Propargyl-Substituted Thiocarbamoylbenzamidines of Technetium and Rhenium: Steps towards Bioconjugation with Use of Click Chemistry

Article abstract of DOI:10.1002/ejic.201601039

A new propargyl-substituted thiocarbamoylbenzamidine has been synthesized. The compound acts as a tetradentate ligand and forms stable complexes with {Re^VO}³⁺and {Tc^VO}³⁺cores. Click couplings of the resulting complexes with benzylazide lead to prototype triazole derivatives, which were analyzed by NMR and IR spectroscopy and mass spectrometry, as well as by X-ray diffraction. A similar coupling procedure has been applied to an azido-modified angiotensin-II peptide, which gives the desired rhenium(V) bioconjugate in good yield. The ease of this coupling and the stability of the conjugate recommend such tetradentate thiocarbamoylbenzamidines also for clinically relevant bioconjugates with^99mTc.

Full text of DOI:10.1002/ejic.201601039

DOI: 10.1002/ejic.201601039
Full Paper
Bioconjugation
Propargyl-Substituted Thiocarbamoylbenzamidines of
Technetium and Rhenium: Steps towards Bioconjugation with
Use of Click Chemistry
Juan Daniel Castillo Gomez,[a][‡] Adelheid Hagenbach,[a] and Ulrich Abram*[a]
Abstract: A new propargyl-substituted thiocarbamoylbenz- as well as by X-ray diffraction. A similar coupling procedure has
amidine has been synthesized. The compound acts as a tetra- been applied to an azido-modified angiotensin-II peptide,
dentate ligand and forms stable complexes with {ReVO}3+ and which gives the desired rhenium(V) bioconjugate in good yield.
{TcVO}3+ cores. Click couplings of the resulting complexes with The ease of this coupling and the stability of the conjugate
benzylazide lead to prototype triazole derivatives, which were recommend such tetradentate thiocarbamoylbenzamidines also
analyzed by NMR and IR spectroscopy and mass spectrometry, for clinically relevant bioconjugates with 99mTc.
Introduction
Thiocarbamoylbenzamidines are excellent chelating agents,
and they form stable complexes with many metal ions.[12] The
development of tri-, tetra-, and pentadentate derivatives makes
them also interesting for the complexation of rhenium and
technetium, since they are suitable candidates for the stabiliza-
tion of their {MVO}3+ or {MVN}2+ cores.[13–19] Recently, we re-
ported on some representatives of such ligands with orthogo-
nal anchor groups in their periphery for bioconjugation and the
corresponding Re and Tc complexes.[20,21] The coupling ap-
proach applied for these examples was the classical peptide
coupling. Another promising coupling method for bioconjuga-
tion is provided by Click chemistry,[22] bearing in mind that az-
ide derivatives of many biomolecules of interest are available
or can be synthesized by well-established procedures.
The metastable technetium nuclide 99mTc has been the most
used radioisotope for diagnostic applications in nuclear medi-
cine in the past decades.[1–7] Its favorable nuclear properties
(virtually pure γ-emitter, Eγ = 143 keV, t1/2 = 6.01 h) and the
ready availability from 99Mo/99mTc generators make it to the
workhorse for most routine applications. 99mTcO4 solutions
–
from such generators normally contain the metal at an approxi-
mately nanomolar level, which is advantageous for clinical ap-
plication, but causes considerable problems during the devel-
opment of new Tc radiopharmaceuticals because of the lack of
reliable structural information.
Therefore, structural studies on technetium compounds are
commonly performed with the long-lived isotope 99Tc (weak
ꢀ–-emitter, Emax = 294 keV, t1/2 = 2.11×105 a) or its heavier
homolog rhenium. The element rhenium itself has two isotopes
with strong ꢀ–-emissions (186Re and 188Re), which make them
interesting for applications in nuclear medical therapy.[8–10]
One goal of current radiochemical research is the search for
suitable chelating agents for radiometals used in nuclear medi-
cine, which build stable complexes and can be attached to bio-
molecules under mild conditions without affecting their biolog-
ical activity. Ligand systems which are not restricted to techne-
tium and also form stable complexes with other radioactive
metal ions having potential for either imaging or therapeutic
applications are of particular interest.[11]
In the present paper, we discuss an approach to bioconju-
gates using propargyl-substituted, tetradentate thiocarbamoyl-
benzamidines as chelating agents for rhenium and technetium
with use of Click chemistry.
Results and Discussion
The synthesis of a suitable propargyl-substituted thiocarbamoyl
benzamidine was performed in the multiple-step synthesis
shown in Scheme 1. The key step is the coupling of propargyl-
aminoglycylglycine 4 to thiocarbamoylbenzimide chloride 5. In
order to avoid undesired cyclization reactions, which we have
recently observed during the attempted synthesis of a related
ligand system,[23] this reaction was scheduled as the last step
of the ligand synthesis.
[a] Institute of Chemistry and Biochemistry, Freie Universität Berlin,
Fabeckstr. 34/36, 14195 Berlin, Germany
E-mail: ulrich.abram@fu-berlin.de
Propargylaminodiglycine hydrochloride (4) can be obtained
through a three-step synthesis. First, the amine function of di-
glycine (1) was protected with di-tert-butyldicarbonate (diBoc).
The protected dipeptide 2 was activated with hydroxybenzotri-
azole (HOBT) and dicyclohexylcarbodiimide (DCC) in order to
attach propargylamine. Finally, 4 was obtained after treatment
http://www.bcp.fu-berlin.de/chemie/chemie/forschung/InorgChem/
agabram/index.html
[‡] Present adress: Cologne University Hospital, Institute for Diagnostic and
Interventional Radiology,
Kerpener Str. 62, 50937 Cologne, Germany
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under http://dx.doi.org/10.1002/ejic.201601039.
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Scheme 1. Synthesis route.
of protected dipeptide 3 with trifluoroacetic acid and HCl. overnight in a freezer at –20 °C, orange-red single crystals of
Benzimidoyl chloride 5 was prepared by a reaction of bis(N,N- the compositions [MO(L)] (M = Tc, Re) were obtained.
diethyl-N′-benzoylthioureato)nickel(II) with SOCl2 following a
standard procedure.[13,24] Then it was coupled to 4 in dry acet-
one with the addition of NEt3 as a supporting base. The result-
Both complexes crystallize in the monoclinic space group
P21/n. The metal atoms are five-coordinate with the tetra-
dentate ligands L3– as the basal planes and the oxido ligands
as the apexes of square pyramids. An ellipsoid representation
ing proligand H3L (6) was obtained a pale yellow solid.
1
The H NMR spectrum of H3L (6) in CDCl3 shows one multi- of the molecular structure of [TcO(L)] is shown in Figure 1. The
plet at 1.22 ppm and two quartets at 3.62 and 3.90 ppm, which structure of the corresponding rhenium complex is virtually
are all assigned to the diethyl residues of the molecule due to identical and is therefore not shown. An ellipsoid plot of this
a hindered rotation around the related C–N bonds. This is not compound can be found in Figure S1 in the Supporting Infor-
unusual for such ligands and has been described before.[13–21] mation. Selected bond lengths and angles of both complexes,
The terminal alkyne proton leads to a triplet with a coupling
constant of J = 2.4 Hz due to a long-range coupling with the
methylene group of the propargyl function. This group itself
displays a doublet of a doublet with coupling constants of 5.4
and 2.4 Hz at 4.02 ppm. Both methylene groups of the glycine
units lead to doublets at 3.97 and 4.12 ppm. The hydrogen
atoms of the NH groups lead to three triplets at 6.46, 6.93, and
7.59 ppm. These signals merge to form a broad singlet depend-
ing on the water content of the solvent CDCl3. All aromatic
signals appear between 7.38 and 7.49 ppm.
A strong absorption band at 3271 cm–1 in the IR spectrum
of compound 6 can be assigned to the C–H stretch of the alk-
yne moiety. Two strong absorptions at 1670 and 1645 cm–1 are
attributed to the carbonyl groups. The ESI+ mass spectrum
shows the expected molecular ion peak [M + H]+ at m/z =
388.1802.
Reactions of 6 with (NBu4)[TcOCl4] or (NBu4)[ReOCl4] in
methanol result in the formation of orange-red solutions. After
the addition of 3–5 drops of water and keeping the solutions
Figure 1. Ellipsoid representation of the molecular structure of [TcO(L)] (7a).
Hydrogen atoms are omitted for clarity. Thermal ellipsoids represent 50 %
probability.
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are compared in Table 1. The labeling scheme of the techne- ascorbate (up to 5 mol-%), the reaction has been performed
tium complex (Figure 1) has also been used for its rhenium with equivalent amounts of the Cu2+ ions and an excess of
analog.
ascorbic acid. Although such conditions might cause problems
during the purification of the products, they have been chosen
since (i) they should accelerate the reactions and (ii) keeping in
mind, that for prospective reactions with 99mTc (and the nano-
molar concentrations of this nuclide in real 99Mo/99mTc genera-
tor eluates) a large excess of copper ions cannot be avoided.
With equivalent amounts of Cu2+ ions also the role of these
ions as competitors in the complex formation with H3L could
be tested, and the possible formation of copper complexes with
the tetradentate ligand should be investigated..
Table 1. Selected bond lengths [Å] and angles [°] in [TcO(L)] (7a) and [ReO(L)]
(7b).
7a
7b
Tc–O10
Tc–S
Tc–N5
Tc–N8
1.654(2)
2.307(1)
2.017(2)
1.956(2)
2.025(2)
1.479(3)
1.455(4)
1.173(4)
108.84(7)
91.31(6)
78.78(8)
78.06(8)
112.0(2)
179.5(3)
Re–O10
Re–S
Re–N5
Re–N8
1.674(4)
2.301(2)
2.021(4)
1.982(4)
2.026(5)
1.481(6)
1.460(9)
1.18(2)
108.4(2)
92.0(2)
78.2(2)
77.8(2)
111.1(5)
179.8(8)
Tc–N11
Re–N11
N11–C12
C12–C13
C13–C14
O10–Tc–S
S–Tc–N5
N5–Tc–N8
N8–Tc–N11
N11–C12–C13
C12–C13–C14
N11–C12
C12–C13
C13–C14
O10–Re–S
S–Re–N5
N5–Re–N11
N8–Re–N11
N11–C12–C13
C12–C13–C14
The reactions between the [MO(L)] complexes 7a and 7b
and benzylazide have been performed in mixtures of dichloro-
methane, methanol, and water (1:3:1 v/v/v). This ensured solu-
bility of all components and a homogenous reaction. After stir-
ring for 5 h at room temperature, dichloromethane was evapo-
rated, which resulted in the precipitation of the orange-red cou-
pling products [TcO(Bnztrz-L)] (8a) and [ReO(Bnztrz-L)] (8b). No
evidence was found for the formation of CuII complexes with
the tetradentate ligand. The completion of the reaction was
The M–O10 bond lengths of 1.654(2) and 1.674(4) Å are in
the typical range of metal-oxygen double bonds. The Tc–S and
Re–S bond lengths are almost identical [2.307(2) and
2.301(2) Å]. The same holds true for the respective M–N bond
lengths. The propargyl groups are almost perfectly linear and
form an angle of approximately 112° with the mean planes of
the tetradentate ligands.
Although the molecular structures of both complexes are
very similar, their 1H NMR spectra show some substantial differ-
ences. The alkyne hydrogen atoms display triplets with cou-
pling constants of J = 2.45 Hz for both [TcO(L)] and [ReO(L)] at
2.27 ppm. These patterns are caused by long-range couplings
with the adjacent methylene group of the propargyl function.
This methylene group leads to two doublets of doublets at 5.34
and 4.86 ppm (coupling constants each J = 2.45 Hz) in the
spectrum of [ReO(L)]. In the spectrum of [TcO(L)], the equivalent
signals can be found at 5.10 and 4.75 ppm. All hydrogen atoms
of the methylene groups are diastereotopic in both complexes.
This leads to a further splitting of the methylene signals due to
geminal couplings, and consequently complicated signal pat-
terns in the range between 3.7 and 5.4 ppm are observed,
which are different in the spectra of the analogous technetium
and rhenium complexes.
1
confirmed by H NMR analysis of the crude products.
Single crystals suitable for the X-ray analyses were obtained
by recrystallization from toluene or acetone/toluene mixtures.
The molecular structure of technetium complex 8a is shown in
Figure 2. The structure of the analogous rhenium compound is
identical and therefore only shown in the Supporting Informa-
tion. Selected bond lengths and angles of both compounds are
given in Table 2.
Figure 2. Molecular structure of 8a. Hydrogen atoms are omitted for clarity.
Thermal ellipsoids represent 50 % probability.
The IR spectra of the studied [MO(L)] complexes are, as ex-
Similar to the structures of the starting materials 7a and 7b,
pected, very similar. They show νM=O stretches at 972 (Tc com- the molecular structures of the products of the Click couplings
pound) and 983 cm–1 (Re complex). Sharp bands at 3261 cm–1 possess five-coordinate metal atoms. The coordination spheres
reflect the presence of the alkyne C–H bonds. Each two intense can best be described as square pyramids with the oxido li-
bands at 1664, 1635 cm–1 (Tc complex) and 1672, 1643 cm–1 gands as their apexes. The coupling reactions in the peripheries
(Re compound) are attributed to the peptide C=O bonds. The of the tetradentate ligands do not change the coordination en-
ESI+ mass spectrum of [ReO(L)] shows the expected molecular vironment of the metal cores in comparison to the starting ma-
ion peak [M + H]+ at m/z = 588.1069. That of the technetium terials. All bond lengths and angles in the coordination spheres
compound has not been recorded for radiation safety reasons.
In order to get information about the robustness of the new
complexes in ongoing reactions, the propargyl-substituted
of the metal atoms remain practically unchanged after the for-
mation of the triazole rings.
A good indication for the progress of the coupling reaction
technetium complex 7a as well as its rhenium analogue 7b is the disappearance of the triplet signals at about 2.27 ppm
have been used for model Click reactions with benzylazide. Un- displayed by the alkyne protons in the 1H NMR spectra. Instead,
like classical Click reactions, which are commonly carried out new singlets, which are assigned to the two protons of the
with small amounts of copper sulfate (ca. 1 mol-%) and sodium methylene groups of the benzyl residues, appear in the spectra
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Table 2. Selected bond lengths [Å] and angles [°] in [TcO(Bnztrz-L)] (8a) and
[ReO(Bnztrz-L)] (8b).
and 4.8 ppm in the spectra of both compounds. The signals of
the aromatic protons of the triazole rings overlap with the other
aromatic signals giving complex multiplets.
8a
8b
The IR spectra of 8a and 8b are expectedly very similar. They
show νTc=O and νRe=O bands at 981 and 993 cm–1, respectively.
Each two sharp absorptions at 1672 and 1651 cm–1 in the spec-
trum of the technetium complex and at 1678 and 1658 cm–1 in
that of 8b originate from by the carbonyl stretches. The triazole
rings display sharp absorption bands at 1417, 1354, and
1328 cm–1 in the spectra of both compounds. The ESI+ mass
spectrum of the rhenium complex shows the molecular ion
peak [M + H]+ at m/z = 721.1716.
Tc–O10
Tc–S
Tc–N5
Tc–N8
1.651(3)
2.306(1)
2.050(3)
1.942(3)
2.014(3)
1.463(5)
1.492(5)
1.357(5)
1.356(5)
1.321(5)
1.357(5)
1.345(4)
1.472(5)
1.518(6)
111.45(9)
89.54(8)
79.3(1)
Re–O10
Re–S
Re–N5
Re–N8
1.671(5)
2.305(2)
2.056(5)
1.945(7)
2.015(5)
1.470(9)
1.50(2)
1.36(2)
1.37(2)
1.33(1)
1.365(9)
1.33(2)
1.47(2)
1.51(2)
110.9(2)
90.3(2)
79.4(2)
78.4(2)
113.1(6)
129.4(7)
113.0(7)
Tc–N11
Re–N11
N11–C12
C12–C13
C13–C14
C13–N15
N15–N16
C14–N17
N16–N17
N17–C18
C18–C19
O10–Tc–S
S–Tc–N5
N5–Tc–N8
N8–Tc–N11
N11–C12–C13
C12–C13–C14
N17–C18–C19
N11–C12
C12–C13
C13–C14
C13–N15
N15–N16
C14–N17
N16–N17
N17–C18
C18–C19
O10–Re–S
S–Re–N5
N5–Re–N11
N8–Re–N11
N11–C12–C13
C12–C13–C14
N17–C18–C19
The successful coupling of 7a and 7b to benzylazide stimu-
lated us to attempt conjugation with a small peptide. For this
experiment, we prepared an azido-substituted angiotensin-II
derivative by a conventional solid-phase peptide synthesis,
GlyN3-AngII-Wang (Scheme 2). Angiotensin-II is a peptide
hormone consisting of eight amino acids (H2N-Asp–Arg–Val–
Tyr–Ile–His–Pro–Phe-COOH). This peptide plays an important
role in the regulation of blood pressure and water balance
in the body and has an effect on the whole cardiovascular
system. Primarily, angiotensin-II causes constriction of blood
vessels.[25–27]
78.9(1)
113.0(3)
130.0(4)
112.5(3)
of 8a and 8b at about 5.45 ppm. The protons of carbon atoms
C12 lead to two doublets at 5.30 and 5.60 ppm in the spectrum
The coupling reaction was intended as a proof-of-principle
of 8a and at 5.39 and 5.88 ppm in that of the rhenium complex. experiment and performed in the solid phase. Wang resin was
All other methylene signals appear as expected between 4.2 used as a solid carrier with the peptides still having protected
Scheme 2. Synthesis of the angiotensin-II derivatives.
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amino acid side chains. This ensures that the functional groups and more convenient reaction conditions can be chosen. An-
of the peptide do not interfere with the coupling procedure, other opportunity for the synthesis of suitable 99mTc derivatives
for example by undesired side-reactions with the Cu2+ ions. De- is the use of transmetalation reactions starting from nonradio-
protection and cleavage from the resin were performed in sub- active rhenium compounds or preferably from copper com-
sequent steps.
plexes. Related studies are currently under investigation in our
research group.
The identity of the coupling product between the azido-sub-
stituted angiotensin-II derivative and [ReO(L)] (7b) was analyzed
with ESI mass spectrometry of the crude product. Signals for
the molecular ion [M + H]+ at m/z = 1716.6488 and the ion [M
+ H]2+ at m/z = 858.8291 are found in the positive-mode ESI
spectrum. The detection of doubly charged ions is not uncom-
mon for the mass spectra of peptides. The corresponding signal
[M – H]– was found at m/z = 1714.6336 in the ESI– spectrum.
The corresponding molecular ion regions of both mass spectra
are shown in Figure 3 together with the calculated isotope dis-
tributions.
Experimental Section
Materials: All chemicals used in this study were reagent grade and
used without further purification. Diethylaminobenzimidoyl chlor-
ide 5,[24] (NBu4)[ReOCl4],[29] (NBu4)[TcOCl4],[30] benzylazide,[31] and
azidoacetic acid (GlyN3)[32] were prepared by published methods.
Solvents were dried with use of standard procedures. Peptides were
synthesized by using modifications of standard procedures of solid
phase peptide synthesis.[33]
Physical Measurements: Infrared spectra were recorded from KBr
pellets with a Shimadzu FTIR-spectrometer between 400 and
4000 cm–1. ESI mass spectra were recorded with an Agilent 6210
ESI-TOF (Agilent Technologies). All MS results are given in the form:
m/z, assignment. Elemental analyses of carbon, hydrogen, nitrogen,
and sulfur were performed with a Heraeus Vario EL elemental analy-
zer. The technetium content was determined with a Beckman
LS6500 liquid szintillation counter. NMR spectra were recorded with
a JEOL 400 MHz multinuclear spectrometer.
Radiation Protection: The radioisotope 99Tc was handled following
standard radiation protection procedures in a laboratory approved
for the work with long-lived beta emitters.
N-Boc-Glycylglycine (2): Diglycine (6.55 g, 50 mmol) and diBoc
(15 g, 50 mmol) were suspended in methanol (50 mL). Triethylamine
(5.05 g, 50 mmol) was added, and the reaction mixture was heated
to reflux for 4 h. After cooling to room temperature, the resulting
solution was filtered and reduced to a volume of about 10 mL with
a rotary evaporator. The resulting solution was added under stirring
to diethylether (200 mL). An oily substance, which separated from
this solution, was isolated, washed several times with diethylether,
and dried under vacuum for a period of several days. Finally, a hard
wax was formed. Yield: 11.01 g (47.5 mmol, 95 %). C9H16N2O5
(232.24): calcd. C 46.55, H 6.94, N 12.06; found C 50.91, H 10.08, N
11.75. IR (KBr): ν = 3325 (s), 2976 (s), 2937 (s), 2802 (w), 2756 (w),
˜
2738 (m), 2677 (s), 2492 (s), 1699 (s), 1668 (s), 1606 (w), 1531 (s),
1394 (s), 1367 (s), 1280 (w), 1251 (s), 1170 (s), 1033 (m), 947 (m),
Figure 3. Molecular-ion regions of the ESI+ and ESI– mass spectra of the
rhenium bioconjugate.
864 (m), 678 (m) cm–1. H NMR ([D6]DMSO): δ = 1.38 (s, 9 H, -CH3),
1
3.53 (d, J = 6.1 Hz, 2 H, -CH2-), 3.61 (d, J = 5.2 Hz, 2 H, -CH2-), 7.05
(t, J = 6.1 Hz, 1 H, -CO–NH-), 7.77 (t, J = 5.2 Hz, 1 H, -CO–NH-) ppm.
ESI-TOF+ (m/z): calcd. [M + Na]+ 255.0951; found 255.0999. ESI-TOF–
(m/z): calcd. [M – H]– 231.0986; found: 231.0859.
Conclusions
Tetradentate thiocarbamoylbenzamidines, which have an alk-
N-Propargylamino Glycylglycine (3): Compound
2 (6.25 g,
ynyl group attached to their periphery, are suitable chelating 26 mmol), propargylamine (2.22 g, 40 mmol), HOBT (5.40 g,
agents for the {TcVO}3+ and {ReVO}3+ cores and can be used
40 mmol), and DCC (8.24 g, 40 mmol) were dissolved in THF (50 mL).
The reaction mixture was stirred for 6 h at room temperature. The
for precomplexation of the metals before bioconjugation. If a
resulting precipitate was filtered off, and the remaining solvents
propargylic group is used, bioconjugation though copper-cata-
were evaporated with a rotary evaporator until an oily residue was
lyzed Click chemistry is possible. A disadvantage of this ap-
obtained. The residue was dissolved in warm acetone (50 mL), and
proach is the fact that interfering functional groups of the bio-
left overnight in a refrigerator for crystallization. The obtained crys-
molecule need to be protected. One way to avoid this incon-
talline compound was filtered off, washed with cold acetone, and
venience is the use of cyclooctyne moieties instead of propargyl
dried. Yield: 2.31 g (8.6 mol, 33 %). C12H19N3O4 (269.30): calcd. C
moieties as a source of the alkyne groups,[28] so that a copper-
53.52, H 7.11, N 15.60; found C 59.37, H 7.75, N 15.98. IR (KBr): ν =
˜
free Click reaction can be performed. This would be of special
3327 (s), 3180 (w), 3122 (w), 3034 (m), 2927 (s), 2850 (s), 2791 (w),
importance for reactions with 99mTc, since time can be saved, 2657 (w), 1625 (s), 1573 (s), 1537 (m), 1462 (w), 1435 (m), 1346 (w),
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1311 (s), 1271 (m), 1244 (s), 1186 (w), 1157 (w), 1087 (s), 1086 (w),
1045 (m), 966 (w), 893 (s), 842 (w), 802 (w), 732 (w), 640 (s) cm–1
3.77–4.10 (m, 4 H, -CH2-), 4.34 (d, J = 17.7 Hz, 1 H, -CH2-), 4.42 (d,
J = 18.6 Hz, 1 H, -CH2-) 4.50 (d, J = 18.6 Hz, 1 H, -CH2-), 4.74 (d, J =
.
1H NMR ([D6]DMSO): δ = 1.38 (s, 9 H, -CH3), 3.11 (t, J = 2.1 Hz, 1 H, 17.8 Hz, 1 H, -CH2-) 4.75 (dd, J = 17.6, 2.4 Hz, 1 H, -CH2-), 5.10 (dd,
-CCH), 3.57 (d, J = 5.8 Hz, 2 H, -CH2-), 3.69 (d, J = 5.6 Hz, 2 H, -CH2-
J = 17.6, 2.5 Hz, 1 H, -CH2-) 7.38–7.41 (m, 2 H, o-Ph) 7.48–7.52 (m,
), 3.86 (dd, J = 5.3, 2.3 Hz, 2 H, -CH2-), 7.03 (t, J = 5.7 Hz, 1 H, -CO– 3 H, m-p-Ph) ppm.
NH-), 8.08 (t, J = 5.3 Hz, 1 H, -CO–NH-), 8.24 (t, J = 5.0 Hz, 1 H, -CO–
[ReO(L)] (7b): [NBu4][ReOCl4] (290 mg, 0.5 mmol) was dissolved in
NH-) ppm. ESI-TOF+ (m/z): calcd. [M + Na]+ 292.1268; found [M +
methanol (5 mL). A solution of 6 (215 mg, 0.55 mmol) in methanol
(5 mL) was added. The reaction mixture was stirred for 15 min at
room temperature. Five drops of water were added, and the result-
ing solution was filtered and kept overnight in the freezer at –20 °C.
Orange-red crystals were formed. They were filtered off, washed
with cold methanol and water, and dried. Yield: 214 mg (0.36 mmol,
73 %). C19H22N5O3ReS (586.67): calcd. C 38.90, H 3.78, N 11.94, S
Na]+ 292.1272, [M + K]+ 308.1010.
Propargylamidoglycylglycine Hydrochloride (4): Compound 3
(2.22 g, 8.2 mmol) was dissolved in trifluoracetic acid (30 mL). The
solution was stirred for 4 h at room temperature, and then the
solvent was slowly evaporated under a flow of argon until an oily
residue was obtained. The residue was isolated and redissolved in
acetone (50 mL). HCl (37 %, 2.5 mL) was added dropwise to this
5.47; found C 38.88, H 3.63, N 11.76, S 5.36. IR (KBr): ν = 3442 (s),
˜
solution. The resulting precipitate was filtered off, washed with 3261 (s), 2978 (w), 2935 (m), 1672 (s), 1643 (s), 1531 (s), 1514 (s),
acetone, and dried. Yield: 1.34 g (6.6 mmol, 80 %). C7H12ClN3O2 1435 (w), 1417 (w), 1381 (m), 1334 (s), 1317 (m), 1290 (w), 1230 (w),
(205.64): calcd. C 40.88, H 5.88, N 20.43; found C 40.51, H 6.56, N
1064 (m), 983 (s), 937 (w), 883 (w), 827 (w), 775 (m), 688 (s), 671 (m)
21.77. IR (KBr): ν = 3246 (s), 3045 (s), 2931 (s), 2850 (w), 2798 (w),
cm–1. 1H NMR (CDCl3:): δ = 1.29 (t, J = 7.1 Hz, 3 H, -CH3), 1.45 (t, J =
˜
2713 (w), 2623 (w), 2121 (w), 2005 (w), 1693 (s), 1660 (s), 1602 (w), 7.2 Hz, 3 H, -CH3), 2.27 (t, J = 2.5 Hz, 1 H, -CCH), 3.80–4.11 (m, 4 H,
1558 (m), 1527 (s), 1496 (s), 1429 (w), 1404 (m), 1342 (s), 1311 (w),
1290 (w), 1259 (s), 1139 (w), 1116 (w), 1087 (m), 1006 (m), 933 (m),
-CH2-), 4.40 (d, J = 18.7 Hz, 1 H, -CH2-), 4.49 (d, J = 18.7 Hz, 1 H,
-CH2-), 4.55 (d, J = 17.7 Hz, 1 H, -CH2-), 4.73 (d, J = 17.7 Hz, 1 H,
-CH2-), 4.86 (dd, J = 17.4, 2.5 Hz, 1 H, -CH2-), 5.34 (dd, J = 17.4,
2.5 Hz, 1 H, -CH2-), 7.42–7.55 (m, 5 H, Ph) ppm. ESI-TOF+ (m/z): calcd.
[M + H]+ 588.1074; found 588.1069.
1
900 (m), 889 (w), 867 (w), 738 (m), 705 (m), 675 (s) cm–1. . H NMR
([D6]DMSO): δ = 1.31 (t, J = 2.5 Hz, 1 H, -CCH), 3.59 (q, J = 5.5 Hz, 2
H, -CH2-), 3.77 (d, J = 5.9 Hz, 2 H, -CH2-), 3.85 (dd, J = 5.5, 2.5 Hz, 2
H, -CH2-), 8.25 (s, br, 3 H, -NH3+), 8.54 (t, J = 5.5 Hz, 1 H, -CO–NH-),
8.79 (t, J = 5.8 Hz, 1 H, -CO–NH-) ppm. ESI-TOF+ (m/z): calcd. [M]+
170.0924; found 170.0932.
[TcO(Bnztrz-L)] (8a): Method 1: Compound 7a (25 mg, 0.05 mmol)
was dissolved in ethanol (10 mL). Subsequently, benzylazide (78 %
in diethyl ether, 58 mg, 0.35 mmol), copper sulfate pentahydrate
(1.24 mg, 0.005 mmol) in water (100 μL), and a solution of sodium
H3L (6): Compound 4 (556 mg, 2.7 mmol) was suspended in dry
acetone (50 mL). Triethylamine (1.01 g, 10 mmol) and solid diethyl- ascorbate (2.47 mg, 0.012 mmol) in water (100 μL) were added. The
aminobenzimidoyl chloride 5 (685 mg, 2.7 mmol) were subse-
quently added to this suspension. The reaction mixture was stirred
for 5 h at room temperature and filtered. The resulting clear solution
was reduced to a volume of about 5 mL and finally added dropwise
reaction mixture was stirred for 8 h at room temperature and left
for slow evaporation of the solvents at room temperature over a
period of several days. The obtained residue was dissolved in a
mixture of acetone/toluene (1:1 v/v, 2 mL). Crystals of the product
under vigorous stirring to diethylether (50 mL). The formed precipi- could be obtained by slow evaporation of this solution. The crystals
tate was filtered off, washed with diethylether, and dried. Yield: were filtered off, washed with cold toluene, and dried. Yield: 15 mg
667 mg (1.72 mmol, 63.8 %). C19H25N5O2S (387.50): calcd. C 58.89, (0.023 mmol, 46 %). Method 2: 7a (50 mg, 0.1 mmol) was dissolved
H 6.50, N 18.07, S 8.27; found C 58.59, H 7.34, N 18.09, S 8.28. IR
in methanol (3 mL). Subsequently, benzylazide (78 % in diethyl
ether, 130 mg, 0.8 mmol), solid copper sulfate pentahydrate
(24.8 mg, 0.1 mmol), and sodium ascorbate (39.6 mg, 0.2 mmol)
were added to the reaction mixture. dichloromethane (1 mL), water
(1 mL), and methanol (3 mL) were added, so that all components
(KBr): ν = 3321 (s), 3271 (s), 3064 (m), 3034 (w), 2976 (s), 2931 (s),
˜
2872 (w), 1670 (s), 1645 (s), 1622 (s), 1597 (m), 1550 (s), 1535 (m),
1519 (w), 1490 (s), 1458 (w), 1419 (m), 1375 (w), 1355 (w), 1338 (m),
1309 (m), 1269 (m), 1240 (s), 1143 (w), 1124 (m), 1093 (m), 1074 (w),
1049 (w), 1022 (m), 993 (w), 889 (m), 779 (m), 702 (s), 682 (s), 648 dissolved and a clear solution without phase separations was ob-
1
(m) cm–1. H NMR (CDCl3:): δ = 1.18–1.26 (m, 6 H, -CH3), 2.20 (t, J =
2.2 Hz, 1 H, -CCH), 3.62 (q, J = 6.8 Hz, 2 H, -CH2-), 3.91 (q, J = 6.8 Hz,
2 H, -CH2-), 3.97 (d, J = 5.7 Hz, 2 H, -CH2-), 4.02 (dd, J = 5.4, 2.4, Hz,
tained. The solution was then stirred for 5 h at room temperature.
The solvents were reduced to a volume of about 3 mL under re-
duced pressure. The obtained precipitate was filtered off, washed
2 H, -CH2-) 4.13 (d, J = 5.1 Hz, 2 H, -CH2-), 6.46 (s, br., 1 H, -NH-) with cold methanol and water, and dried. Single crystals of the
6.93 (s, br., 1 H, -CO–NH-), 7.38–7.49 (m, 5 H, Ph) 7.59 (s, br., 1 H,
-CO–NH-) ppm. ESI-TOF+ (m/z): calcd. [M + H]+ 388.1802; found [M
+ H]+ 388.1802, [M + Na]+ 410.1627, [M + K]+ 426.1361.
product were obtained from slow evaporation of a solution of the
complex in toluene. Yield: 36 mg (0.057 mmol, 57 %).
C
26H29N8O3STc (632.53): calcd. Tc 15.6; found Tc 15.4. IR (KBr): ν =
˜
3441 (s), 3136 (m), 3064 (w), 2976 (m), 2935 (m), 2906 (m), 1672 (s),
1651 (s), 1508 (s), 1417 (s), 1355 (s), 1328 (m), 1294 (m), 1255 (w),
1211 (w), 1145 (w), 1130 (w), 1072 (m), 1049 (m), 1012 (m), 981 (s),
[TcO(L)] (7a): [NBu4][TcOCl4] (192 mg, 0.49 mmol) was dissolved in
methanol (5 mL). A solution of 6 (224 mg, 0.57 mmol) in methanol
(5 mL) was added, and the reaction mixture was stirred for 15 min
at room temperature. After the addition of five drops of water, the
solution was filtered and left overnight at –20 °C in the freezer.
Orange-red crystals were formed. They were filtered off, washed
with cold methanol and water, and dried. Yield: 142 mg (0.3 mmol,
62 %). C19H22N5O3STc (499.38): calcd. Tc 19.8; found Tc 19.4. IR (KBr):
869 (m), 823 (w), 775 (w), 729 (w), 700 (w), 669 (w) cm–1 1H NMR
.
(CDCl3): δ = 1.23 (t, J = 7.1 Hz, 3 H, -CH3), 1.41 (t, J = 7.1 Hz, 3 H,
-CH3), 3.71–4.10 (m, 4 H, -CH2-), 4.31 (d, J = 17.7 Hz, 1 H, -CH2-),
4.39 (d, J = 18.6 Hz, 1 H, -CH2-), 4.48 (d, J = 18.6 Hz, 1 H, -CH2-),
4.70 (d, J = 17.8 Hz, 1 H, -CH2-), 5.31 (d, J = 15.5 Hz, 1 H, -CH2-),
5.46 (s, 2 H, -CH2-), 5.60 (d, J = 15.5 Hz, 1 H, -CH2-), 7.21–7.49 (m,
11 H, Harom) ppm.
ν = 3442 (s), 3261 (s), 2978 (w), 2937 (w), 1664 (s), 1635 (s), 1529
˜
(s), 1508 (s), 1448 (m), 1433 (m), 1415 (m), 1394 (m), 1382 (w), 1334
(s), 1315 (m), 1288 (w), 1064 (m), 1008 (w), 972 (s), 775 (m), 698 (s),
[ReO(Bnztrz-L)] (8b): Compound 7b (58 mg, 0.1 mmol) was sus-
pended in methanol (3 mL). Subsequently, benzylazide (78 % in
1
690 (s), 669 (m) cm–1. H NMR (CDCl3:): δ = 1.25 (t, J = 7.1 Hz, 3 H,
-CH3), 1.45 (t, J = 7.1 Hz, 3 H, -CH3), 2.27 (t, J = 2.4 Hz, 1 H, -CCH), diethyl ether, 130 mg, 0.8 mmol), solid copper sulfate pentahydrate
Eur. J. Inorg. Chem. 0000, 0–0
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(24.8 mg, 0.1 mmol), and sodium ascorbate (39.6 mg, 0.2 mmol) Fmoc–Pro-OH (539 mg, 1.6 mmol), Fmoc–His(trt)-OH (992 mg,
were added to the reaction mixture. Finally, dichloromethane
(1 mL), water (1 mL), and methanol (3 mL) were added, so that all
1.6 mmol), Fmoc–Ile-OH (565 mg, 1.6 mmol), Fmoc–Tyr(tBu)-OH
(755 mg, 1.6 mmol), Fmoc–Val-OH (543 mg, 1.6 mmol), Fmoc–
components dissolved and a clear solution without phase separa- Arg(Pbf)-OH (779 mg, 1.6 mmol), Fmoc–Asp(OtBu)-OH (657 mg,
tions was obtained. The solution was stirred for 5 h at room temper- 1.6 mmol). The still protected and resin-bound peptide was dried
ature, and dichloromethane was evaporated under reduced pres- and used in this form for further reactions. For the characterization
sure. Water (50 mL) was added to the reaction mixture, which was
then extracted with dichloromethane (3 × 50 mL). The combined
organic phases were dried with magnesium sulfate, filtered, and
of the product, the resin-bound peptide (10 mg) was treated with
piperidine (2 mL) for 1 h, washed several times with DMF and di-
chloromethane, and cleaved from the resin and the protecting
reduced to a volume of about 5 mL. This solution was added under groups by treatment with a mixture of trifuoroacetic acid/dichloro-
vigorous stirring to n-hexane (50 mL). The obtained precipitate was
filtered off, washed with n-hexane, and dried. The resulting solid
methane (95:5, v/v, 1 mL) over a period of 3 h. The resin was filtered
off, and the residue was concentrated and analyzed by ESI mass
was dissolved in a mixture of acetone/toluene (1:1, 2 mL). Good- spectrometry. Yield of Fmoc-AngII-Wang: 1.11 g (resin-bound and
quality crystals of the product, suitable for X-ray analysis, were ob-
tained by slow evaporation of this solution at room temperature.
The crystals were filtered off, washed with cold toluene, and dried.
Yield: 37 mg (0.051 mmol, 51 %). C26H29N8O3ReS (719.83): calcd. C
43.38, H 4.06, N 15.57, S 4.45; found C 43.43, H 4.08, N 15.47, S 5.91.
protected). Mass spectra of the unprotected peptide AngII: ESI-TOF+
(m/z): calcd. [M + H]+ 1046.5418; found 1046.5429. ESI-TOF– (m/z):
calcd. [M – H]– 1044.5272; found 1044.5478.
(GlyN3-AngII)-Wang (10) and GlyN3-AngII: (Fmoc-AngII)-Wang
(200 mg, about 0.03 mmol) was suspended in DMF (5 mL) and
shaken for 30 min. After filtration, the resin was treated with piper-
idine (5 mL) over a period of 1 h and washed several times with
DMF. The resin was then treated with a solution of HOBT (108 mg,
0.8 mmol), HBTU (302 mg, 0.8 mmol), DIPEA (103 mg, 0.8 mmol),
and GlyN3·2Et2O (200 mg, 0.8 mmol) in DMF (2 mL) over a period
of 4 h. The resulting resin was washed several times with DMF and
dichloromethane, and dried. The product (GlyN3-AngII)-Wang was
used in this form for further reactions. For the characterization of
the product, part of the product (10 mg) was treated with a mixture
of trifluoroacetic acid/dichloromethane (95:5, v/v, 1 mL) for 3 h. The
resin was filtered off, and the residue was concentrated and ana-
lyzed by ESI mass spectrometry. Yield of (GlyN3-AngII)-Wang:
187 mg (resin-bound and protected). Mass spectra of the unpro-
IR (KBr): ν = 3442 (s), 3138 (m), 3064 (w), 3978 (m), 3935 (m), 3912
˜
(m), 1678 (s), 1658 (s), 1516 (s), 1417 (s), 1354 (s), 1328 (m), 1296
1
(m), 1132 (w), 1051 (w), 993 (s), 871 (w) cm–1. H NMR (CDCl3): δ =
1.25 (t, J = 7.1 Hz, 3 H, -CH3), 1.40 (t, J = 7.1 Hz, 3 H, -CH3), 3.77–
4.10 (m, 4 H, -CH2-), 4.38 (d, J = 18.7 Hz, 1 H, -CH2-), 4.46 (d, J =
18.8 Hz, 1 H, -CH2-), 4.51 (d, J = 17.7 Hz, 1 H, -CH2-), 4.69 (d, J =
17.7 Hz, 1 H, -CH2-), 5.39 (d, J = 15.2 Hz, 1 H, -CH2-), 5.45 (s, 2 H,
-CH2-), 5.88 (d, J = 15.2 Hz, 1 H, -CH2-), 7.20–7.51 (m, 11 H, Harom
)
ppm. ESI-TOF+ (m/z): calcd. [M + H]+ 721.1714; found [M + H]+
721.1716, [M + Na]+ 743.1540, [M + K]+ 759.1273.
Fmoc-AngII-Wang (9) and AngII: Fmoc-Phe-Wang resin (711 mg,
0.4 mmol) was shaken for 30 min in DMF (5 mL). After filtration, the
resin was suspended in piperidine (2 mL) and shaken for further
30 min. The resin was filtered off and washed several times with
DMF. The resin was then treated with a solution of HOBT (217 mg,
1.6 mmol), DIPEA (207 mg, 1.6 mmol), HBTU (606 mg, 1.6 mmol),
and Fmoc–Pro-OH (509 mg, 1.6 mmol) in DMF (5 mL) over a period
tected peptide GlyN3-AngII: ESI-TOF+ (m/z): calcd. [M
+
H]+
1129.5543; found 1129.5544. ESI-TOF– (m/z): calcd. [M – H]–
1127.5387; found 1127.5402.
of 3 h. After filtration, the resin was washed several times with DMF [ReO(L-TrzGly-AngII)] (11): (GlyN3-AngII)-Wang (20 mg) was sus-
and treated again with piperidine (2 mL) for 30 min. The same
pended in DMF (5 mL) and shaken for 30 min. After filtration, the
procedure was repeated with all other amino acid derivatives:
resin was treated with a solution of [ReO(L)] (58 mg, 0.1 mmol),
Table 3. Crystal data and details of the structure determinations.
[ReO(L)]
[TcO(L)]
[ReO(Bnztrz-L)]
[TcO(Bnztrz-L)]
Formula
C19H22N5O3ReS
586.69
C19H22N5O3STc
498.48
monoclinic
12.832(2)
9.683(1)
16.592(2)
90
C26H29N8O3ReS
719.83
C26H29N8O3STc
631.63
Mw [g mol–1
]
Crystal system
a [Å]
b [Å]
c [Å]
α [°]
monoclinic
12.853(1)
9.661(1)
16.617(2)
90
orthorhombic
14.789(1)
14.737(1)
25.113(2)
90
orthorhombic
14.856(2)
14.732(1)
25.092(2)
90
ꢀ [°]
γ [°]
91.58(1)
90
2062.6(4)
P21/n
91.22(1)
90
2061.1(5)
P21/n
90
90
5473.3(7)
Pbcn
8
1.747
4.561
integration
0.5373/0.7422
45955
90
90
5491.6(9)
Pbcn
8
1.528
0.644
integration
0.7280/0.9353
29023
V [Å3]
Space group
Z
4
4
ρcalcd. [g cm–3
]
1.889
6.023
integration
0.4858 / 0.8215
15645
5558
264
0.0619/0.0822
0.854
1.606
0.831
none
–
14288
5498
264
0.0476/0.0672
0.995
μ [mm–1
Absorption correction
min/Tmax
]
T
No. of reflections
No. of independent
No. parameters
R1/wR2
5364
354
0.1019/0.0701
0.802
7396
348
0.1069/0.0943
1.542
GOF
CCDC nos.
CCDC-1499406
CCDC-1499405
CCDC-1499408
CCDC-1499407
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[8] P. J. Blower, S. Prakash in Perspectives on Bioinorganic Chemistry (Eds.:
R. W. Hay, J. R. Dilworth, K. B. Nolan), JAI Press Inc., CT, 1999, Vol. 4, pp.
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copper sulfate pentahydrate (24.8 mg, 0.1 mmol), and sodium
ascorbate (39.6 mg, 0.2 mmol) in DMF/ethanol/water mixture (1:1:1,
v/v/v, 3 mL) over a period of 3 h. The resin was filtered off and
washed several times with water, DMF, ethanol, and dichloro-
methane. For cleavage of the bioconjugate the resin was treated
with a mixture of trifluoroacetic acid/dichloromethane (95:5, v/v,
1 mL) for 3 h, filtered off, and washed several times with dichloro-
methane. The washing solution was added to the trifluoroacetic
acid solution, and the solvents were removed under reduced pres-
sure. The residue was analyzed by ESI mass spectrometry without
further purification. Yield (crude product): 17 mg (ca. 0.01 mmol).
ESI-TOF+ (m/z): calcd. [M + H]+ 1716.6544; found [M + H]+
1716.6488, [(M + 2 H)/2]+ 858.8291. ESI-TOF– (m/z): calcd. [M – H]–:
1714.6387; found 1714.6336.
X-ray Crystallography: The intensities for the X-ray determinations
were recorded with a STOE IPDS 2T with Mo Kα radiation (λ =
0.71073 Å). Standard procedures were applied for data reduction
and absorption correction. Structure solution and refinement were
performed with SHELXS and SHELXL.[34] Hydrogen atom positions
were calculated for idealized positions and treated with the “riding
model” option of SHELXL. More details on data collections and
structure calculations are summarized in Table 3. Additional infor-
mation on the structure determinations has been deposited with
the Cambridge Crystallographic Data Centre (Cambridge, UK).
[16] H. H. Nguyen, J. D. Castillo Gomez, U. Abram, Inorg. Chem. Commun.
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2012, 43, 123–130.
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CCDC 1499406 (for ReO(L)]), 1499405 (for [TcO(L)]), 1499408 (for
[ReO(Bnztrz-L)]), and 1499407 (for [TcO(Bnztrz-L)]) contain the sup-
plementary crystallographic data for this paper. These data can be
obtained free of charge from The Cambridge Crystallographic Data
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Keywords: Technetium · Rhenium · Click chemistry ·
Bioconjugation · Structure determination
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[34] G. M. Sheldrick, SHELXS-97 and SHELXL-97 – Programs for the solution
and refinement of X-ray crystal structures, University of Göttingen, Gött-
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[2] J. R. Dilworth, S. J. Parrott, Chem. Soc. Rev. 1998, 27, 43–55.
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Received: August 23, 2016
Published Online: ■
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Bioconjugation
J. D. Castillo Gomez, A. Hagenbach,
U. Abram* ............................................. 1–9
Propargyl-Substituted
Thiocarb-
amoylbenzamidines of Technetium
and Rhenium: Steps towards Bio-
conjugation with Use of Click Chem-
istry
Ligands with propargyl-substituted couplings. Successful conjugation of
thiocarbamoylbenzamidines form sta- the rhenium compound with the pept-
ble complexes with {99TcO}3+ and ide angiotensin-II was performed and
{ReO}3+ cores. Prototype Click coupling represents a proof-of-principle solu-
products with benzylazide confirm the tion for ongoing experiments with
suitability of such units for orthogonal 99mTc.
DOI: 10.1002/ejic.201601039
Eur. J. Inorg. Chem. 0000, 0–0
www.eurjic.org
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim