedding of Tc(CO)3+ cation in a peptide chain. Therefore the study of tricarbonyltechnetium (TCTc) chemistry and its complexes, and also their biologicalbehavior is extremely actual task for the development of newtechnetium-99m radiopharmaceuticals (RPh). The similarworks with radionuclides of rhenium-186 and 188 are guided
on the development of RPh for radionuclide therapy.
Practically all foreign publications of the last years describe
the method firstly offered by Alberto et al.1, 2 which based on
reduction of sodium pertechnetate, 99mTc in an alkaline medium
in the presence of tartrates-ions. It is supposed that the tartrate is
the stabilizer of an intermediate pentavalent state of technetium.
The commercial lyophilized kit “IsoLink”™ (MallinckrodtMedical) has been developed. TC-99mTc was prepared by simple
introduction of sodium pertechnetate solution in a kit vial withthe subsequent heating of the vial in a boiling water bath during
20 minutes.
Alternative method for high-pressure (160 – 200 atm) carbonylation of sodium pertechnetate under elevated temperature(150–180 ˚C) in acidic media was developed in Khlopin Radium
Institute3 (Russia). The main advantage of this procedure isabsence of organic impurities in preparation, however it requires
special microautoclave unit.
The goal of this work was to study products obtained inbasic and acidic media upon atmospheric pressure and highpressure carbonylation and to propose probable mechanism ofreaction.
2. Material and Methods
The commercially produced generators of Tc-99m (Obninsk,Russia) were used in this work.
Synthesis of the TC-99mTc in acid medium was carried out byautoclave carbonylation according.3 After cooling of the reaction mixture it was neutralized by sodium carbonate or citrate.
TC-99mTc in an alkaline medium was prepared using commercial kit “IsoLink” or potassium boranocarbonate.
*Corresponding author. E-mail: vkorsuns@rcibph.dol.ru. FAX:
+7-495-190-3590.

Reaction mixtures and obtained radiopharmaceuticals wereanalyzed by the following methods:
1. TLC on silica gel (Merck 5554) in methanol/HCl conc. 99/1
and Whatman 3 MM in acetone.
2. HPLC was carried out on a fluid chromatograph “Analist7800” (LDC, USA); terms of separation:
Column: Ultrasphere-ODS (Altex, USA), (250 × 4.6 mm, 5 µ)
Mobile phase: 55% of acetonitrile, 45% of 0.1% solution ofphosphoric acid. Flow rate: 1 mL/min.
The retention time (Rt) for KTcO4 was 1.9 – 2.1 min and forTc(CO)3(H2O)3Cl was 3.6 – 3.9 min.
Chloride of pentacarbonyltechnetium-99 (PC-Tc) and solution of TC-99Tc, prepared from the PC-Tc with the subsequent
NMR identification, were used as the standards. K99TcO4 wasalso used for the preparation of the artificial mixtures. A position of components on chromatograms was determined by anautoradiography. The position of 99 Tc-components was also
identified in UV light (F254).
3. Results and Discussion
According to the literature data TC-99mTc obtained in an
alkaline media has Rf 0.4 (silica gel in methanol/HCl conc.
99/1), whereas the pertechnetate-ions move with solvent front.
On our data Rf for TC-99mTc (obtained with or without of technetium-99, and also for artificial mixtures) was from 0.3 up to0.6 in dependence on the grade of silica gel and conditions ofexperiment. Rf for TcO4 − was 0.8 – 1.0.
Practically in all experiments on autoclave carbonylitationof technetium we observed a component (which originally considered as free TcO4 −) with Rf close, but not identical, with Rffor TcO4 − -ions.
In Figure 1 the chromatograms of reaction mixtures andsolution of Na99mTcO4 (eluate of the generator) are submitted.
Two components with R f 0.5 and 0.8 are observed in initialsolution of a reaction mixture (pH 1). It is possible to find up
to 10% of activity on start of a chromatogram. After dilution
and neutralization up to pH 6 in solution there is mainly onecomponent TC-99mTc.
Two components with Rf 0.0 – 0.1 and 0.4 is also possible toobserve on chromatogram of initial solution of reaction mixture(pH 11) after carbonylitaion of technetium in alkaline media.
There is one component in solution after neutralization (0.1 MHCl) up to pH 6 – 7. In both cases the addition of sodium citrate(5 × 10−3 mole/L) to the neutral solution of TC-99mTc results information of component with Rf 0.8 – 1.0. However the real con-© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 12/28/2005

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

KodinaFigure 2. HPLC: 2a -artificial mixture of K99TcO4 and TC-99Tc; 2b reaction mixtures, obtained upon autoclave carbonylation at the carrier presence; 2c -reaction mixture of TC-Tc, [Tc(CO) 4 (H2O) 2] + andPC-Tc; 2d -neutralized solution of [99mTc(CO)3(H2O)3]+, pH ~7

Figure 1. Chromatograms of reaction mixtures and solution ofNa99mTcO4

tent of TcO4 − -ions does not exceed 10% according to the chromatography of citrate solution on Whatman 3 MM in acetone.
The HPLC results of artificial mixture of K99TcO4 and TC99
Tc are submitted in Figure 2a. At analysis of reaction mixtures, obtained upon autoclave carbonylation at the carrier
presence we observed the appearance of the third component,
the retention time of which is 4.6 – 5.0 min (Figure 2b). The
solution of PC-Tc chloride was prepared for identification of thiscomponent. The retention times for TC-Tc, [Tc(CO) 4 (H2O) 2] +and PC-Tc were 3.7 min, 4.0 min and 4.9 min correspondingly(Figure 2c). Such interpretation was assumed on the data.3 We
did not observe the presence of a component with retention
time 4.0 min in reaction mixtures. At dilution and heating of areaction mixture the signal of PC-Tc disappeared. Besides during TLC of neutralized reaction mixtures we always observedsome of activity on start of chromatograms, we have assumed,that these impurities (probably hydrolyzed reduced technetium)
are retained in a column during HPLC. As a result of column
treatment with 0.1 M HCl after elution of TcO4 − -ions and products of carbonylation we obtained one more fraction containing
Tc in an amount ~10% from total concentration in initial sample. Thus it is possible to conclude, that upon autoclave carbonylation of technetium in amounts (1 – 5) × 10−3 mole/L weobtained mixture of TcO4 −, [Tc(CO)3 (H2O)3]+, [Tc(CO)5]+ andpartially reduced Tc (IV) or Tc (V).
Mainly one peak of TC-99mTc (no carry added) is observed
during HPLC of the reaction products, obtained in an alkaline
media after neutralization to pH 7 (Figure 2c). The data of HPLC
practically coincide with the data TLC. Quantitative yield ofTC-99mTc is possible only in presence of tartrate-ion. Presumably
it stabilizes intermediate five-valence state of technetium, whichis reduced to univalent state under elevated temperature andexcess of carbonylation agent (CO, boranocarbonate). The same
effect was observed by us in the case of preparation of Tc(I)isocyanide complexes at the presence of L-cysteine.4 Last one
is also known as powerful tridentate ligand for linking ofTc(CO)3+ species and corresponding complex was isolated andcharacterized earlier.5
For comparative experiments on Tc-99m carbonylation wechose sodium citrate and L-cysteine, which introduced into areaction mixture instead of tartrate in equlmolar amounts.
In Figure 3a,b the chromatograms of products, obtained dur-Figure 3. Chromatograms of products, obtained during carbonylation in the following conditions: 3a- at the presence of K, Na-tartratewith the subsequent neutralization of mixture by solution of sodiumcitrate; 3b- at the presence of sodium citrate with the subsequent neutralization by 0.1 M HCl; 3c- reaction mixture containing L-cysteineand sodium citrate

ing carbonylation in the following conditions, are submitted: 1)
at the presence of K, Na-tartrate with the subsequent neutralization of mixture by solution of sodium citrate; 2) at the presence of sodium citrate with the subsequent neutralization by
0.1 M HCl. The behavior of radioactive components of thereaction mixture at pH 11 is completely different. We alreadymentioned, that citrate complex of TC-99mTc has Rf 0.8 – 1.0 on
silica gel in methanol/HCl conc, (99/1). The chromatogramsof both mixtures are identical after neutralization. Thus, it ispossible to assume, that the citrate-ions substitute molecules ofwater or hydroxyl-ions in TC-99mTc, synthesized at the presenceof tartrate-ions. The functional groups of citrate at once areincluded into coordination core of technetium (I), when carbonylation is carried out in the presence of citrate-ions.
The results of chromatography of reaction mixture containing
L-cysteine and sodium citrate are submitted in Figure 3c. At
use of L-cysteine as the stabilizing agent in a reaction mixture,on the data of TLC, the complex of TC-99mTc with cysteine ismainly formed, which at the subsequent adding of sodium
citrate does not variate the chromatographic behavior.
Hydrolyzed forms of technetium-99m are not formed practicallyboth in alkaline, and in neutral mediums. It is possible to conclude, that in the reactions of carbonylation cysteine behaves
similarly to citrate, however the chromatographic mobility ofcomplex TC-99mTc-cysteine (Rf 0.3) differs from mobility ofcomplex TC-99mTc-citrate (Rf 0.8–1.0). The results of biologicaltests of these radiopharmaceuticals confirm this suppose. Bothligands are more “strong” complex agents in relation to tricarbonyltechnetium, as against of tartrate-ions, which capability
to supersede water from the coordination core of Tc (I) isdoubtfully.

J. Nucl. Radiochem. Sci., Vol. 6, No. 3, 2005 185

Mechanism of Carbonylation Reactions

It is possible to assume the various mechanism for carbonylation reactions in acidic and alkaline mediums on the basis of
the obtained data. The comparison of TLC and HPLC results
for reaction products and artificial mixtures, containing Tc-99,
specifies probability of formation PC-99mTc as an intermediate
product upon autoclave carbonylation. PC-99mTc- chloride turns
into TC-99mTc at dilution (neutralization) of a reaction mixture.
The probability of step reduction of technetium in acid medium
basically exists (by analogy to the reaction carried out in an
alkaline media). So the following sequence of reactions during
autoclave carbonylation of technetium in acid medium can beproposed:
HTcOCl4

TcO4 −COH +, COTc(CO)5Cl

H2O[Tc(CO)3(H2O)3]Cl0t , p (160 atm)

In an alkaline media at the presence of tartrate-ions the following reactions probably take place:
TcO4−

OH− ,CO, Tart
t 0, p (1 atm)

[TcO(OH)]Tart
or[TcOCl5] −2

CO, H2O[Tc(CO)3(H2O)2(OH)] H2O, HClor/and[Tc(CO)3(H2O)3]Cl[T c(CO)3(H2O)(OH)2 ] −If the citrate is introduced into a reaction mixture instead oftartrate, schematically the reaction of carbonylation can be presented as follows:
OH−, CO, Cit
CO, H2O
TcO4 − → [TcOCit] → [Tc(CO)3Cit]t˚, p (1 atm)
Three carbonyl groups of citric acid or two carbonyl groups
and oxy-group can participate in formation of pentavalent technetium complex with citrate in alkaline media according to.5
Therefore it is possible to consider the submitted above formulas symbolic.
We isolated and characterized complex with overall structure [Tc(CO) 3 (Cit)] with tridentate coordination of ligand
which have the same Rf in TLC. In the 99Tc NMR we observed
singlet at –906 ppm and in PMR spectrum we observed complex multiplet, centered at 2.85 ppm, which assigned tounequivivalent methylene groups. This fact along with the dataof IR spectroscopy, which show coordination of two COO
groups of the ligand and absence of OH stretch, allows us toconsider the tridentate coordination of citrate-anion Thus we
can conclude that. 99mTc(CO)3+ converted to [99mTc(CO)3 (Cit)]at concentration of citrate 5 × 10 −3M. Proposed structure isshown below.
Spectral characteristics of Tc(CO) 3 (Cit) complexOOTc NMP (ppm) –906
1H NMR (ppm, D2O) 2.85 (dd, dd (unequiv. CH2))

OCCOTc+O

IR (cm−1, nujol) 2030, 1905 (νCO –fac coord.),1710, 1605 (νCOO coord. stretch),
1550 (νCOO free stretch), νOHval is absent

COO-

CH2
CH2O

O
O-

Proposed structure

In conclusion we can say, that tricarbonyltechnetium(I)fragment forms strong complexes with charged ligands. The
polydentate anions (like citrate anion), which occupies threecoordination places in octahedral technetium arrangement
forms strongest complexes. So Tc(CO)3 moiety in aqueous solutions exhibits properties of a typical d-element and a “soft” acid.
Acknowledgements. This work was financially supportedby ISTC (Project N 1723). We acknowledge, TYKO Healthcare(Mallinckrodt) for the “IsoLink” kits.
References(1) R. Alberto and R. Schibli, Patent WO 98/48848, A61K
51/04.
(2) S. Liu and D. S. Edwards, Chem. Rev. 99, 2235 (1999).
(3) N. I. Gorshkov, A. A. Lumpov, and A. E. Miroslavov et al.,Radiochemistry, 42, 213 (2000) (in Rusian).
(4) G. E. Kodina, A. O. Malysheva, and T. A. Babushkina etal., Coordination Chem. 23, 682 (1996) (in Russian).
(5) B. Johansson, H. Spice Technetiumverbindugen Chemischeund radiopharmakologischeunter-suchengen unter besoderer Berucksichtigung von Technetium-Thiol-Komplexen,(Rosendorf, 1981), p 213.