Vitamin B12 as a ligand for technetium and rhenium complexes

Article abstract of DOI:10.1002/anie.200460923

Robust complexes with a central {Co-CN-Re(Tc)} feature are formed when the cyanide ligand in vitamin B12 acts as bridging ligand between rhenium and technetium carbonyl complexes (see picture). This concept paves the way for radiolabeling of vitamin B12 or metal-mediated coupling of bioactive molecules.

Full text of DOI:10.1002/anie.200460923

Angewandte
Chemie
Bioinorganic Chemistry
Vitamin B12 as a Ligand for Technetium and
Rhenium Complexes**
Susanne Kunze, Fabio Zobi, Philipp Kurz,
Bernhard Spingler, and Roger Alberto*
Vitamin B12 is a biomolecule that is fundamental for most
living organisms despite being produced by only a few
bacteria. It plays a key role in enzymatic processes in the
[*] S. Kunze, F. Zobi, P. Kurz, Dr. B. Spingler, Prof. Dr. R. Alberto
Institute of Inorganic Chemistry
University of Zürich, Winterthurerstrasse 190
8057 Zürich (Switzerland)
Fax: (+41)1-635-6803
E-mail: ariel@aci.unizh.ch
[**] This work has been supported by Solidago AG, Bern, Switzerland,
and Mallinckrodt Med. Inc., Petten, The Netherlands
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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DOI: 10.1002/anie.200460923
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Communications
mitochondria, cell nucleus, and cytoplasm. Its uptake in
Our interest lies in radiopharmaceuticals containing the
fac-[99mTc(CO)3]+ moiety.[20–22] The water ligands are readily
exchanged in [99mTc(OH2)3(CO)3]+, to yield complexes of
high kinetic stability even with monodentate donors.[23] The
concept of our study involved B12 acting as a monodentate
ligand while the other two sites of fac-[M(CO)3]+ are bound to
a bidentate ligand L2. The complex [99mTc(OH2)(L2)(CO)3] is
then coordinated to B12. The ligand L2, introduced prior to
B12 coordination, is variable and allows the biological
authenticity of the final conjugate to be fine-tuned. The use
of a bidentate and a monodentate ligand on the fac-
humans is very complex and requires at least three different
transport proteins: intrinsic factor (IF), transcobalamin, and
haptocorrin. The human organism uses vitamin B12 very
efficiently: the daily requirement is only about 10 mg. Its
chemistry, biochemistry, and biology has been comprehen-
sively reviewed.[1–4]
The demand for vitamin B12 (abbreviated as “B12”) is
concentrated at sites of enhanced proliferation, in particular
in cancer cells or at sites of bacterial infections. An organismꢀs
need for B12 makes it an attractive targeting agent. Appli-
cations of B12 to the delivery of radioisotopes[5,6] or as various
cytotoxic agents to cancer cells, for which it can be used as a
Trojan horse, have been the most actively studied areas of
research.[7–9] Both of these strategies require derivatization of
B12, and so the introduction of ligands or receptor-binding
molecules has been reported.[5,6,9,10] Chelators for radiometals
have been conjugated to peripheral acid groups (prepared by
controlled amide hydrolysis) and coordinated to the 5’-OH
group in the ribose ring of the backloop. Alternatively, a
CoIII center can be reduced to CoI and a molecule introduced
by oxidative alkylation.[8,11]
[
99mTc(CO)3]+ moiety is consistent with a mixed ligand
approach.[24]
The reaction of [M(OH2)3(CO)3]+ (M = 99Tc, 1; Re, 2) in
water with the monoanionic ligands (L2), imidazolecarboxylic
acid (Himc), picolinic acid (Hpic), 2,4-dipicolinic acid
(Hdipic) or serine (Hser), and N,N-dimethylglycine (Hdmg)
yields [M(L2)(OH2)(CO)3]. The corresponding 99mTc com-
plexes are prepared directly from [99mTcO4]ꢀ. The bidentate
ligand is tightly bound but the remaining water ligand can be
replaced by the nitrogen atom from the cyanide anion in B12.
[M(L2)(OH2)(CO)3] exhibits a strong affinity for imidazole-
type ligands but coordination to intermediately released
benzimidazole from B12 was not observed. The reaction of
[Re(imc)(OH2)(CO)3] (3), [Re(dipic)(OH2)(CO)3] (4),
[Re(ser)(OH2)(CO)3] (5), [Re(dmg)(OH2)(CO)3] (6), or
their 99mTc analogues with B12 in methanol or water afforded
The only functionality in B12 that has not received much
attention is the CoIII-coordinated cyanide group. It is well
ꢀ
established that M CN moieties tend to bridge two metal
centers to form a M-C-N-M’ unit. Numerous examples have
been published and reviewed to date,[11] but examples with
porphyrin-like systems are rare[12–14] and, to the best of our
knowledge, unknown for B12. The reverse situation in which
[Fe(CN)6]3ꢀ or nitroprusside [Fe(NO)(CN)5]ꢀ are coordi-
nated to CoIII or CoII centers of aqua–cobalamin has been
studied in detail.[15–19]
ꢀ
ꢀ
ꢀ
ꢀ
[(3 B12)] (7), [(4 B12)] (8), [(5 B12)] (9), and [(6 B12)]
(10). High-performance liquid chromatographic (HPLC)
analysis after coordination of Re or 99(m)Tc centers gave only
two well-separated signals in a ratio of about 1:1. The
comparable retention times from the UV/Vis analysis of, for
example, 7 and the corresponding radioactivity trace of the
99mTc complex indicate the identity of the Re and Tc com-
plexes (Figure 1). The signals can be understood by the two
possible orientations adopted by the bidentate N,O ligand L2
relative to the corrin ring ([M(L2)(OH2)(CO)3] complexes are
racemic) and, thus, the presence of two diastereomers. The
It is intriguing to use the cyanide anion in B12 as a ligand
and to introduce a metal complex either by direct coordina-
tion to B12 or by conjugation of an organic molecule through
mediation by a metal complex (pathways 1 and 2 in
Scheme 1). Since Co-CN-M’ is not very stable, M’ represents
an inert complex fragment.
Scheme 1. a) Structure of B12 with assignment of the side arms. b) Coordination of [99(m)Tc(OH2)(L2)(CO)3] to B12: 1) for radiopharmaceuticals
and 2) metal-mediated coupling of biomolecules (BM).
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Figure 1. HPLCtraces of the reaction between B12 and 3 showing the
two diastereomers. Lower trace: UV/Vis spectrum at l=360 nm,
upper trace: g-detection of the 99mTc analogue of 7.
two compounds could be separated by preparative HPLC and
showed the same mass (m/z = 869.1 [M2+]; m/z = 1736.9 [M+])
in ESI-MS; 1H, 13C, and 31P NMR spectroscopic analysis
confirmed the proposed composition (see Supporting Infor-
mation).
Figure 3. Hydrogen-bonding interactions in 7 and 10 (above) and ori-
The orientation of B12 relative to the bidentate ligand was
investigated by ROESY experiments, as recently performed
for a (methoxycarbonyl)methyl derivative.[25] The spectra did
not show cross peaks even at low-temperature, thus indicating
fast rotation of the complex. Since the equatorial planes of the
complexes are relatively far away from the corrin framework,
they are not locked in a fixed position. For all the complexes
7–10, the two isomers could be readily separated by prepa-
rative HPLC. X-ray quality crystals for one of the isomers of
both 7 and 10 could be grown, and so their structures could be
elucidated (Figure 2).[26]
entation of the imc and dmg ligands relative to the corrin ring (below).
C2 represents the carbon atom to which the a-side chain is attached.
that the respective diastereomer of each complex might be
represented by the orientations as observed for 7 and 10.
We observed that the diastereomerically pure complexes
[M(L2)(L’1)(CO)3] with one labile ligand (for example, l-
serine (L2) and guanine L’1) always return to equilibrium in
water within hours, probably through formation of a five-
coordinate intermediate by dissociation of the monodentate
ligand. The pure diastereomers of 7–10 do not interconvert
into each other even after days in water at room temperature,
which confirms the high kinetic stability of the monodentately
bound B12. Acetonitrile is a strong ligand for complexes
[Re(L2)(OH2)(CO)3]. Dissolving 7 or 9 in a water/acetonitrile
mixture did not result in cleavage of 3 from B12 in 7 and only
about a 10% cleavage of 9 from 10, which confirms again the
stability of the Co-CN-Re(Tc) bonds.
The electronic spectra of the two diastereomers of 10 both
have essentially the shape of B12, but the shoulder at about
482 nm in the spectrum of B12 becomes a distinct peak at
475 nm (see Suppporting Information). Furthermore, B12
Figure 2. X-ray crystal analysis of 7 (left) and 10 (right). Important
bond lengths [Š] and angles [8] are for 7: Co-CN: 1.846(13), Co-
N(benzimidazole): 2.010(8), C-N(cyanide): 1.113(14), CN-Re1A:
2.171(9), Co-C-N: 169(1), C-N-Re1A: 166(1); and for 10: Co-CN:
1.90(1), Co-N(benzimidazole): 2.032(7), C-N(cyanide): 1.180(12), CN
Re: 2.14(1), Co-C-N: 167.7(9) , C-N-Re: 169.2(9).
and 7 behave different electrochemically. Whereas B12
+
1
exhibits a reduction wave (E = = ꢀ670 mV versus Ag /
2
AgCl) with about 60% reversibility, the same process with 7
ꢀ
1
(E = = ꢀ625 mV) is about 80% reversible (Figure 4). The
2
CoIII center in 7 is easier to reduce since coordination of the
cyanide group through the nitrogen atom to the rhenium
center reduces the electron density at the CoIII center. The
increased ease of the reversibility can be understood by the
more facile accommodation of the two additional electrons in
the Co!C backbond. Complex 7 can be considered as an
(inorganic) isocyanide complex of a CoIII center. Since
isocyanides bind to low-oxidation states better than the
cyanide anion, the CoI center becomes stabilized.
The relative orientation of the N,O ligands L2 imc in 7 and
dmg in 10 are reversed for the two structures. Both show a
hydrogen bridge between the terminal carbonyl oxygen O74
in 7 and the coordinating O72 atom in 10 to the amide
nitrogen atom of the a-side chain in B12 (Figure 3). The
bridge is shorter in 7 than in 10 and probably determines the
orientation of the Re complex in the solid state. We assume
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Communications
[1] B. Kräutler, D. Arigoni, B. T. Golding, Vitamin B12 and B12-
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Figure 4. Cyclovoltamograms of complex 7 and B12. The shoulder
results from the reduction of imc in 7.
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1989, 28, 4314.
[16] H. Kunkely, A. Vogler, Z. Naturforsch. B 1996, 51, 245.
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Inorg. Chem. 1986, 25, 970.
[18] A. R. Butler, C. Glidewell, Chem. Soc. Rev. 1987, 16, 361.
[19] M. Wolak, G. Stochel, R. van Eldik, J. Am. Chem. Soc. 2003, 125,
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[20] R. Alberto, R. Schibli, A. Egli, U. Abram, T. A. Kaden, P. A.
Schubiger, J. Am. Chem. Soc. 1998, 120, 7987.
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113, 3152.
[23] N. Aebischer, R. Schibli, R. Alberto, A. E. Merbach, Angew.
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2004, 1320.
[25] M. Puchberger, R. Konrat, B. Kräutler, U. Wagner, C. Kratky,
Helv. Chim. Acta 2003, 86, 1453.
[26] Crystallographic analysis: 7 (C70H106CoN16O28PRe), red plates,
0.57 ” 0.15 ” 0.04 mm3, orthorhombic, space group P212121, a =
15.958(1), b = 21.2328(12), c = 27.9776(13) Š, V= 9479.6(9) Š3,
Z = 4, 1calcd = 1.328 gcmꢀ3, R1(I ꢁ 2s(I)) = 0.0990, wR2(F2, I ꢁ
2s(I)) = 0.2370 for 16557 independent data (10604 observed
(I ꢁ 2s(I))), 1026 parameters, 40 restraints, numerical absorption
correction, m = 1.545 mmꢀ1, Tmin = 0.5508, Tmax = 0.9331, Flack
parameter= ꢀ0.007(11). 10 (C73H112.6CoN15O27.3PRe), red nee-
dles, 0.46 ” 0.08 ” 0.07 mm3, orthorhombic, space group P212121,
Biological applications of these compounds require them
to be stabile in serum and have an affinity for the various B12-
binding proteins. It is known that haptocorrin recognizes
derivatives of B12 whereas the transcobalamins are more
sensitive towards structural changes.[27–29] Derivatizations at
the CoIII center are more readily tolerated by both proteins
and direct labeling of B12 with [99mTc(L2)(OH2)(CO)3] is
feasible. Complexes
[
99mTc(L2)(OH2)(CO)3] coordinated
within
quantitatively to B12 between 10ꢀ2 and 10ꢀ3
m
60 minutes at 378C. Once formed and separated by HPLC,
the isomers were stable for at least 24 hours at 378C. Complex
10 was treated with human serum albumin at 378C (1% in
phosphate buffer) but no transmetalation to proteins could be
observed. This opens a convenient way for studying the
biological behavior of labeled native B12 by varying the
nature of L2. The intracellular B12-dependent enzymes might
not recognize these derivatives anymore but then the radio-
nuclide has already reached its target. We emphasize that the
Re center is likely to mediate L2-coupled biologically active
molecules and B12 (for example, through the free carboxylic
acid functionality in dipic).
In conclusion, we have shown that CNꢀ in B12 bridges to
ReI and TcI centers to yield robust complexes with the central
structural feature {Co-CN-Re(Tc)}. This concept allows direct
labeling of B12 with complexes [99mTc(OH2)(L2)(CO)3] for
radiodiagnosis or with rhenium as a mediator between B12
and additional biomolecules. The observed kinetic stability
implies that coordination of other fragments with the d6 or d8
configurations is also possible. The use of B12 as an
enantiomerically pure and stereochemically demanding
ligand provides water solubility, which could be useful for
enantioselective synthesis with an appropriate catalyst. Bind-
ing studies with different B12 transporters and the coordina-
tion of other metal complexes are currently under investiga-
tion.
a = 15.8758(7),
b = 21.845(1),
c = 26.3673(14) Š,
V=
9144.4(8) Š3, Z = 4, 1calcd = 1.390 gcmꢀ3, R1(I ꢁ 2s(I)) = 0.0662,
wR2(F2, I ꢁ 2s(I)) = 0.1588 for 68628 collected data (17853
independent), 1071 parameters, 2 restraints, numerical absorp-
tion correction, m = 1.601 mmꢀ1, Tmin = 0.7060, Tmax = 0.9257,
Flack parameter= ꢀ0.014(8). CCDC-240399 (for 7) and
CCDC-240398 (for 10) contain the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from www.ccdc.cam.ac.uk/conts/retrieving.html (or from
Received: June 9, 2004
Keywords: bridging ligands · cyanides · radiochemistry ·
.
technetium · vitamins
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Chemie
the Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB21EZ, UK; fax: (+ 44)1223-336-033; or deposit@
ccdc.cam.ac.uk).
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Quadros, P. McLoughlin, A. C. Morgan, Bioconjugate Chem.
1997, 8, 161.
[29] P. M. Pathare, D. S. Wilbur, S. Heusser, E. V. Quadros, P.
McLoughlin, A. C. Morgan, Bioconjugate Chem. 1996, 7, 217.
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