A new [2 + 1] mixed ligand concept based on [99(m)Tc(OH 2)3(CO)3]+: A basic study

Article abstract of DOI:10.1039/b400220b

Mixed ligand fac-tricarbonyl complexes of the general formula [M(L ¹)(L²)(CO)₃] (M = Re, ^99(m)Tc, L¹ = imidazole, benzyl isocyanide, L² = 1H-imidazole-4-carboxylic acid, pyridine-2,4-dicarboxylic acid, pyridine-2,5-dicarboxylic acid) have been prepared starting from the precursors [M(OH₂₎₃(CO)₃]⁺. The complexes can be obtained in good yield and purity in a two-step procedure by first attaching the bidentate ligand followed by addition of the monodentate. ^99mTc compounds can also be prepared at the tracer level in one-pot procedures with L¹ and L² being concomitantly present. This [2 + 1] approach allows the labeling of bioactive molecules containing a monodentate or a bidentate donor site. Examples given in here are N-(tert-butoxycarbonyl) glycyl-N-(3-(imidazol-1-yl)propyl)phenylalaninamide, 5-((3-(imidazol-1-yl) propyl)aminomethyl)-2′-deoxyuridine and 4-(5-isonitrilpentyl)-1-(2- methoxyphenyl)-piperazine as L¹ and N-((6-carboxypyridine-3-yl) methyl)glycylphenyl-alanine as L². The corresponding second ligand can be used to influence the physico-chemical properties of the conjugate. The crystal structures of [⁹⁹Tc(OH₂)(imc)(CO)₃], [Re(OH₂)(2,4-dipic)(CO)₃], [Re(bic)(2,4-dipic)(CO) ₃] and [Re(im)(2,5-dipic)(CO)₃] are reported.

Full text of DOI:10.1039/b400220b

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A new [2 ؉ 1] mixed ligand concept based on
99(m)Tc(OH2)3(CO)3]؉: a basic study
[
Stefan Mundwiler, Monika Kündig, Kirstin Ortner and Roger Alberto*
Institute of Inorganic Chemistry, University of Zürich, 8057 Zürich, Switzerland.
E-mail: ariel@aci.unizh.ch; Fax: ϩ 41 1 635 68 03; Tel: ϩ 41 1 635 46 31
Received 7th January 2004, Accepted 22nd March 2004
First published as an Advance Article on the web 2nd April 2004
Mixed ligand fac-tricarbonyl complexes of the general formula [M(L1)(L2)(CO)3] (M = Re, 99(m)Tc, L1 = imidazole,
benzyl isocyanide, L2 = 1H-imidazole-4-carboxylic acid, pyridine-2,4-dicarboxylic acid, pyridine-2,5-dicarboxylic
acid) have been prepared starting from the precursors [M(OH2)3(CO)3]ϩ. The complexes can be obtained in good
yield and purity in a two-step procedure by first attaching the bidentate ligand followed by addition of the
monodentate. 99mTc compounds can also be prepared at the tracer level in one-pot procedures with L1 and L2
being concomitantly present. This [2 ϩ 1] approach allows the labeling of bioactive molecules containing a
monodentate or a bidentate donor site. Examples given in here are N-(tert-butoxycarbonyl)glycyl-N-
(3-(imidazol-1-yl)propyl)phenylalaninamide, 5-((3-(imidazol-1-yl)propyl)aminomethyl)-2Ј-deoxyuridine and
4-(5-isonitrilpentyl)-1-(2-methoxyphenyl)-piperazine as L1 and N-((6-carboxypyridine-3-yl)methyl)glycylphenyl-
alanine as L2. The corresponding second ligand can be used to influence the physico-chemical properties of the
conjugate. The crystal structures of [99Tc(OH2)(imc)(CO)3], [Re(OH2)(2,4-dipic)(CO)3], [Re(bic)(2,4-dipic)(CO)3]
and [Re(im)(2,5-dipic)(CO)3] are reported.
accumulation in targets and organs clearly emphasizing the
Introduction
need for rational complex design by tuning the chelators to
The radionuclide 99mTc is one of the most important for nuclear
medicine due to its availability from generators, favorable decay
characteristics and cost which becomes a more and more
important issue in clinical application.1,2 Since Tc is a transition
metal, its conjugation with a targeting biomolecule is demand-
ing. Technetium has to be stabilized by chelators containing
functionalities with particular and often dominant physico-
chemical features. The resulting biomolecules vary to a substan-
tial degree e.g. in size, dipole moment and solubility from the
native biomolecule of interest.
achieve optimal in vivo behavior.15–17
We reported about the synthesis of [M(OH2)3(CO)3]ϩ (M =
99Tc, 1; 99mTc, 2; Re 3) directly from water.18,19 One of the most
intriguing features of the fac-[M(CO)3]ϩ core is its d6 low spin
configuration based high kinetic stability with many types of
ligands. Although the thermodynamic stability constants of
monodentate ligands bound to the fac-[M(CO)3]ϩ moiety are
not likely to exceed 104 MϪ1, such complexes are very robust in
aqueous solution.
We want to present herein a basic study for the introduction
of a new mixed ligand concept based on the [99mTc(CO)3]ϩ core
in which the three water ligands in [M(OH2)3(CO)3]ϩ are substi-
tuted by one bidentate and one monodentate ligand. As biden-
tate ligands we have chosen 4-imidazolecarboxylic acid (imc),
2,4-pyridinedicarboxylic acid (2,4-dipic) and 2,5-pyridinedi-
carboxylic acid (2,5-dipic), respectively. Monodentate ligands
are imidazole (im) and benzyl isocyanide (bic) as well as deriv-
atives thereof (Scheme 2). A biomolecule can be linked either to
the monodentate ([2 ϩ 1B] concept) or to the bidentate ligand
([2B ϩ 1] concept). Since [M(OH2)3(CO)3]ϩ accepts many types
of ligands it is possible to design complexes whose properties
such as hydro/lipophilicity are adapted to those of the bio-
molecule. We present in this study the synthesis and character-
ization of some mixed ligand rhenium and technetium com-
plexes based on the model ligands and on some derivatized
biologically active molecules. The new mixed ligand approach
presented herein is shown in Scheme 1.
Ligands providing sufficient stability to a 99mTc core under
physiological conditions are limited. Basic frameworks are e.g.
tetradentate N,S,O chelators for the [Tc()᎐O]3ϩ core.3
᎐
Although powerful, tuning the resulting complexes properties
by varying the ligands is not routine. Keeping the donors
constant requires the introduction of substituents at the
C-backbone which frequently gives rise to the formation of
stereoisomers. Mixed ligand approaches enable smooth tuning
of the ligand properties and, thus, screening of their biological
behavior. The best known mixed ligand approach is [3 ϩ 1] by
Pietzsch and co-workers.4 The tridentate ligand can link to
the biomolecule and is kept constant while the monodentate
coligand is varied continuously or vice versa.5 Clear structure–
activity-relationships are feasible. Recently, it was reported that
[3 ϩ 1] complexes suffer sometimes from trans-metallation by
ligand exchange reactions in biological media which limits their
application.6,7 Other mixed ligand systems have been proposed
but are less convenient.8 Lipophilic Tc() complexes with
umbrella-like tetradentate NSSS ligands and monodentate iso-
cyanides showed excellent stability in vitro towards plasma
components.9–11 The hynic approach can also be considered as a
kind of mixed ligand approach. Biomolecules are linked to
6-hydrazinonicotinamide which acts most likely as a mono-
dentate ligand. The remaining coordination sites at the Tc core
are then occupied by co-ligands. Comparing the different label-
ing techniques for e.g. peptides, it has been stated that a major
advantage of the hynic approach is the variability of the co-
ligands over quite a broad range according to the chemical
nature of the metal center.12,13 It could be shown that the co-
ligand has a decisive influence on the biological properties
of the peptide.12,14,15 Altering the coligand entailed different
Results and discussion
The bidentate ligands studied in this work and a typical reac-
tion sequence are depicted in Scheme 2. For the mixed ligand
[2 ϩ 1B] concept the bidentate coligand represents the vari-
able portion of any radiolabeled biomolecule. It allows to tune
physico-chemical properties and has to fulfil two requirements.
First, it should be anionic in order to leave one labile H2O at the
last coordination site (the later binding site of the monodentate
ligand and the biomolecule). Complexes with neutral bidentate
ligands contain in general a ClϪ/BrϪ at this position which is for
electrostatic reasons more difficult to substitute. Secondly, steric
D a l t o n T r a n s . , 2 0 0 4 , 1 3 2 0 – 1 3 2 8
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
1320

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of a second ligand L2 via monodentate coordination of the
aromatic amino group to the remaining coordination site as the
non-coordinating carboxylic group of the second L2-unit would
sterically interfere with the ligands in cis-position. We never
observed even traces of a hypothetical complex [Re(η2-L2)-
(η1-L2)(CO)3] throughout this study.
To gain experimental evidence for the basic structures and for
HPLC comparison with 99mTc, complexes of general com-
position [Re(OH2)(L2)(CO)3] have been synthesized in water by
mixing 3 with the corresponding bidentate ligands imc, 2,4-
dipic and 2,5-dipic, respectively. The complexes 4, 5 and 6 were
formed in good to excellent yields in short time. In analogy,
[99Tc(OH2)(imc)(CO)3] 4b was prepared from 1. Even a large
excess of L2 did not allow further substitution and one and only
one ligand was incorporated in the complexes. To confirm the
presence of a remaining H2O ligand, crystals were grown by
slow vapor diffusion procedure and the X-ray structures elucid-
ated for the Tc analogue of 4 and for 5. ORTEP presentations
of the two complexes are given in Figs. 1 and 2, respectively. The
crystal data and refinement parameters of compounds 4b, 5, 10
and 11 are summarized in Table 1. Both complexes are rare
examples of organometallic complexes comprising a water
ligand. The distances to the H2O ligands are for both complexes
about 2.2 Å. This is significantly longer than other Re–O bond
lengths in this work and in literature indicating a relatively weak
interaction and explaining their versatile substitution with
other ligands.20 Mono-anionic ligands such as ClϪ or BrϪ have
no affinity for these neutral complexes and do not coordinate
even in the presence of a large excess of halides. This clearly
contrasts the behavior of complexes with neutral bidentate
ligands in which coordination of a halide is a thermodynamic-
ally favored process, resulting in an overall neutral complex.
The electron donating nitrogen atom and the carboxylate push
enough electron density to the metal center to decrease the
affinity for additional negatively charged (π-)donors.
Scheme 1 Mixed ligand concept illustrating the [2B ϩ 1] (left part) or
the [2 ϩ 1B] (right part) pathway.
Fig. 1 ORTEP of 4b. Important bond lengths (Å): Tc–C(5) 1.920(3),
Tc–C(6) 1.911(3), Tc–C(7) 1.915(3), Tc–N(1) 2.154(2), Tc–O(3)
2.200(2), Tc–O(1) 2.216(2). Ellipsoids are drawn at 50% probability.
Scheme
2
Formation of [M(OH2)(L2)(CO)3] complexes, typical
reaction sequence and the bidentate ligands used.
reasons have to make sure that only one bidentate ligand can
coordinate. Many bidentate ligands meet these criteria and we
have chosen for a first study of bidentate ligands based on one
aromatic heterocycle, either pyridine or imidazole and a carb-
oxylic acid as second and charge neutralizing coordinating
functionality. The ligands imc, 2,4-dipic and 2,5-dipic are vers-
atile and commercially available. The basic framework of the
corresponding complexes is small and allows the variation of
lipo-/hydrophilicity in a convenient way. The carboxylic acid in
ortho-position to the aromatic N-donor prevents coordination
Fig. 2 ORTEP of 5. Important bond lengths (Å): Re–C(8) 1.931(7),
Re–C(9) 1.930(7), Re–C(10) 1.924(7), Re–N(1) 2.186(5), Re–O(1)
2.163(4), Re–O(5) 2.198(5). Ellipsoids are drawn at 50% probability.
In the [2 ϩ 1B] concept, complexes of the general com-
position [99mTc(OH2)(L2)(CO)3] can be considered as new pre-
cursors for the labeling of biomolecules through a monodentate
anchor group (Table 2). Complexes 4a, 5a and 6a, the 99mTc-
analogs of 4, 5 and 6, are examples of such precursors and
their synthesis was achieved along two different routes. In the
D a l t o n T r a n s . , 2 0 0 4 , 1 3 2 0 – 1 3 2 8
1321

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Table 1 Crystal data for complexes 4b, 5, 10 and 11
4b
5
10
11
Formula
Mr
C7H5N2O6Tc
311.13
C10H10NO10Re
490.39
C18H11N2O7Re
553.49
C13H8N3O7Re
504.42
T /K
Space group
183(2)
P21/n
183(2)
P21/c
183(2)
C2/c
183(2)
P1
¯
a/Å
b/Å
c/Å
α/Њ
11.7875(9)
7.3430(6)
12.8952(10)
90
114.894(8)
90
1012.45(14)
4
2.041
10.3056(7)
23.3953(10)
24.7252(16)
90
98.380(8)
90
5897.7(6)
16
2.209
20.232(11)
13.240(5)
14.296(6)
90
102.13(6)
90
3744(3)
8
1.964
7.5005(8)
8.6008(9)
12.6462(13)
95.120(12)
94.378(12)
106.362(11)
775.24(14)
2
β/Њ
γ/Њ
V/Å3
Z
Dc/g cmϪ3
2.161
7.879
476
µ(Mo-Kα)/mmϪ1
F(000)
1.437
608
8.292
3712
6.534
2112
Crystal size/mm
Crystal description
θ Range/Њ
0.40 × 0.35 × 0.25
Colorless block
3.37–25.90
1817
1817
1560
0.7153/0.5972
1817/0/165
0.989
0.0253
0.0639
0.24 × 0.20 × 0.07
Yellow plate
2.41–28.02
45230
14181
9218
0.5945/0.2409
14181/0/835
0.847
0.0326
0.0722
0.30 × 0.14 × 0.02
Yellow plate
2.49–28.03
18625
4500
3194
0.8804/0.2446
4500/59/280
0.908
0.0378
0.0866
0.16 × 0.08 × 0.05
Yellow column
2.81–30.39
11003
4253
2852
0.6941/0.3654
4253/0/221
0.821
0.0345
0.0640
Total no. of data
No. of unique data
Observed dataa
Max./min. transmission
Data/restraints/parameters
Goodness of-fit on F 2
Rab
wR2 ac
Max, min peaks/e ÅϪ3
1.068, Ϫ0.574
2.245, Ϫ1.008
2.689, Ϫ0.754
1.556, Ϫ1.287
2
a Observation criterion: I > 2σ(I ). b R = Σ||Fo| Ϫ |Fc||/Σ|Fo|. c wR2 = {Σ[w(Fo2 Ϫ Fc2)2]/Σ[w(Fo )2]}1/2
.
Table
2
Yields and reaction conditions for the conversion of
[
99mTc(OH2)3(CO)3]ϩ to [99mTc(OH2)(L2)(CO)3]
L2
Complex
t/min
T /ЊC
L2/mM
Yield (%)
imc
imc
4a
4a
30
45
90
90
0.1
0.1
87
98
2,4-dipic
2,4-dipic
5a
5a
30
45
90
90
0.1
0.1
98
>99
2,5-dipic
2,5-dipic
20
6a
6a
21a
30
45
30
90
90
90
0.1
0.1
0.01
98
>99
93
consecutive approach as depicted in Scheme 3 routes (i) and
(ii), 2 is prepared first and then reacted with the bidentate
ligand. The yields are very good and at concentration of 10Ϫ4 M
in 2,4-dipic or 2,5-dipic the corresponding 99mTc complexes
[
99mTc(OH2)(L2)(CO)3] (5a, 6a) are formed in >95% yield after
30 min at 90 ЊC. Concentrations of L2 can be as low as 10Ϫ5 M
and the reaction with e.g. 20 (see Scheme 2) gave 93% yield.
The rate increases at higher concentrations without later affect-
ing the coordination of the monodentate ligand. In the con-
certed approach (Scheme 3, pathway (iii), the bidentate ligand
can be added with [99mTcO4]Ϫ of the generator eluate to the vial
containing the carbonylation kit. The ligands used in this study
are not reduced in the kit and at 10Ϫ4 M ligand concentration
and 30 min at 90 ЊC afforded the complexes [99mTc(OH2)(L2)-
(CO)3] in one single step in >98% yield. The identity of all
99mTc complexes was confirmed by comparative HPLC with the
Re complexes. Other ligands can be used provided they are
stable under reductive conditions. Thus, beside the common
precursor 2, many novel cores can be generated with the poten-
tial of systematic variation of the last coordinatively “active”
position.
Scheme 3 Pathways to mixed ligand complexes: (i) preparation of 2,
(ii) [2 ϩ 1B] concept, coordination of L2 and then L1 in separate steps;
(iii) one-pot synthesis of the [L2 ϩ H2O] complex from [TcO4]Ϫ,
(iv) one-pot synthesis of the [2 ϩ 1B] complex from 2, or (v) one-pot
synthesis of the [2 ϩ 1B] complex directly from [TcO4]Ϫ.
monodentate ligands, we focused on imidazole derivatives and
isocyanides. Substitution rates and required concentrations for
quantitative complex formation with the free ligand and the
derivatized biomolecules allows an estimation of the potential
of monodentate ligands. With respect to coordinating proper-
ties and orbital participation, imidazole and isocyanide repre-
sent two “extremes” since the former is essentially a σ-donor
whereas the latter has significant π-acceptor properties. Both
ligand groups have a practical significance since they are
easily introduced in biomolecules, isocyanide via dehydration
of formamides and imidazole e.g. by commercially available
3-aminopropyl-N-imidazole. The monodentate ligands used
inhere are shown in Scheme 4.
Substitution of the H2O ligand by a monodentate ligand
represents the labeling step in the [2 ϩ 1B] concept. Preferen-
tially, the monodentate ligand is bound to the biomolecule due
to the more facile synthesis of such derivatives. Following the
arguments given above for electronic properties of potent
D a l t o n T r a n s . , 2 0 0 4 , 1 3 2 0 – 1 3 2 8
1322

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Fig. 4 ORTEP of [Re(im)(2,5-dipic)(CO)3] (11). Important bond
lengths (Å): Re–C(11) 1.927(7), Re–C(12) 1.918(9), Re–C(13) 1.885(8),
Re–N(1) 2.186(5), Re–N(2) 2.196(5), Re–O(1) 2.185(4). Ellipsoids are
drawn at 50% probability.
complex 4b 3.9 × 10Ϫ3 MϪ1 sϪ1. We found that the reaction with
bic was significantly faster with all precursors [Re(OH2)-
(L2)(CO)3]. The same tendency was observed with 99mTc at the
tracer level. Reaction of a 1 : 1 mixture of bic and im (each
0.05 mM) with [99mTc(OH2)(2,4-dipic)(CO)3] at 50 ЊC for 1 h
gave a mixture of 62% of [99mTc(bic)(2,4-dipic)(CO)3] and
26% of [99mTc(im)(2,4-dipic)(CO)3] with 10% of remaining
[
99mTc(OH2)(2,4-dipic)(CO)3]. The ratio of the rate constants
bic/im is therefore about 2.5–3. We emphasize that the reactions
went to completion at elevated temperature and extended reac-
tion time. Under comparable conditions π-acceptors are better
monodentate ligands than σ-donors. Since H2O is a very weak
σ-donor its easy replacement is further rationalized by these
observations. IR spectroscopy reveals an unusually high CO
stretching frequency at 2039 cmϪ1 for neutral complexes as in
e.g. 4.21 Steric interactions between the coordinated imidazole
and the ligands in cis-position may play an important role as
well. As evident from the X-ray structure analysis, minimization
of sterical interactions is achieved by rotation of imidazole by
about 45Њ with respect to the equatorial CO ligands and the
bidentate chelator. This situation is not optimal from an elec-
tronic point of view and leads to reduced rate of formation.
The isocyanide does not encounter such a steric interaction due
to its linear arrangement and binds rapidly with maximum
overlap explaining its high efficiency and rate of coordination.
Besides model monodentate ligands, an imidazole group
was attached to the dipeptide H-Gly-Phe-OH which allowed
the study of the influence of additional groups on complex
formation rate. Commercially available 1H-(3-aminopropyl)-
imidazole (api) was coupled to the BOC-protected dipeptide
Boc-Gly-Phe-OH via amide bond formation at the C-terminus
affording compound 13. The same approach can be used for
imidazole derivatization at the N-terminus by starting from
the corresponding carboxylic acid derivative. The complexes
[Re(Boc-Gly-Phe-api)(imc)(CO)3] 14 and [Re(Boc-Gly-Phe-
api)(2,5-dipic)(CO)3] 15 were synthesized in methanol and
water from 4 and 6 in the presence of 13 at elevated temperature
and good yields of 84 and 90%, respectively. One single product
was found for all reactions and amides did not interfere with the
mixed ligand complex formation.
Scheme 4 Monodentate ligands for the [2 ϩ 1B] concept.
Imidazole (im) and benzyl isocyanide (bic) reacted at elevated
temperature with the complexes 4–6 in methanol and in water
to yield the corresponding complexes [Re(L1)(L2)(CO)3] with a
mixed [2 ϩ 1] coordination sphere. The complexes [Re(bic)(2,4-
dipic)(CO)3] 10 and [Re(im)(2,5-dipic)(CO)3] 11 were prepared
in quantitative yield along this pathway. X-Ray crystal structure
analysis confirmed the formulation of these complexes. ORTEP
presentations are given in Figs. 3 and 4.
Fig. 3 ORTEP of [Re(bic)(2,4-dipic)(CO)3] (10). Important bond
lengths (Å): Re–C(8) 2.113(6), Re–C(29) 1.957(7), Re–C(27) 1.948(6),
Re–C(28) 1.912(8), Re–N(1) 2.189(4), Re–O(1) 2.159(5). Ellipsoids are
drawn at 50% probability. Only one configuration of the phenyl ring is
shown.
The reactions at the macroscopic level with rhenium served
as a base for 99mTc. Conditions and yields of mixed ligand 99mTc
complex formation are summarized in Table 3. The concen-
tration of 99mTc depends on the generator condition and is in
the 10Ϫ7 M range. Extrapolation of the kinetic data received in
the 99Tc experiments gives an estimate for the observed product
formation. The reaction of [99mTc(OH2)(imc)(CO)3] with 1 mM
imidazole gave about 60% of the corresponding complex after
60 min at 80 ЊC (entry 1 in Table 3). With k1 = 3.9 × 10Ϫ3 MϪ1 sϪ1
as determined at 20 ЊC for imidazole and 4b (see earlier) and
the rate-temperature rule (double rate for 10 ЊC increase in
The rate of formation as a function of the entering mono-
and the present bidentate ligand has been investigated. A solu-
tion containing the corresponding [Re(OH2)(L2)(CO)3] and a
monodentate ligand was stirred at r.t. and the amount of prod-
uct formation was analyzed by HPLC under pseudo-first order
conditions. Assuming a pre-equilibrium mechanism, the rate
constant k1 of H2O replacement can then be estimated. The rate
constant k1 for the second order reaction of 4 with imidazole
was about 1.1 × 10Ϫ3 MϪ1 sϪ1 and for the reaction with the 99Tc
D a l t o n T r a n s . , 2 0 0 4 , 1 3 2 0 – 1 3 2 8
1323

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Table 3 Conditions and yields for the labelling with 99mTc following the [2 ϩ 1] concept. (Missing numbers in the Product column means that the
complexes have only be prepared with 99mTc)
Entry
L2
L1
Product
t/min
T /ЊC
L1/mM
Yield (%)
1
2
3
4
5
6
7
8
imc
imc
imc
imc
imc
imc
imc
imc
im
im
13
7a
7a
60
30
30
30
30
30
60
30
80
50
50
50
80
80
80
50
1
1
1
1
0.1
0.1
0.1
1
62
51
71
64
65
88
87
83
14a
16
18
19a
8a
8a
8a
bic
bic
bic
9
10
11
12
13
14
15
16
2,4-dipic
2,4-dipic
2,4-dipic
2,4-dipic
2,4-dipic
2,4-dipic
2,4-dipic
2,4-dipic
im
13
9a
30
30
60
30
30
30
60
30
50
50
80
50
50
60
80
80
1
1
0.1
1
1
0.1
0.1
0.1
61
73
62
67
95
81
88
96
13
16
bic
bic
bic
22
10a
10a
10a
17
18
19
20
21
2,5-dipic
2,5-dipic
2,5-dipic
2,5-dipic
2,5-dipic
im
13
bic
bic
bic
11a
15a
12a
12a
12a
30
30
30
30
60
50
50
50
80
80
1
1
1
0.1
0.1
60
63
93
85
89
22
20
im
30
90
1
74
temperature) a half life time for the reaction at 80 ЊC of about
2800 s or 75% yield after 3600 s can be estimated. This is in the
same order as observed. The reaction with bic afforded between
80 and 90% product under these conditions. Reducing the con-
centration to 100 µM retained this yield. Increasing temper-
ature or reaction time for both monodentate ligands completed
the reaction.
The stability of the complexes [99mTc(L1)(L2)(CO)3] was
tested by challenge reactions with the very potent tridentate
ligands cysteine and histidine. All of the compounds with bic as
monodentate ligand turned out to be completely stable at 37 ЊC
within 24 h even at a high excess of the amino acids (10Ϫ2 M,
L2 = 10Ϫ4 M, L1 = 10Ϫ3 M). The complexes with imidazole as L1
exhibited ligand exchange under these conditions but showed
reasonable stability at 1 mM amino acid concentrations (about
10% ligand exchange after 6 h at 37 ЊC).
The reaction of the derivatized dipeptide 13 with 4a–6a
(
99mTc) gave [99mTc(Boc-Gly-Phe-api)(L2)(CO)3] at 50 ЊC after
30 min in good yields (entries 3, 10, 11 and 18 in Table 3). A
typical HPLC trace is given in Fig. 5. The yields are significantly
better than with free imidazole which is probably due to a better
interaction between 13 and the precursors or the higher stability
of the encounter complex. Rising the reaction temperature to
80 ЊC caused some Boc deprotection but showed only the two
products with and without Boc protecting group. The nature of
[
99mTc(H–Gly–Phe–api)(L2)(CO)3] was proven by the direct
reaction of [99mTc(OH2)(2,4-dipic)(CO)3] with 16 affording one
single product. The reaction with 16 exemplifies the potential of
the [2 ϩ 1B] principle for peptides.
Apart from the model studies with bic, the isocyanide group
was introduced in the serotonergic receptor ligand WAY
to yield 4-(5-isonitrilepentyl)-1-(2-methoxyphenyl)piperazine
(WAY-NC, 22).22,23 The reaction of the precursor 5a with 22
gave the mixed ligand complex [99mTc(2,4-dipic)(22)(CO)3] at
low concentration in almost quantitative yield (entry 16). Even
under strong conditions, only one species is formed which is
stable under the synthetic conditions. This is an advantage over
e.g. the [3 ϩ 1] concept, where separation from other mixed
ligand species is required. The [2 ϩ 1B] approach is straight
forward and derivatization of a biomolecule with a mono-
dentate ligand is in general convenient. Imidazole is already
present in the side chain of histidine, thus, this functionality
can be used for a direct [2 ϩ 1B] labeling. This behavior could
Fig. 5 Typical HPLC traces for the labeling of peptides following the
mixed ligand concept: (a) synthesis of [99mTc(OH2)(2,4-dipic)(CO)3]
(5a); (b) 5a ϩ 13
min: [TcO4]Ϫ, 19.40 (a) and 20.82 min (b): [99mTc(OH2)(2,4-dipic)(CO)3],
22.07 min:
99mTc(H-Gly-Phe-Api)(2,4-dipic)(CO)3], 24.28 min:
99mTc(Boc-Gly-Phe-Api)(2,4-dipic)(CO)3]. The identity of the peaks at
[
99mTc(Boc-Gly-Phe-api)(2,4-dipic)(CO)3]. 3.72
[
[
19.40 and 20.82 min was confirmed by an injection of a mixture of the
samples.
however also lead to the formation of isomers in peptides with
more than one histidine residue or affecting the receptor affinity
of the labeled peptide. For such cases, the introduction of a
bidentate ligand or a more powerful isocyanide ligand into
the biomolecule is preferred.
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We further extended the [2 ϩ 1B] approach to thymidine, a
more complex bioactive molecule. Conjugation to an imidazole
group is shown in Scheme 4. The acetyl protected thymidine
derivative 17 was synthesized from 5-(bromomethyl)-2Ј-deoxy-
3Ј,5Ј-di-O-acetyluridine.24 Deprotection gave 18 which readily
formed the mixed ligand rhenium complex [Re(18)(imc)(CO)3]
(19) in quantitative yield upon reaction with 4. No side prod-
ucts were observed, the further donor groups of thymidine did
not interact with the Re core. The reaction of 4a with 18 gave
19a in good yield (Table 3, entry 5). Obviously, imidazole
coupled to a larger molecule reacts more readily than free imid-
azole as is obvious from comparison with the corresponding
data (Table 3, entries 1 and 2). This observation is equally true
for the dipeptidyl derivative 16 and for the discussed thymidine
derivative 18.
As an alternative to the mixed ligand [2 ϩ 1B] concept, the
biomolecule can be conjugated to a bidentate ligand as outlined
in Scheme 1. This introduces the mixed ligand [2B ϩ 1]
approach. With this approach, higher specific activities can be
expected since bidentate ligands (and thus the corresponding
bioconjugates) are more powerful and required concentrations
are usually less than for monodentate ligands. The mono-
dentate ligand on the other hand can be present in high concen-
trations since it does not interfere with receptor binding. In the
[2B ϩ 1] method, the variable parameter is the monodentate
ligand. The bidentate ligand 5-bromomethyl-2-carboxy-
pyridine25 represents an attractive starting material for this
approach. Its direct reaction with the unprotected dipeptide
H-Gly-Phe-OH gave the bioconjugate N-((6-carboxypyridine-
3-yl)methyl)glycylphenylalanine 20 which contains picolinic
acid as a bidentate ligand at the N-terminus. The reaction of
20 with 3 in methanol afforded the corresponding complex
[Re(OH2)(20)(CO)3] (21) in quantitative yields within 4 h. The
reaction of 2 with 20 (10Ϫ4–10Ϫ5 M) gave 93% yield after 30 min
at 90 ЊC. To complete the mixed ligand coordination sphere, the
further reaction with imidazole was then comparable to any of
the model ligands discussed above. At 90 ЊC and 10Ϫ3 M con-
centration, 74% was converted to the final product (Table 3,
entry 22) and after 1 h the reaction went to completion. Higher
imidazole concentrations enhance the reaction rate. In agree-
ment with the mixed ligand concept, the H2O ligand can be
replaced by different monodentate ligands. which allows fine
tuning of the bioconjugate.
Ideally the synthesis of mixed ligand complexes is performed
directly in the presence of both the bidentate and the mono-
dentate ligand (the latter biomolecule in the [2 ϩ 1B] approach)
as depicted in Scheme 3 pathway (iv). To illustrate this possibil-
ity we added a 10/1 mixture of monodentate 13 and 2,4-dipic
to a freshly prepared solution of [99mTc(H2O)3(CO)3]ϩ to verify
the one step [2 ϩ 1B] concept. The complex [99mTc(13)(2,4-
dipic)(CO)3] formed in quantitative yield (Scheme 5). The only
difference between the consecutive and the one-pot method was
that a higher amount of Boc deprotection of 13 occurred in the
later one. Assessment of the [2B ϩ 1] concept was performed by
one-pot reaction of 2 with 20 and imidazole. Again, the com-
plex [99mTc(im)(20)(CO)3] formed quantitatively after 30 min at
90 ЊC. HPLC analysis exhibited no byproducts in both cases
and no complexes containing e.g. two monodentate ligands
were observed in particular. Obviously, the bidentate ligand
can not be replaced anymore and the reaction leads with high
selectivity to the [2B ϩ 1] mixed ligand complexes.
Scheme 5 One-pot preparation of [99mTc(13)(2,4-dipic)(CO)3] from
99mTc(OH2)3(CO)3] (2), 13 and 2,4-dipic.
[
plexes of general composition [99mTc(L1)(L2)(CO)3] can be
prepared in one single step.
Conclusion
In conclusion, we introduced with this study a new mixed
ligand [2 ϩ 1] approach as an alternative to the common [3 ϩ 1]
and [4 ϩ 1] concepts. It is more convenient since all the steps
can be performed in water and the mixed [2 ϩ 1] ligand com-
plexes are in general of high kinetic stability. This extends the
range of possible co-ligands significantly and therefore enables
a fine tuning of physico-chemical properties of labeled bio-
molecules. The complexes [M(OH2)(L2)(CO)3] 4–6 with the
bidentate ligands based on imidazole and pyridine carboxylic
acids had different properties as evident from the HPLC reten-
tion times. The remaining water ligand could then be replaced
by a coordinating group from a biomolecule. Binding of
the precursor to the biomolecule consists simply in the co-
ordination of a monodentate ligand. Imidazole and isocyanide
have been shown to be very efficient as monodentate ligands. In
analogy to 6-hydrazinenicotinic acid (hynic) imidazole and iso-
cyanides can conveniently be attached to both the N- or to
the C-terminus of a peptide. The concept can be extended to the
[2B ϩ 1] concept in which the biomolecule is coupled to the
bidentate ligand and variation of the properties of the final
complex is achieved by changing the monodentate ligand. Both
concepts have been verified on the macroscopic level with
rhenium complexes as well as on the microscopic level with
99mTc. Systematic studies with receptor binding biomolecules
are currently on the way.
Experimental
Materials and methods
Reagent grade chemicals from Merck, Aldrich and Fluka,
Switzerland were used without further purification. The pre-
cursors 1, 2 and 3 were synthesized according to previously
published methods.18,20 4-(5-Isonitrilpentyl)-1-(2-methoxy-
phenyl)piperazine (22) was a gift from H.-J. Pietzsch, For-
schungszentrumRossendorf,Dresden,Germany.HPLCanalyses
were performed on a Merck-Hitachi L-7000 system equipped
with a EG&G Berthold LB 508 radiometric detector, using a
Macherey-Nagel C-18ec reversed-phase column (5 µm particle
size, 100 Å pore size, 250 × 3 mm) for separation. HPLC sol-
vents consisted of 0.1% trifluoroacetic acid in water (solvent A)
and methanol (solvent B). HPLC gradient: 0–3 min 100% of
solvent A; 3.1–9 min: 75% A and 25% B. At 9 min switch to
The possibility of preparing the mixed ligand complexes
in one step is useful for potential applications. Biomolecules
derivatized with a monodentate ligand can systematically be
screened for their biological behavior as a function of the
bidentate co-ligand. Furthermore, preliminary experiments
showed the possibility of performing the complete synthesis of
[
99mTc(im)(2,4-dipic)(CO)3] in one step directly from [99mTcO4]Ϫ,
imidazole and 2,4-dipic. Provided that the biomolecule is stable
under reductive conditions (and e.g. peptides usually are), com-
D a l t o n T r a n s . , 2 0 0 4 , 1 3 2 0 – 1 3 2 8
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66% A and 34% B, followed by a linear gradient to 100% B from
9 to 20 min; 20–22 min: 100% B then switching back to 100% A.
The flow rate was 0.5 ml minϪ1. Semipreparative HPLC separ-
ations were performed on an LDC Analytical system equipped
with a Consta Matric 4100 pump and a Spectro Monitor 4100
UV-detector, measuring at 250 nm, using a Merck LiChro-
Spher C-18 reversed-phase column (5 µm particle size, 100 Å
pore size, 250 × 10 mm). IR spectra were recorded on a Bio-Rad
FTS-45 spectrometer with the samples in compressed KBr-
pellets. Electrospray ionisation mass spectra (ESI-MS) were
recorded on a Merck Hitachi M-8000 spectrometer, reported
are the values of the 187Re isotope. NMR spectra were recorded
on Varian Gemini 300 MHz and Bruker DRX 500 MHz
spectrometers. The chemical shifts are reported relative to
residual solvent protons as a reference.
285 mg of 6 (96%). 1H NMR (dmso-d6): δ 9.09 (s, 1H, pyridine),
8.68 (dd, 1H, pyridine), 8.21 (d, 1H, pyridine); 13C NMR
(methanol-d4): δ 197.8, 197.5, 194.4, 174.5, 165.6, 154.5, 153.8,
143.2, 133.2, 128.4; ESI-MS: m/z 438 [M Ϫ H2O]ϩ; IR νC᎐O(st)/
cmϪ1: 2044, 1899. Anal. found: C, 26.43; H, 1.70; N,᎐3.34.
C10H6NO8Re requires C, 26.45; H, 1.33; N, 3.07%.
[Re(im)(imc)(CO)3)] (7). A solution of 4 (45.1 mg, 0.113
mmol) and imidazole (7.67 mg, 0.113 mmol) in MeOH (5 ml)
was stirred at 50 ЊC for 5 h. HPLC analysis showed complete
product formation. The solvent was removed in vacuo to give
the crystalline product 7 in a yield of 50.6 mg (100%). 1H NMR
(dmso-d6): δ 8.38 (s, 1H, imc or im), 7.80 (s, 1H, imc or im), 7.58
(s, 1H, imc or im), 7.18 (s, 1H, imc or im), 6.77 (s, 1H, imc or
im). 13C NMR (dmso-d6): δ 198.3, 197.3, 196.5, 169.3, 138.2,
138.0, 134.9, 127.9, 119.7, 118.1; ESI-MS: m/z 921 [2M Ϫ 2 ϩ
Suitable crystals were covered with Paratone N oil, mounted
on top of a glass fibre and immediately transferred to a Stoe
IPDS diffractometer. Data was collected at 183(2) K using
graphite-monochromated Mo radiation (0.71073 Å). Data was
corrected for Lorentz and polarisation effects as well as for
absorption. Structures were solved with direct methods using
SHELXS-9726 or SIR9727 and were refined by full-matrix least-
Na]ϩ, 472 [M Ϫ 1 ϩ Na]ϩ, 450 [M]ϩ; IR νC᎐O(st)/cmϪ1: 2035,
᎐
1910; Anal. found: C, 26.57; H, 2.00; N, 12.11. C10H7N4O5Re
requires C, 26.67; H, 1.79; N, 12.44%.
Complexes 8 and 9 can be prepared in an analogous way
starting from 4 and 5, respectively. Analytical data for 8: yield:
1
90%; H NMR (methanol-d4) δ 8.16 (d, 1H, imc), 7.66 (d, 1H,
2
squares methods on F with SHELXL-97.28 The crystal data
imc), 7.35 (m, 3H, phenyl), 7.18 (m, 2H, phenyl), 4.89 (s, 2H,
and refinement parameters of compounds 4b, 5, 10 and 11 are
CH2); ESI-MS: m/z 499 [M]ϩ; IR νC᎐O(st)/cmϪ1: 2023, 1908;
᎐
summarized in Table 1.
CCDC reference numbers 228229–228232.
See http://www.rsc.org/suppdata/dt/b4/b400220b/ for crystal-
lographic data in CIF or other electronic format.
Anal. found: C, 36.01; H, 1.91; N, 8.34. C15H10N3O5Re requires
C, 36.14; H, 2.02; N, 8.43. Analytical data for 9: Yield: 80%,
1H NMR (methanol-d4) δ 9.12 (dd, 1H, pyridine), 8.48 (m, 1H,
pyridine), 8.21 (dd, 1H, pyridine), 7.82 (t, 1H, imidazole),
7.07 (t, 1H, imidazole), 6.94 (t, 1H, imidazole); 13C NMR
(methanol-d4) δ 197.9, 197.9, 195.6, 174.8, 165.9, 154.7, 152.2,
143.7, 139.5, 129.7, 129.6, 127.6, 119.3; ESI-MS: m/z 505 [M]ϩ;
[Re(OH2)(imc)(CO)3] (4). (NEt4)2[ReBr3(CO)3] (3) (488 mg,
0.63 mmol) was dissolved in water (5 ml) and added to a solu-
tion of 4-imidazolecarboxylic acid (imc) (71 mg, 0.63 mmol) in
H2O (3 ml). After 2 h under reflux, the product precipitated as a
white powder at r.t. Filtration, washing with diethyl ether and
drying at high vacuum gave 186 mg of 4 (73%). 1H NMR
(dmso-d6): δ 8.40 (s, 1H, imc), 7.68 (s, 1H, imc), 7.08 (br s, 1H,
IR νC᎐O(st)/cmϪ1: 2024, 1890.
᎐
[Re(bic)(2,4-dipic)(CO)3] (10). A solution of 5 (30 mg, 0.07
mmol) and benzyl isocyanide (8 µl, 0.07 mmol) in methanol
(3 ml) was stirred at room temperature for 12 h. The solvent was
removed in vacuo, and the product was dried at high vacuum to
imc); IR νC᎐O(st)/cmϪ1: 2039, 1936. ESI-MS: m/z 764 (2M Ϫ
᎐
2H2O)ϩ, 382 [M Ϫ H2O]ϩ. Anal. found: C, 21.15; H, 1.41; N,
give 10 in a yield of 37 mg (96%). H NMR (dmso-d6): δ 8.94
1
6.97. C7H5N2O6Re requires C, 21.05; H, 1.26; N, 7.02%.
(d, 1H, pyridine), 8.31 (s, 1H, pyridine), 8.13 (d, 1H, pyridine),
7.29 (m, 3H, phenyl), 7.08 (m, 2H, phenyl); 5.12 (s, 2H, CH2);
[99Tc(OH2)(imc)(CO)3] (4b). A solution of 1 (71 mg, 0.13
mmol) and imc (14.5 mg, 0.13 mmol) in water was stirred for
4 h. Sodium carbonate (6.2 mg, 0.06 mmol) was added to neu-
tralize the protons released from imc. After 12 h at r.t. the
reaction was complete and the product precipitated from water.
X-Ray quality crystals were obtained from MeOH–H2O.
ESI-MS: m/z 555 [M ϩ 1]ϩ; IR νC᎐O(st)/cmϪ1: 2033, 1914; Anal.
᎐
found: C, 38.87; H, 2.17; N, 5.07. C18H11N2O7Re requires C,
39.06; H, 2.00; N, 5.07%. Crystals for X-ray diffraction analysis
were obtained by slow diffusion of n-hexane into a solution
of 10 in acetone. The phenyl ring of the isocyanide group is
disordered, only one conformation is shown.
[Re(OH2)(2,4-dipic)(CO)3] (5). 3 (101 mg, 0.13 mmol) was
dissolved in water (10 ml). After addition of AgNO3 (70 mg,
0.4 mmol) and stirring at r.t. for 3 h, AgBr was removed by
filtration and pyridine-2,4-dicarboxylic acid (2,4-dipic) (24 mg,
0.13 mmol) was added to the colorless solution, followed by
stirring at 50 ЊC for 2 h. The now yellow solution was cooled
down and the product was allowed to crystallize at 4 ЊC for
12 h. The yellow crystals were collected by filtration, washed
with water and dried at high vacuum to give the product in a
yield of 38 mg (65%). 1H NMR (dmso-d6): δ 8.96 (d, 1H, pyrid-
ine), 8.34 (s, 1H, pyridine), 8.15 (d, 1H, pyridine), 7.50 (s, 1H,
[Re(im)(2,5-dipic)(CO)3] (11). A solution of 6 (80 mg, 0.17
mmol) and imidazole (12 mg, 0.17 mmol) in methanol (2 ml)
was heated to reflux for 2 h and then allowed to cool down. An
orange product precipitated. The solvent was removed in vacuo,
and the product was dried at high vacuum to give 11 in an yield
1
of 82 mg (95%). H NMR (dmso-d6): δ 9.20 (s, 1H, pyridine),
8.58 (d, 1H, pyridine), 8.06 (d, 1H, pyridine), 8.00 (s, 1H, imid-
azole), 7.18 (s, 1H, imidazole), 6.85 (s, 1H, imidazole); ESI-MS:
m/z 505 [M]ϩ. Crystals for X-ray diffraction analysis were
obtained by slow evaporation of a methanolic solution of 11.
Complex 12 can be synthesized in the same way. Analytical data
1
pyridine); ESI-MS: m/z 438 [M Ϫ H2O]ϩ; IR νC᎐O(st)/cmϪ1: 2036,
for 12: H NMR (methanol-d4) δ 9.26 (m, 1H, pyridine), 8.70
᎐
1920. Anal. found: C, 26.15; H, 1.67; N, 3.07. C10H6NO8Re
requires C, 26.45; H, 1.33; N, 3.07%. Crystals for X-ray diffrac-
tion analysis were obtained by slow evaporation of an aqueous
solution.
(dd, 1H, pyridine), 8.22 (d, 1H, pyridine), 7.32 (m, 3H, phenyl),
7.17 (m, 2H, phenyl), 4.96 (s, 2H, CH2); ESI-MS: m/z 554 [M]ϩ;
IR νC᎐O(st)/cmϪ1: 2036, 1919; Anal. found: C, 38.80; H, 2.15; N,
᎐
5.12. C18H11N2O7Re requires C, 39.06; H, 2.00; N, 5.06%.
[Re(OH2)(2,5-dipic)(CO)3] (6). 3 (500 mg, 0.65 mmol) was
dissolved in water (50 ml), and pyridine-2,5-dicarboxylic acid
(110 mg, 0.65 mmol) was added. The yellow colored solution
was stirred at 80 ЊC for 3 h and then cooled down to r.t. The
product precipitated as a yellow powder. It was collected by
filtration, washed with water and dried at high vacuum to give
N-(tert-Butoxycarbonyl)glycyl-N-(3-(imidazol-1-yl)propyl)-
phenylalaninamide (Boc-Gly-Phe-api, 13). Boc-Gly-Phe-OH
(845 mg, 2.6 mmol) was dissolved in dry THF (15 ml), and
N-hydroxysuccinimide (361 mg, 3.14 mmol) was added. The
solution was cooled in an ice-bath, and dicyclohexane-
carbidiimide (650 mg, 3.15 mmol) was added. After stirring the
D a l t o n T r a n s . , 2 0 0 4 , 1 3 2 0 – 1 3 2 8
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solution at room temperature for 30 min and letting it stand at
4 ЊC for 12 h the colorless precipitate was removed by filtration
and the solvent of the mother-liquid was removed in vacuo. The
residue was washed with diethyl ether and dissolved in
dimethylformamide (20 ml). 1H-(3-Aminopropyl)imidazole
(313 µl, 2,6 mmol) was added and the mixture stirred for
30 min. After removing of the dimethylformamide in vacuo, the
white powder was washed with diethyl ether and dried at
high vacuum to give a crude product in a yield of 1.10 g
(79%). Further purification was achieved by semipreparative
allowed to warm up to r.t. for another hour, the solvent was
removed in vacuo, and the product was purified by chromato-
graphy (silica, methanol). Excess 1-(3-aminopropyl)imidazole
was removed by stirring a suspension of the product in a mix-
ture of ethyl acetate (5 ml) and diethyl ether (5 ml). Filtration
and washing of the residue with the same solvent mixture gave
1
84 mg of 17 (42%). H NMR (methanol-d4): δ 7.65 (s, 1H,
imidazole), 7.61 (s, 1H, thymine), 7.12 (s, 1H, imidazole), 6.95
(s, 1H, imidazole), 6.34 (t, 1H, ribose), 5.24 (m, 1H, ribose),
4.1–4.2 (m, 3H, ribose), 4.12 (m, 2H, CH2), 3.45 (s, 2H, CH2),
2.52 (t, 2H, CH2), 2.37 (m, 2H, ribose), 2.09 (s, 3H, CH3), 2.07
(s, 3H, CH3), 1.97 (m, 2H, CH2); ESI-MS: m/z 450 [M ϩ 1]ϩ,
488 [M ϩ K]ϩ.
1
HPLC. H NMR (dmso-d6): δ 8.03 (m, 2H, NH), 7.57 (s, 1H,
imidazole), 7.29 (m, 5H, phenyl), 7.13 (s, 1H, imidazole),
6.96 (m, 1H, NH), 6.88 (s, 1H, imidazole), 4.45 (m, 1H, CH),
3.81 (m, 2H, CH2), 3.53 (m, 2H, CH2), 2.89 (m, 4H, CH2), 1.75
(m, 2H, CH2); 1.35 (s, 9H, Boc); IR ν/cmϪ1: 3328m, 2977w,
2931w, 2852w, 1781w, 1705s, 1654s, 1628m, 1521m, 1456w,
1437w, 1392w, 1367w, 1283w, 1243m, 1167w, 1109w, 1087w,
1051w, 1031w, 998w, 918w, 892w, 862w, 818w, 749w, 702w,
662w; ESI-MS: m/z 429 [M]ϩ. Anal. found: C, 57.42; H, 8.06;
N, 15.15. C22H31N5O4ؒ1.5H2O requires C, 57.88; H, 7.51; N,
15.34%.
5-((3-(Imidazol-1-yl)propyl)aminomethyl)-2Ј-deoxyuridine
(18). Acetyl deprotection was achieved by stirring a solution of
17 (3.5 mg, 7.8 µmol) in a mixture of water (5 ml) and aqueous
NaOH (200 µl, 2 M) for 1 h at r.t. The solution was then neu-
tralized with aqueous HCl and the solvent removed in vacuo.
The product was purified by analytical HPLC and was used in
subsequent reactions without removing the NaCl salt. 1H NMR
(methanol-d4): δ 8.15 (s, 1H, thymine), 7.74 (br s, 1H, imid-
azole), 7.22 (br d, 1H, imidazole), 6.99 (br s, 1H, imidazole),
6.26 (t, 1H, ribose), 4.41 (m, 1H, ribose), 4.17 (m, 2H, CH2),
3.93 (m, 1H, ribose), 3.80 (m, 2H, ribose), 3.72 (s, 2H, CH2),
2.91 (t, 1H, CH2), 2.79 (m, 1H, CH2), 2.27 (m, 2H, ribose),
2.16 (m, 2H, CH2), the signals were assigned with the aid of
COSY and HETCOR spectra; 13C NMR: δ 165.9, 152.2, 142.0,
137.5, 127.3, 120.0, 106.4, 86.7, 85.6, 70.2, 61.0, 44.1, 43.9,
39.0, 36.6, 27.4; ESI-MS: m/z 250 [M Ϫ ribose]ϩ, 366 [M]ϩ, 388
[M ϩ Na]ϩ; IR ν/cmϪ1:3421s, 2931m, 1684s, 1568s, 1438m,
1409m, 1281w, 1088w, 1054w, 760w, 667w.
[Re(Boc-Gly-Phe-api)(imc)(CO)3] (14). A solution of 4 (24
mg, 0.06 mmol) in methanol (2 ml) was given to a solution of
13 (26 mg, 0.06 mmol) in H2O (3 ml). The mixture was stirred
at r.t. for 13 h, the solvent removed in vacuo and the resi-
due washed with diethyl ether and dried at high vacuum to give
1
14 as a white powder in a yield of 41 mg (84%). H NMR
(dmso-d6): δ 8.28 (s, 1H, imidazole), 8.05 (d, 2H, N–H), 7.88 (s,
1H, imidazole), 7.54 (s, 1H, imidazole), 7.20 (m, 6H, imidazole
and phenyl), 6.94 (m, 1H, N–H), 6.69 (s, 1H, imidazole), 4.40
(m, 1H, CH), 3.75 (m, 2H, CH2), 2.93 (m, 2H, CH2), 2.81 (m,
2H, CH2), 1.65 (m, 2H, CH2), 1.32 (s, 9H, Boc).
[Re(18)(imc)(CO)3] (19). A solution of 18 (116 µmol) and 4
(30.1 mg, 75.4 µmol) in methanol (8 ml) was stirred at 45 ЊC.
After 4 and 24 h, portions of 4 (3.2 mg, 8.0 µmol) were added.
After 28 h, the solvent was removed in vacuo and the residue
washed with dichloromethane (10 ml) and decanted twice. Then
it was dissolved in methanol and separated from undissolved
salt. The solvent was removed in vacuo, the residue was partly
dissolved in water and applied to a small RPC18ec column. For
complete removal of remaining salts, the column was washed
with water. The product was isolated by elution with methanol,
removing the solvent in vacuo and drying at high vacuum
to obtain 19 as an off-white solid in a yield of 46.4 mg (68.3%).
1H NMR (methanol-d4): δ 8.21 (m, 1H, imidazole), 8.09 (s,
1H, imidazole or ribose), 7.82 (s, 1H, imidazole or thymine),
7.55 (m, 1H, imidazole), 7.12 (s, 1H, imidazole or thymine),
6.88 (s, 1H, imidazole or thymine), 6.26 (t, 1H, ribose), 4.41
(br s, 1H, ribose), 4.07 (m, 2H, CH2), 3.93 (m, 1H, ribose), 3.77
(m, 2H, ribose or CH2), 3.62 (m, 2H, ribose or CH2), 2.56 (m,
1H, CH2), 2.22 (m, 1H, CH2), 1.96 (m, 4H, ribose and CH2);
[Re(Boc-Gly-Phe-api)(2,5-dipic)(CO)3] (15). was prepared
analogously and purified by column on silica to give a yellow
powder in a yield of 93.5%. H NMR (methanol-d4): δ 9.38 (s,
1
1H, pyridine), 8.66 (dd, 1H, pyridine), 8.17 (d, 1H, pyridine),
7.83 (s, 1H, imidazole), 7.21 (m, 5H, phenyl), 7.19 (s, 1H, imid-
azole), 9.61 (s, 1H, imidazole), 4.50 (t, 1H, CH), 3.68 (m, 4H,
CH2), 2.98 (m, 4H, CH2), 1.73 (m, 2H, CH2), 1.38 (s, 9H, Boc);
13C NMR (methanol-d4) δ 198.0, 197.8, 195.6, 174.6, 173.6,
173.6, 172.8, 166.0, 154.4, 153.3, 142.7, 141.4, 138.5, 133.6,
130.5, 130.3, 129.8, 128.4, 128.1, 122.4, 81.0, 56.4, 46.0, 44.8,
38.5, 36.6, 31.3, 28.7; ESI-MS: m/z 866 [M]ϩ; IR νC᎐O(st): 2023,
᎐
1893.
Glycyl-N-(3-(imidazol-1-yl)propyl)phenylalaninamide (H-Gly-
Phe-api, 16). Boc-Gly-Phe-api (13) (97 mg, 0.23 mmol) was dis-
solved in trifluoroacetic acid (2 ml) and stirred for 30 min at
room temperature. The solvent was removed in vacuo, the
product was suspended in diethyl ether, isolated by filtration,
washed with tetrahydrofuran and dried at high vacuum to give
the bis-trifluoroacetate salt of 16 as a white powder in a yield of
ESI-MS: m/z 747 [M]ϩ, 770 [M ϩ Na]ϩ; IR νC᎐O(st)/cmϪ1: 2022,
᎐
1891.
1
75 mg (60%). H NMR (dmso-d6): δ 9.05 (s, 1H, imidazole),
8.76 (d, 1H, N–H), 8.30 (s, 1H, N–H), 7.97 (s, 2H, N–H), 7.73
(d, 2H, imidazole), 7.24 (m, 5H, phenyl), 4.51 (m, 1H, CH),
4.04 (m, 2H, CH2), 3.39 (m, 4H, CH2), 1.88 (m, 2H, CH2);
13C NMR (methanol-d4): δ 173.7, 167.6, 138.2, 136.7, 130.3,
129.6, 128.0, 123.2, 121.2, 57.1, 47.6, 41.5, 38.8, 36.5, 30.8;
ESI-MS: m/z 330 [M ϩ 1]ϩ; IR ν/cmϪ1: 3435m, 3292m, 3079m,
2964w, 2873w, 1676s, 1550m, 1437w, 1204m, 1132m, 835w,
800w, 721w.
N-((6-Carboxypyridine-3-yl)methyl)glycylphenylalanine (20).
5-Bromomethyl-2-carboxypyridine (236.2 mg, 1.03 mmol) was
stirred in aqueous NaOH (11.3 ml, 0.1 M) for 40 min at r.t. A
solution of H-Gly-Phe-OH (1.14 g, 5.13 mmol) and triethyl-
amine (715 µl, 5.03 mmol) in water (10 ml) was cooled to 0 ЊC,
and the bromide solution was slowly added during 1 h. The
mixture was stirred at 0 ЊC for 10 h, the solvent was removed
in vacuo and the crude product was purified by preparative
HPLC to give the trifluoroacetate salt of 20 as a white powder
in a yield 344.8 mg (71.0%). 1H NMR (methanol-d4): δ 8.74 (s,
1H, pyridine), 8.14 (m, 2H, pyridine), 7.20 (m, 5H, phenyl),
4.72 (m, 1H, CH), 4.32 (m, 2H, CH2), 3.91 (m, 2H, CH2), 3.21
(m, 1H, CH2), 2.94 (m, 1H, CH2); 13C NMR (methanol-d4):
δ 174.5, 167.1, 166.5, 151.8, 149.8, 141.9, 138.3, 132.6, 130.5,
129.8, 128.2, 126.7, 55.4, 48.8, 48.8, 38.4; ESI-MS: m/z 358
5-((3-(Imidazol-1-yl)propyl)aminomethyl)-2Ј-deoxy-3Ј,5Ј-di-
O-acetyluridine (17). 5-(Bromomethyl)-2Ј-deoxy-3Ј,5Ј-di-O-
acetyluridine (200 mg, 0.444 mmol) in dry acetonitrile (8 ml),
was added dropwise over 1.5 h to 1-(3-aminopropyl)imidazole
(159 µl, 1.33 mmol) and diisopropylamine (114 µl, 0.666 mmol)
in dry acetonitrile (10 ml) at 0 ЊC. The mixture was stirred and
D a l t o n T r a n s . , 2 0 0 4 , 1 3 2 0 – 1 3 2 8
1327

View Article Online
[M ϩ 1]ϩ; IR ν/cmϪ1: 3431s, 3068m, 3034m, 2960m, 1733m,
1717m, 1670s, 1442w, 1197s, 1139m, 798w, 722w, 701w.
Acknowledgements
We acknowledge financial support from the Bundesamt für
Bildung und Wissenschaft BBV under contract C 99.0055
within the COST-B12 framework and from Mallinckrodt Med.
B.V. Petten NL.
[Re(OH2)(20)(CO)3] (21). 3 (72.4 mg, 0.094 mmol) and 20
(44.3 mg, 0.094 mmol) were stirred in methanol (5 ml). The
solution turned yellow shortly after the addition of the react-
ants. After 2 h, HPLC exhibited full conversion of the starting
materials. Water (1 ml) was added, and after another hour of
stirring the solvent was removed in vacuo, the yellow residue
was washed with CHCl3 and dried at high vacuum to give 21 in
an yield of 35.3 mg (58%). 1H NMR (methanol-d4): δ 8.935 (m,
1H, pyridine), 8.22 (d, 1H, pyridine), 8.12 (d, 1H, pyridine),
7.25 (m, 5H, phenyl), 4.78 (m, 1H CH), 4.30 (m, 2H, CH2), 3.89
(m, 2H, CH2), 3.28 (m, 1H, CH2), 2.98 (m, 1H, CH2); ESI-MS:
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99mTc(OH2)(L2)(CO)3]. Aliquots of the bidentate ligands
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