Selective Displacement of a Scorpionand Triazole Ligand from Metallocyclam Complexes Visualised with NMR Spectroscopy

Article abstract of DOI:10.1002/ejic.201601474

Target-activated metal complexes (TAMCs) – complexes that remain benign until reaching a specific biomolecular target, binding to which then effects structural change that turns on cytotoxicity or other activity – hold considerable allure. The successful

Full text of DOI:10.1002/ejic.201601474

A Journal of
Accepted Article
Title: Selective displacement of a scorpionand triazole ligand from
metallo-cyclam complexes visualised using NMR spectroscopy
Authors: Joseph Wong, Nicholas Proschogo, Matthew Todd, and Peter
J. Rutledge
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To be cited as: Eur. J. Inorg. Chem. 10.1002/ejic.201601474
Link to VoR: http://dx.doi.org/10.1002/ejic.201601474

10.1002/ejic.201601474
European Journal of Inorganic Chemistry
Selective displacement of a scorpionand triazole ligand from metallo-
cyclam complexes visualised using NMR spectroscopy.
Joseph K.-H. Wong, Nicholas Proschogo, Matthew H. Todd* and Peter J. Rutledge*
School of Chemistry, The University of Sydney, Sydney, New South Wales 2006, Australia
* Corresponding authors: Dr Peter J. Rutledge, peter.rutledge@sydney.edu.au, +61 2 9351
5020 and Dr Matthew H. Todd, matthew.todd@sydney.edu.au, +61 2 9351 2180.
Abstract
Target-activated metal complexes (TAMCs) – complexes that remain benign until reaching a
specific biomolecular target, binding to which then affects structural change that turns on
cytotoxicity or other activity – hold considerable allure. The successful development of
TAMCs requires analytical methods that allow clear and unequivocal visualisation of
changes in the coordination geometry of these systems in solution. Towards this goal, we
report an NMR-based method to monitor coordination/ de-coordination of a pendant triazole
ligand to/ from metal-cyclam complexes of zinc(II) and mercury(II). This scorpionand ligand
can be displaced from the metal, which remains bound to the macrocyclic ligand, using an
appropriate competing ligand: chloride for the mercury(II) complexes, piperidine or citrate at
1
zinc(II). Triazole displacement may be visualised by monitoring the H NMR resonance of
2
the single triazole C–H proton environment. Using H NMR with a specifically deuterated
1
complex enables the same change to be monitored in a noisy H landscape, as would be
encountered at a protein binding site or other biological context. MALDI-TOF mass
spectrometry experiments provide confirmation that the changes observed by NMR
spectroscopy are due to changes in triazole coordination, rather than stripping of the metal
ion from the complex.
Keywords
azamacrocycles; click chemistry; scorpiand; scorpionand complexes; target activation
1
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European Journal of Inorganic Chemistry
Introduction
Metal complexes have become an increasingly important part of modern medicine as imaging
agents and chemotherapeutics.[1-6] However the modes by which metal complexes interact
with biological systems are often non-specific, leading to serious side effects in the
therapeutic setting and limiting their use.[7] ‘Target-activated’ metal complexes are thus an
enticing prospect: complexes that remain benign and unreactive until reaching their specific
biomolecular target, whereupon binding renders structural and functional change in the
complex to turn on cytotoxicity or other activity (Figure 1a).[8, 9]
While no such ‘target-activated’ complexes have yet been reported, we have previously
described the prototype biotinylated cyclam-CuII complex 1 and its target-activated behaviour
upon interacting with avidin (Figure 1b).[8] When the biotin component of 1 interacts with
avidin, a change in the geometry of the metal ion is triggered, which can be observed using
EPR and ENDOR spectroscopy; i.e. the biotin-avidin binding event can be monitored by
following changes to the coordination sphere of cyclam-bound CuII.
Figure 1: a. The concept of a ‘target-activated’ metal complex (TAMC): a metal complex that is benign and
unreactive until it binds to a specific biomolecular target, which effects a structural change in the complex to turn on
activity (represented by the blue lightning bolt). b. Structures of biotinylated cyclam complexes previously used to
study ‘target activated’ behaviour: triazole coordination to CuII in complex 1 was visualised using EPR and ENDOR
spectroscopy,[8] while the naphthalimide fluorophore in ZnII complex 2 enabled characterisation of biotin:avidin
binding using fluorescence spectroscopy.[9]
The behaviour of such cyclam-triazole-biotin conjugates was further characterised using
fluorescence, by incorporating a naphthalimide fluorophore as well as biotin in the 1,8-
2
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European Journal of Inorganic Chemistry
disubstituted cyclam conjugate 2.[9] Significant quenching of the fluorescence emission of
ZnII complex 2 was observed at up to 4:1 ratio of 2:avidin (Figure 2). This quenching is
quantitatively associated with the biotin-avidin binding event. The observed fluorescence
changes are consistent with altered coordination of the biotinylated triazole to ZnII affecting
the fluorescence mechanisms in operation.
Figure 2: Fluorescence response of 2 in response to the addition of avidin or controls. (Reprinted with permission
from ChemBioChem 2013, 14 (2), 224–229. Copyright © 2013 WILEY-VCH)
However the EPR/ENDOR approach was hampered by the poor signal to noise ratio of these
spectroscopic techniques and the consequent low resolution of the data obtained, while the
fluorescence strategy does not directly visualise metal coordination, so does not specifically
elucidate changes in coordination environment. To create and definitively characterise a
target-activated metal complex, we need an analytical method that enables unequivocal
visualisation of changes in the coordination geometry in these systems.
In separate fluorescence studies of coumarin-substituted cyclam chemosensors for CuII and
HgII, it was observed that adding certain anions to the mercury complex 3 brought about a
revival of the quenched fluorescence.[10] Further investigation revealed that the addition of
chloride to mercury complex 3 triggered displacement of the pendant triazole from the metal
ion, which remained bound to the macrocycle, whereas adding thiosulphate anion caused
demetallation of the same cyclam complex (Figure 3a).
Can we use chloride and related species to selectively displace just the triazole from the metal
in complexes such as 4, 5 and 6 (Figure 3b), and monitor changes in the triazole environment
by NMR spectroscopy? This would then serve as a reference system and enable the direct
visualisation of changes in coordination geometry as is required to definitively characterise
these scorpionand-triazole systems as target-activated metal complexes. NMR spectroscopy
3
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10.1002/ejic.201601474
European Journal of Inorganic Chemistry
could provide a more versatile and operationally simple method to further study the metal
coordination geometry in cyclam conjugates upon ligand-biomolecule binding, and thus
enable validation of the proposed target-activated ‘allosteric scorpionand’ mechanism that is
thought to operate with complexes 1 and 2.[8, 9]
Herein we report the synthesis of metal-cyclam complexes 4, 5 and 6 as model systems to
evaluate the potential of NMR spectroscopy for visualising biological binding events, and
experiments to achieve and characterise anion- and ligand-induced triazole displacement.
2
Triazole-deuterated versions of 4 and 6 were also synthesised for H NMR spectroscopic
evaluation.
Figure 3: a. The anion induced displacement of triazole or demetallation of 3 reported previously;[10] b. structures of
metal-cyclam complexes 4, 5 and 6 used in this study.
Results and Discussion
Synthesis of Ligands and Metal Complexes
The synthetic route to the required phenyl-triazolyl-cyclam ligands 7 and 8 and metal
complexes 4–6 was adapted from published methods, proceeding from tri-Boc-cyclam 9, via
the tri-Boc-azidoethyl-cyclam 10 or tri-Boc-propargyl-cyclam 11 as precursors for the
copper-catalysed azide/ alkyne cycloaddition (CuAAC) or ‘click’ reaction (Scheme 1).[10-13]
For the synthesis of the complexes 4 and 5, in which the pendant phenyl group is connected
via the triazole C4, 2-azidoethyl toluenesulfonate 12 was obtained by first refluxing 2-
bromoethanol with NaN3 in H2O in moderate yield (51%), then used to alkylate tri-Boc
cyclam 9 to give tri-Boc-azidoethyl-cyclam 10 in good yield (67%).[10] An alternative route
4
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European Journal of Inorganic Chemistry
from 9 to 10 was also explored, via alkylation of 9 with bromoacetonitrile, reduction of the
nitrile, and diazotransfer to the resulting amine (Scheme 1 and Scheme S1, Supporting
Information); this route proved inferior, and is discussed in more detail in the Supporting
Information. The azide 10 was reacted with phenylacetylene under the modified click
conditions reported previously,[14-16] to afford the Boc-protected ligand 13 in good yield
(68%). HCl-mediated Boc-deprotection gave the HCl salt of 7 which was neutralised with
excess Ambersep® 900 hydroxide form resin in ethanol to give the cyclam-phenyl ligand 7 in
quantitative yield. Adapting a method reported previously for related systems,[17-19] stirring
with mercury or zinc perchlorate under room temperature followed by filtration afforded the
metal complexes 4 and 5 in good yields (69% and 57% respectively).
Scheme 1: Synthesis of the complexes 4 and 5 containing the ‘reversed’ C4-phenyl triazole connectivity and
mercury complex 6 containing the N1-phenyl triazole connectivity: (i) 2-azidoethyl toluenesulfonate 12, K2CO3,
MeCN, reflux, 72 h, 67%; (ii) BrCH2CN, K2CO3, MeCN, 50 ºC, 16 h, 92%; (iii) H2, Raney-Ni, NH3 in EtOH, rt, 48
h, 54%; (iv) 1H-imidazole-1-sulfonyl azide, K2CO3, CuSO4·5H2O, MeOH, rt, 16 h, 57%; (v) PhC≡CH,
CuSO4·5H2O, sodium ascorbate, THF/H2O (7:3), 50 °C, 16 h, 68%; (vi) a) HCl, dioxane, rt, 16 h, b) Ambersep® 900
hydroxide form resin, MeOH, rt, 10 min, 99%; (vii) M(ClO4)2·xH2O, EtOH, rt, 16 h, 4: 69%, 5: 57%; (viii)
propargyl bromide, K2CO3, MeCN, 86%; (ix) PhN3, CuSO4·5H2O, sodium ascorbate, THF/H2O (7:3), 50 °C, 16 h,
88%; (x) a) HCl, dioxane, rt, 16 h, b) Ambersep® 900 hydroxide form resin, MeOH, 100%; (xi) Hg(ClO4)2·4H2O,
EtOH, rt, 16 h, 75%.
An analogous approach was followed to synthesise the mercury complex 6 which contains
the N1 triazole connectivity to the phenyl group (Scheme 1). Tri-Boc cyclam 9 was alkylated
with propargyl bromide to give tri-Boc-propargyl-cyclam 11 in excellent yield (86%), and
this was reacted with phenyl azide to give the Boc-protected ligand 14 in very good yield
(88%). HCl-mediated deprotection followed by neutralisation with Ambersep® 900
hydroxide form resin afforded ligand 8 in quantitative yield. Complexation of 8 with mercury
perchlorate was initially attempted at 50 ºC to ensure complete reaction; however, droplets of
5
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European Journal of Inorganic Chemistry
elemental mercury were observed, likely arising from the decomposition of the mercury
perchlorate at the elevated temperature, and the resulting mixture could not be purified.
Complexation at room temperature was successful and gave the mercury complex 6 in good
yield (75%).
2
Additional NMR experiments were devised using H NMR spectroscopy to enable easier
monitoring in biological systems, where it is anticipated that overlap with protein-derived 1H
signals may obscure the key triazole proton signal. Given the low natural abundance of
deuterium, deuteration of the triazole would provide a highly selective method to visualise
changes in triazole environment. Two methods were considered for the synthesis of
deuterated triazoles, both of which avoid the need to synthesise new intermediates or starting
materials. The first route would see lithiation of the triazole in protected ligands 13 and 14,
followed by D2O quench; the second approach was to replace the H2O component of the
solvent mixture used for the CuAAC reaction with D2O so that the final protonation[20] step in
2
1
the mechanism delivers H in place of H. By virtue of its simplicity and mild reaction
conditions the second option was preferred; this protocol adapts the procedure reported by
Akula and Lakshaman who have synthesised deuterated triazoles in a biphasic system.[21]
Scheme 2: Synthesis of the deuterated complexes 19 and 20. a. Complex 19 containing the C4-phenyl triazole
connectivity: (i) PhC≡CH, CuSO4·5H2O, sodium ascorbate, THF/D2O (7:3), 50 °C, 16 h, 82%; (ii) a) HCl, dioxane,
rt, 16 h, b) Ambersep® 900 hydroxide form resin, MeOH, rt, 10 min, 67%; (iii) Hg(ClO4)2·4H2O, EtOH, rt, 16 h,
34%. b. Complex 20 containing the N1-phenyl triazole connectivity: (iv) PhN3, CuSO4·5H2O, sodium ascorbate,
THF/D2O (7:3), 50 °C, 16 h, 78%; (v) a) HCl, dioxane, rt, 16 h, b) Ambersep® 900 hydroxide form resin, MeOH, rt,
10 min, 100%.; (vi) Hg(ClO4)2·4H2O, EtOH, rt, 16 h, 31%.
Thus click precursors 10 and 11 were combined with their corresponding coupling partners,
phenyl azide and phenylacetylene, under the same click conditions but using a THF/D2O
solvent system (Scheme 2). These reactions proceeded in very good yields (82% and 79%
6
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respectively) and deuterium incorporation (90% and 92% D-incorporation, determined by
HRMS) to give Boc-protected ligands 15 and 16. Acid-mediated Boc-deprotection gave the
deuterated ligands 17 and 18 in good (67%) and quantitative yield respectively.
Complexation of these ligands with mercury perchlorate afforded the deuterated complexes
19 and 20 in poor but tolerable yields (34% and 31% respectively). HRMS analysis indicated
low levels of deuterium leakage during the conversion of 15 and 16 to metal complexes 19
and 20, which retain ≥80% D-incorporation following the deprotection and metalation steps.
Visualisation of Triazole Displacement by NMR Spectroscopy
Control Experiments with Free Ligands
To establish a baseline for the analysis of these complexes, the free ligands 7 and 8 were
subjected to the high concentrations of chloride needed to induce triazole displacement. Thus
each ligand was dissolved in D2O and titrated with NaCl (in D2O) in the NMR tube.
For the titration of the C4 ligand 7 (Figure S3, Supporting Information), as the concentration
of Cl- was increased, all of the peaks of 7 gradually moved slightly downfield, to a final
change of ca. 0.1 ppm after the addition of 100 equivalents of Cl-; no changes to individual
peak shapes or multiplicities were evident. For the titration of the N1 ligand 8 (Figure S4,
Supporting Information), as the concentration of Cl- increased, the majority of peaks
gradually moved very slightly downfield, to a final change of ca. 0.03 ppm after the addition
of 100 equivalents of Cl-. One peak moved further: the multiplet at ca. 2.80 ppm, which
moved downfield to 2.92 after 100 equivalents of Cl-, and in so doing became deconvoluted
from other signals at 2.73 and 2.85 ppm.
1H NMR Characterisation of Triazole Displacement in Mercury Complex 4
1
The H NMR spectroscopic titration of the C4 HgII complex 4 revealed important changes
upon the addition of Cl- (Figure 4 and Figure S1). Notably, the peaks in the spectra
corresponding to the triazole (8.70) and phenyl group (7.53 to 7.93 ppm) in 4 (Figure 4,
black) moved upfield with increasing concentration of Cl-, in contrast to equivalent signals of
the control 7 (Figure S3). A significant movement of the triazole proton was observed, from
8.70 for the Hg complex 4, to 8.48 after the addition of 5 equivalents of Cl-; further addition
of Cl- had a negligible impact on the triazole proton signal, although a small downfield
movement of this signal was detected at very high equivalents of Cl- (> 50 equivalents). That
five equivalents of Cl- are required to move the triazole proton to its point of greatest
7
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difference suggests a competitive equilibrium process for coordination to mercury between
the triazole and added Cl-, rather than direct substitution of the coordinated triazole by Cl-,
which would presumably require only a stoichiometric amount of Cl-. The observed upfield
movement of the triazole proton signal corresponds to an increase in electron density in the
triazole-phenyl pendant group, and is thus consistent with displacement of the triazole from
the Lewis acidic metal ion. Overall a small upfield movement of ca. 0.03 ppm in the phenyl
signals was observed after the addition of 100 equivalents of Cl- (Figure 4, red); this
comparatively small change was not unexpected as the phenyl group is distant from the
mercury coordination site. The positions of the triazole and phenyl peaks in the final
spectrum of 4 + 100 equiv. Cl- (Figure 4, red) corresponded closely to those of the control 7
+ 100 equiv. Cl- (Figure 4, blue), supporting the hypothesis that the triazole is no longer
coordinated to the metal centre.
In contrast, the cyclam-derived signals at ~1.5–4.0 ppm appeared quite differently in these
two spectra (Figure 4, blue and red). Of the remaining methylene signals, the multiplet at
4.79–4.95 ppm (the upfield portion of the multiplet which is obscured by the residual solvent
signal) is due to the linker CH2 adjacent to the triazole. This signal coalesced into a triplet
after 5 equivalents of Cl- had been added, consistent with greater freedom of movement
gained at this position after triazole displacement, which would render these protons
equivalent on an NMR time-scale. The remaining upfield peaks (δ < 3.6) correspond to
protons of the cyclam CH2 groups and the linker CH2 nearest the cyclam in 4. These signals
were complicated, due to the different geometries that cyclam can adopt after metal binding,
and the consequent chirality of the coordinating nitrogens.[22] The splitting patterns of these
methylenes did change a little at high concentrations of Cl-, however these peaks remained at
essentially the same positions.
Most importantly, this region of the spectrum was clearly very different to the spectrum of
the metal-free sample (Figure 4, blue), suggesting that the metal ion remained bound to the
1
macrocycle even after addition of 100 equiv. Cl- (Figure 4, red). In comparison with the H
NMR spectrum of the free ligand control 7 (Figure 4, blue), the cyclam-derived methylene
signals in the spectra of both the chloride-free mercury complex 4 (Figure 4, black) and 4 +
100 equiv. Cl- (Figure 4, red) appeared significantly downfield (ca. 1 ppm). Additionally, in
the spectra of the complex 4, these signals remained convoluted and overlapping after
chloride addition, compared to the well resolved methylene signals of 7 + 100 equiv. Cl-.
8
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This suggests strongly that HgII remained complexed to cyclam in 4, even after addition of
100 equiv. Cl-.
Thus the change in the 1H NMR shift of the triazole signal from 8.70 to 8.48 ppm, combined
with the minimal changes to the methylene signals indicate that (i) mercury remained bound
to the cyclam, but (ii) the pendent triazole had been displaced by the addition of Cl-, and (iii)
triazole displacement from the metal manifested in a clearly discernible upfield shift in the 1H
signal due to the triazolyl CH.
Figure 4: 1H NMR spectroscopic titration of the C4 HgII complex 4 with Cl-: free ligand 7 + 100 equiv. Cl- (Blue), 4 + 100
equiv. Cl-‘ (Black), 4 (Red); : Triazole CH, : Phenyl CH, : Linker CH2 adjacent to phenyl, : Remaining CH2.
1H NMR Characterisation of Triazole Displacement in Zinc Complex 5
Initial attempts to effect the displacement of the ZnII-coordinated triazole in the C4 complex 5
2-
using anions (Cl-, SCN- and S2O3 ) or imidazole as the competing ligand were unsuccessful,
with no significant changes to peak positions observed (Figure S5). These negative results
were not surprising given that zinc is considered marginally hard, while mercury is a soft
metal according to the Hard and Soft Acids and Bases Principle, and the strength of their
binding to a variety of ligands differs accordingly.[23] This is apparent in the stability
constants for complex formation of HgII with Cl- (log K1 = 6.7) compared to those of ZnII with
9
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Cl- (0.5), SCN- (0.9), S2O32- (2.3) and imidazole (2.6).[24] The stability constant for complex
formation between aqueous ZnII and citrate is 5.0.[24]
)
Instead displacement of the triazole within complex 5 was achieved using either piperidine or
1
citrate as the competing ligand. In the H NMR spectrum of the ZnII complex 5 (Figure 5,
black) the triazole proton appeared as several peaks of varying intensity, presumed to arise
from the different geometries the macrocycle can adopt after metal binding.[22] (This is a
contrast to spectra of HgII complex 4, in which the signal due to this proton appeared as a
1
Figure 5: H NMR spectroscopic titration of the C4 ZnII complex 5 with piperidine: free ligand 7 + 100 equiv. piperidine
(Blue), 5 + 100 equiv. piperidine (Red), 5 (Black); : Triazole CH, : Phenyl CH, : Linker CH2 adjacent to phenyl,
Remaining CH2, : Piperidine CH2.
:
singlet.) After addition of 100 equivalents of piperidine these triazole signals had moved
upfield by ca. 0.15 ppm, and the two largest peaks at 8.67 and 8.64 ppm coalesced into one
major singlet peak at 8.49 (Figure 5, red). In the complex 5, the peaks corresponding to the
pendant phenyl group appeared as multiplets in the range 7.50–7.65 and an apparent triplet at
7.98 ppm; after addition of 100 equivalents of piperidine, the multiplets had moved upfield
by ca. 0.03 ppm and resolve into two distinct triplets at 7.58 and 7.49, while the apparent
triplet at 7.98 moved to become an apparent doublet at 7.88 ppm. Of the higher field
methylene protons, the peaks corresponding to the linker CH2 adjacent to the triazole were
10
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obscured by the residual solvent while the various overlapping multiplets due to cyclam-CH2
groups did not move significantly, even after addition of 100 equivalents of piperidine.
Titration of the ZnII complex 5 with trisodium citrate (Figure S6) revealed changes in the
peaks consistent with those observed in the piperidine titration, and with the Cl- titration of
the HgII complex 4. The two major triazole peaks moved upfield from 8.67 and 8.63 ppm in
the spectrum of 5 without added citrate, to 8.58 and 8.50 in the spectrum of 5 + 100 equiv.
citrate. Of the peaks corresponding to the pendant phenyl group, the multiplets (7.50–7.65)
moved upfield by ca. 0.03 ppm, while the previously assigned apparent triplet at 7.97
presented as a double-doublet (Figure S6, black), but became an apparent triplet at 7.89 after
the addition of 100 equivalents of citrate (Figure S6, red).
As in the piperidine experiment, the peak corresponding to the linker CH2 adjacent to the
triazole was obscured by the residual solvent signal and the complex signals arising from
cyclam environments remained significantly different to the spectrum of the free ligand 7,
after the addition of 100 equivalents of citrate.
1H NMR Characterisation of Triazole Displacement in Mercury Complex 6
Ligand 8 (phenyl group at N1, cyclam at C4, 1 CH2 group between cyclam and triazole) has
the opposite triazole connectivity to 7 (phenyl group at C4, cyclam at N1, 2 CH2 groups
between cyclam and triazole), and thus presents a subtly different coordination environment
1
to a ligating metal ion (Figure 3). As a consequence, the H NMR titration of mercury
complex 6 with increasing concentrations of Cl- (Figure S7) was less clear cut.
The change in the position of the signal due to the triazole proton was noticeably less
pronounced than with complex 4: this signal comes at 8.70 ppm for the N1 complex 6
(Figure S7, black) and moves upfield to 8.63 after the addition of 100 equivalents of Cl-
(Figure S7, red), a change of only 0.07 ppm. Contrast this to the response of the C4 complex
4, in which this proton moved by 0.22 ppm after only 5 equivalents of Cl- were added. While
the triazolyl and phenyl protons of the N1 complex 6 moved upfield as Cl- was added, there
remained a significant difference between the position of these shifts in the spectra of 6 + 100
equiv. Cl- (Figure S7, red) and of the control 8 + 100 equiv. Cl- (Figure S7, blue).
The two doublets of the N1 complex 6 at 3.89 and 4.37 ppm – each due to one proton in the
CH2 linker between cyclam and triazole – coalesced at 4.27 after the addition of 100 equiv. of
Cl-, mirroring the behaviour of the CH2-triazole signals of the C4 complex 4. The remaining
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peaks (δ < 3.5) correspond to the cyclam CH2 groups and displayed complex changes to
splitting patterns while remaining at the same chemical shifts throughout the titration, as seen
with the C4 complex 4.
Overall this NMR titration did not deliver unequivocal evidence that the addition of Cl- to the
mercury complex 6 displaces the coordinated triazole. More significantly, it does not offer a
clear means to monitor triazole displacement by NMR spectroscopy. Consequently, further
investigations focused on the C4 connectivity, with HgII complex 4, ZnII complex 5, and
deuterated HgII complex 15.
Visualisation of Triazole Displacement by 2H NMR
1
Having ascertained using H NMR that chloride-mediated triazole displacement from
complex 4 manifests in a 0.22 ppm shift in the triazole CH signal, we wished to confirm that
a similar change occurs in the corresponding deuterated complex 19. Comparing the 2H NMR
spectrum of 19 + 100 equiv. Cl- (Figure 6, red) with the spectrum of complex 19 (Figure 6,
black), it was evident that the deutero-triazole C–D signal had undergone a corresponding
shift upfield, from 8.69 to 8.49 ppm after the addition of 100 equiv. of Cl-.
Figure 6: 2H NMR spectra of: 19 + 100 equiv. Cl- (red), 19 (black); spectra obtained in H2O with D2O as reference.
1
These 2H chemical shifts are in excellent agreement with those seen in the H NMR spectra:
8.69 vs. 8.70 for the complexes, 8.49 vs. 8.48 for the complexes after Cl- addition. Given this
close correspondence with the 1H NMR results, and the ease in which the deuterated triazoles
2
1
were generated, H NMR offers a useful complement to the H NMR investigation in more
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complicated systems, such as biotin-avidin binding and other ligand-biomolecule
interactions.
Mass Spectrometric (MS) Investigations
To validate conclusions drawn from the NMR experiments – i.e. that the added competing
species (chloride, piperidine or citrate) displace the triazole from the metal in these
complexes without disrupting metal:cyclam ligation – mass spectra were recorded for
complexes 4, 5 and 6 in the absence and presence of added competing species.
Initial experiments using electrospray ionisation (ESI) MS were unsuccessful, on account of
two complicating factors. First, the conditions employed triggered a significant degree of
metal displacement from both of the C4-linked complexes 4 and 5: the base peak in the
spectra of both complexes (Figures S8 and S9)was the signal at m/z 372.2 corresponding to
-
the demetallated ligand [7+H]+. Secondly, some of the ClO4 counterions of complexes 4, 5
and 6 were reduced under ESI conditions, generating Cl- and thence forming chloride adducts
of these metal complexes even in the absence of extraneous chloride. For C4 HgII complex 4,
a set of peaks centred at m/z 608.2 showed the characteristic isotope pattern for mercury and
correspond to [4–2ClO4+Cl]+ (Figure S8); in the ESI spectrum of C4 ZnII complex 5, a signal
at m/z 470.2 corresponds to Cl- adduct [5–2ClO4+Cl]+ (Figure S9); with N1 HgII complex 6,
a set of peaks at m/z 594.1 are due to Cl- adduct [6–2ClO4+Cl]+) (Figure S10). Attempts to
inhibit Cl- adduct formation by changing ionisation voltages on the spectrometer were not
successful.
While ESI is considered a soft ionisation technique, evidently the spectrometer environment
is sufficiently high in energy to initiate reactions that disrupt or destroy these coordination
-
complexes, and promote reduction of the ClO4 counterion to Cl-. Colton and Dakternieks
previously observed formation of Cl- adducts from perchlorates under ESI+ MS conditions,
during a study of mercury phosphine complexes with perchlorate counterions.[25] In the
current context, this outcome presents a problem for our experimental design, given the
intended use of Cl- to displace the triazole from mercury complexes 4 and 6. Indeed the ESI
spectrum of 4 + 100 equiv. Cl- (Figure S11) looks very similar to the spectrum of 4 alone
(Figure S8), while the spectrum of 6 + 100 equiv. Cl- (Figure S12) is dominated by the
demetallated species [8+H]+, though there is a cluster of very weak peaks centred at m/z
595.1 due to the Cl- adduct [6–2ClO4+Cl]+. The ESI MS of complex 5 + 100 equiv.
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piperidine (Figure S13) presents different challenges, as it does not show any ion
recognisable as corresponding to the complex 5, piperidine adducts of 5, or even the free
ligand 8.
The problems encountered with decomposition of the metal complexes and reduction of
perchlorate counterions were overcome by using MALDI-TOF MS. Using α-cyano-4-
hydroxycinnamic acid (α-CCA) as the MALDI matrix, the MALDI-TOF MS of the C4 HgII
complex 4 (Figure S14) showed sets of peaks corresponding to the loss of two perchlorates
(m/z 572.3, [4–2ClO4–H]+) and to loss of one perchlorate (m/z 672.2, ([4–ClO4]+);
importantly, no Cl- adducts were observed in the absence of added extraneous chloride.
Upon the addition of 100 equiv. of Cl- (Figure S15), a new set of strong signals
corresponding to the Cl- adduct of complex 4 ([4–2ClO4+Cl]+) were apparent at m/z 608.2.
The original set of peaks at m/z 672.2 ([4–ClO4]+) had disappeared, while the signal centred
at m/z 572.3 ([4–2ClO4–H]+)) remained. These two sets of MALDI-TOF data for the complex
4 substantiate the successful Cl-mediated triazole displacement observed in the NMR
experiments, and the proposed mechanism involving Cl- coordination to the HgII centre while
the metal remains bound to cyclam.
In the MALDI-TOF MS of ZnII complex 5 (Figure S16), the most important signals were a
set of peaks at m/z 534.2 ([5–ClO4]+), and an adduct species formed between 5 and the α-
CCA matrix at m/z 623.3 ([5–2ClO4–H+αCCA]+). However, the spectra arising from this
complex with added piperidine (100 equiv., Figures S17 and S18) did not provide
confirmatory evidence for the 5:piperidine complex that had been anticipated on the basis of
the NMR data.
Working instead with the 2′,4′,6′-trihydroxyacetophenone matrix (THAP), with and without
added ammonium citrate, the ‘THAP-only’ MS of 5 (Figure S19) showed signals for [5–
2ClO4–H]+ at m/z 434.3, and [5–ClO4]+ at m/z 534.2; there were also a Cl- adduct [5–
2ClO4+Cl]+ at m/z 470.2, and a matrix adduct [5–2ClO4–H+THAP]+ at m/z 602.3. In contrast,
the MS of 5 run with the THAP matrix and added citrate (Figure S20) only held two of these
signals: [5–2ClO4–H]+at m/z 434.2, and the THAP adduct at 602.2. The base peak set in this
spectrum was centred at m/z 626.1 which corresponds to the dibasic citrate adduct [5–
2ClO4+H+citrate2-]+. This result confirmed that citrate is able to coordinate to the ZnII
complex 5 without displacing it from the macrocycle, and supports the conclusions drawn
14
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10.1002/ejic.201601474
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from the NMR experiments that citrate displaces the triazole from ZnII in 5 as Cl- does with
the corresponding HgII complex 4.
The MALDI-TOF MS spectrum of the N1 HgII complex 6 (Figure 21) also showed sets of
signals arising from the loss of two perchlorates (m/z 558.3 [6–2ClO4–H]+) and loss of one
perchlorate (m/z 658.2 ([6–ClO4]+), with no evidence of unwanted Cl- adducts. As was the
case in the NMR experiments with complex 6 detailed above, the changes wrought by adding
Cl- to this complex were less clear cut. The MALDI-TOF MS spectrum of 6 + 100 equiv. Cl-
(Figure S22) showed a variety of species, but did include peaks that correspond to the Cl-
adduct [6–2ClO4+Cl]+ centred at m/z 594.2, corroborating the triazole displacement observed
by NMR spectroscopy, along with the original set of peaks for [6–2ClO4–H] at m/z 558.2.
Conclusion
These experiments demonstrate that it is possible to displace the triazole ligand from these
mercury and zinc complexes without disrupting macrocycle coordination, if the competing
ligand is matched to the metal complex. Chloride anion triggers triazole displacement in
mercury complexes 4 and (at least partially) 6, piperidine and citrate do similarly with zinc
complex 5. The resulting displacement can be visualised by a change in the NMR resonance
of the triazole C–H (1H NMR) or, in the specifically deuterated analogue 19, the triazole C–
D. Using 2H NMR offers orthogonality for work with biomolecular systems, in which the key
1
triazole H signal would likely be obscured by protein or nucleic acid-derived 1H signals.
MALDI-TOF mass spectrometry verifies the conclusion that the metal-cyclam complex
remains intact after addition of chloride (to 4 and 6) or citrate to 5; the 5:piperidine complex
proved more elusive and less stable under the MS conditions investigated.
While spectral changes are observed with all metal complexes tested, the C4-linked mercury
complex 4 offers the best combination of operational simplicity and clear, discernible change
1
in the key H resonance. The triazole proton appears as a distinct singlet in spectra of
mercury complex 4 before and throughout titration experiments, and in the free ligand 7; it
undergoes an upfield shift of 0.22 ppm upon triazole displacement from the metal. This
2
change is replicated in the H spectra and provides a clear reference marker for triazole
coordination/ decoordination in subsequent work with ligand/ biomolecule binding events
like those detailed in Figure 1. The corresponding proton in the C4-linked zinc complex 5
undergoes similar change in position and might reasonably be utilised as a marker in a similar
15
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10.1002/ejic.201601474
European Journal of Inorganic Chemistry
manner. However this proton presents as a comparatively complicated mixture of several
signals in spectra of the complex, which brings additional complexity to analysis.
Changes in the spectra of N1-linked mercury complex 6 upon addition of Cl- are less
pronounced, such that this system does not enable clear monitoring of triazole displacement
by NMR. In discussing differences in the behaviour of the mercury complexes 4 and 6, it is
interesting to consider structural data previously reported for similar complexes. Crystal
structures of complexes that include ligands with triazole connectivity as in 4 and 5 (phenyl
group at C4, cyclam at N1) show that the pendant triazole coordinates to the metal through its
central nitrogen atom (N2) and thus forms a six-membered metallocycle (as shown for 4 and
5 above, Figure 3).[10, 16] Whereas in structures of complexes that contain ligands with the
triazole connectivity of 6 (phenyl group at N1, cyclam at C4), the triazole coordinates through
nitrogen atom N3 (derived originally from the terminal N of the azide coupling partner)
forming a five-membered metallocycle (as shown for 6 above, Figure 3).[8, 16] This difference
in chelate size is presumably the reason behind the different response of the triazole to added
Cl- in complexes 4 and 6.
2
While the H NMR experiments demonstrate the concept of orthogonality designed to
2
simplify detection in biological environments, the inherently low sensitivity of H NMR is
likely to be problematic with regard to the concentrations of proteins and other biomolecules
required for binding studies. To extend this concept, the next step is to prepare selectively
19
fluorinated triazoles,[26, 27] then exploit the improved sensitivity of F NMR to advance the
method.
1
2
In parallel, work is underway to apply this method, using H and H NMR in the first
instance, to characterise the biotin/ avidin couple previously investigated (Figure 1). Using
both the original and reversed triazole connectivities, the aim is to verify the changes in
triazole:metal coordination upon biomolecule binding previously proposed on the basis of
EPR/ENDOR and fluorescence experiments. The ultimate goal of this work is to apply
insights gleaned from these model studies to the development of target-activated metal
complexes for therapeutic applications.
16
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10.1002/ejic.201601474
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Experimental
Synthetic Procedures
Safety note: Sodium azide, organic azides and perchlorate salts of metal complexes with
organic ligands are potentially explosive. Only small amounts of material should be prepared
and these should be handled with caution.
Tri-tert-butyl 11-(2-(4-phenyl-1H-1,2,3-triazol-1-yl)ethyl)-1,4,8,11-tetraazacyclotetradecane-
1,4,8-tricarboxylate 13
Tri-Boc-azidoethylcyclam 10 (108 mg, 0.19 mmol) and phenylacetylene (25 µL, 0.23 mmol)
were reacted using general synthetic procedure A and purified by automated flash column
chromatography (10 g cartridge, 30% EtOAc in P.E. over 3 CV, 55% over 2 CV and 84%
over 9 CV) to afford click product 13 as a white foam (109 mg, 87%). 1H NMR (CDCl3, 300
MHz): δ 1.44 (s, 9H), 1.46 (s, 18H), 1.62 – 1.80 (m, 4H), 2.46 – 2.56 (m, 2H), 2.62 – 2.73
(m, 2H), 2.98 (t, 2H, J 6.5), 3.09 – 3.38 (m, 12H), 4.41 (t, 2H, J 6.6), 7.29 – 7.37 (m, 1H),
7.38 – 7.47 (m, 2H), 7.78 – 7.87 (m, 3H). LRMS (ESI+): m/z 672.1 ([M+H]+, 13%), 694.2
([M+Na]+, 100%), 1364.9 ([2M+Na]+, 61%). HRMS (ESI+): m/z Calcd. for C35H57N7NaO6+
[M+Na]+ 694.4263, found 694.4261. The spectroscopic data were in agreement with those in
the literature.[10]
1-(2-(4-Phenyl-1H-1,2,3-triazol-1-yl)ethyl)-1,4,8,11-tetraazacyclotetradecane 7
Protected ligand 13 (77 mg, 0.12 mmol) was deprotected using general synthetic procedure C
1
to afford the Boc-deprotected target 7 as a sticky off-white solid (42 mg, 99%). H NMR
(D2O, 500 MHz): δ 1.45 – 1.53 (m, 2H), 1.53 – 1.59 (m, 2H), 2.09 (t, 2H, J 6.3), 2.20 (t, 2H,
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J 5.5), 2.25 – 2.36 (m, 4H), 2.43 (t, 2H, J 5.6), 2.53 (t, 2H, J 5.7), 2.54 – 2.64 (m, 4H), 2.88
(t, 2H, J 5.2), 4.51 (t, 2H, J 5.4), 7.43 (t, 1H, J 7.6), 7.51 (t, 2H, J 7.6), 7.79 (d, 2H, J 7.7),
8.29 (s, 1H). 13C NMR (D2O, 75 MHz): δ 25.6, 26.5, 44.2, 45.2, 45.3, 45.6, 45.9, 46.6, 48.4,
50.3, 52, 52.5, 123.1, 125.7, 129.3, 129.6, 129.7, 147.5. LRMS (ESI+): m/z 372.2 ([M+H]+,
+
100%). HRMS (ESI+): m/z Calcd. for C20H34N7 372.2870, found 372.2870. The
spectroscopic data were in agreement with those in the literature.[10]
Mercury perchlorate complex of 1-(2-(4-phenyl-1H-1,2,3-triazol-1-yl)ethyl)-1,4,8,11-tetra-
azacyclotetradecane 4
Ligand 7 (66 mg, 0.18 mmol) was complexed with Hg(ClO4)2·4H2O (84 mg, 0.18 mmol) at
rt using general synthetic procedure E to afford mercury complex 4 as a white solid (94 mg,
69%). m.p.: 135 ºC (decomp.). LRMS (ESI+): m/z 372.2 ([M–Hg–2ClO4+H]+, 100%),
608.2 ([M–2ClO4+Cl]+, 50%). LRMS (MALDI-TOF, α-CCA): m/z 572.3 ([M–2ClO4–H]+,
+
100%), 672.2 ([M–ClO4]+, 13%). HRMS (ESI+): m/z Calcd. for C20H33ClHgN7 [M–
2ClO4+Cl]+ 608.2187, found 608.2190. FTIR (ATR) νmax/cm-1: 3265, 1470, 1453, 1076, 930,
884, 842, 770, 699, 621.
Zinc perchlorate complex of 1-(2-(4-phenyl-1H-1,2,3-triazol-1-yl)ethyl)-1,4,8,11-tetraaza-
cyclotetradecane 5
Ligand 7 (42 mg, 0.11 mmol) was complexed with Zn(ClO4)2·6H2O (46 mg, 0.12 mmol) at
rt using general synthetic procedure E to afford zinc complex 5 as an off-white solid (41 mg,
57%). m.p.: 173 ºC (decomp.). LRMS (ESI+): m/z 217.5 ([M–2ClO4]2+, 100%), 375.2 (([M–
Hg–2ClO4+H]+, 73%), 434.1 ([M–2ClO4–H]+, 43%), 470.2 ([M–2ClO4+Cl]+, 21%), 534.1
([M–ClO4]+, 45%); LRMS (MALDI-TOF, α-CCA): m/z 534.2 ([M–ClO4]+, 100%); LRMS
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(MALDI-TOF, THAP + dibasic ammonium citrate): m/z 626.2 ([M–2ClO4+citrate-]+. HRMS
(ESI+): m/z Calcd. for C20H33ClN7O4Zn+ [M–ClO4]+ 534.1569, found 534.1572. FTIR
(ATR) νmax/cm-1: 3236, 2879, 1456, 1066, 1000, 978, 929, 886, 848, 770, 740, 697, 620, 508.
Tri-tert-butyl 11-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-1,4,8,11-tetraazacyclotetradecane-
1,4,8-tricarboxylate 14
Tri-Boc-propargylcyclam 11 (379 mg, 0.67 mmol) and phenyl azide (102 mg, 1.00 mmol)
were reacted using general synthetic procedure A and purified by automated flash column
chromatography (10 g cartridge, 12% EtOAc in P.E. over 1 CV, 12 to 100% over 10 CV and
1
100% over 1 CV) to afford click product 14 as an off-white foam (385 mg, 88%). H NMR
(CDCl3, 500 MHz): δ 1.41 (s, 9H), 1.44 (s, 18H), 1.75 (qn, 2H, J 6.3), 1.86 – 1.97 (m, 2H),
2.46 – 2.55 (m, 2H), 2.62 – 2.71 (m, 2H), 3.23 – 3.52 (m, 12H), 3.87 (s, 2H), 7.40 – 7.46ma
(m, 1H), 7.49 – 7.56 (m, 2H), 7.77 (d, 2H, J 7.8), 7.82 – 8.03 (m, 1H). 13C NMR (CDCl3, 100
MHz): δ 26.7, 28.5, 28.5, 28.5, 45.6, 47.7, 49, 51.2, 79.6, 120.5, 128.6, 129.7, 137.1, 155.9.
LRMS (ESI+): m/z 658.5 ([M+H]+, 23%), 680.4 ([M+Na]+, 100%). HRMS (ESI+): m/z
+
Calcd. for C34H56N7O6 [M+H]+ 658.4287, found 658.4288. FTIR (ATR) νmax/cm-1: 2975,
2932, 1684, 1503, 1478, 1414, 1366, 1245, 1163, 1043, 761, 732.
1-((1-Phenyl-1H-1,2,3-triazol-4-yl)methyl)-1,4,8,11-tetraazacyclotetradecane 8
Protected ligand 14 (110 mg, 0.12 mmol) was deprotected using general synthetic procedure
C to afford Boc deprotected 8 as a sticky off-white solid (60 mg, 100%). 1H NMR (DMSO-
d6, 500 MHz): δ 1.51 – 1.64 (m, 2H), 1.71 – 1.82 (m, 2H), 2.46 (t, 2H, J 5.5), 2.48 – 2.55 (m,
2H), 2.55 – 2.74 (m, 12H), 3.83 (s, 2H), 7.48 (t, 1H, J 7.4), 7.60 (t, 2H, J 7.9), 7.89 (d, 2H, J
7.6), 8.69 (s, 1H). 13C NMR (DMSO-d6, 125 MHz): δ 25.4, 27.8, 45.9, 46.5, 47.2, 47.8, 48,
49.4, 50.6, 53.3, 56, 62.8, 119.9, 121.9, 128.5, 129.9, 136.7, 143.3. LRMS (ESI+): m/z 358.2
19
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([M+H]+, 100%). HRMS (ESI+): m/z Calcd. for C19H31N7Na+ 380.2533, found 380.2533.
FTIR (ATR) νmax/cm-1: 2975, 2932, 1687, 1502, 1478, 1465, 1414, 1391, 1366, 1296, 1246,
1164, 861, 761, 733, 688.
Mercury perchlorate complex of 1-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-1,4,8,11-
tetraaza-cyclotetradecane 6
Ligand 8 (50 mg, 0.14 mmol) was complexed with Hg(ClO4)2·4H2O (73 mg, 0.16 mmol) at
rt using general synthetic procedure E to afford mercury complex 6 as a white solid (87 mg,
75%). m.p.: 190 ºC (decomp.). LRMS (ESI+): m/z 279.5 ([M–2ClO4]2+, 60%), 594.1 ([M–
2ClO4+Cl]+, 100%). LRMS (MALDI-TOF, α-CCA): m/z 558.3 ([M–2ClO4–H]+, 100%),
658.2 ([M–ClO4]+, 41%). HRMS (ESI+): m/z Calcd. for C19H31ClHgN7O4+ [M–ClO4]+
658.1827, found 658.1827. FTIR (ATR) νmax/cm-1: 3266, 1502, 1468, 1451, 1426, 1260,
1200, 1076, 1028, 944, 929, 886, 870, 852, 768, 693, 621, 532.
Tri-tert-butyl 11-(2-(4-phenyl-1H-1,2,3-triazol-1-yl-5-d)ethyl)-1,4,8,11-tetraazacyclotetra-
decane-1,4,8-tricarboxylate 15
Tri-Boc-azidoethylcyclam 10 (515 mg, 0.904 mmol) and phenylacetylene (119 µL, 1.09
mmol) were reacted using modified general synthetic procedure A (temperature = rt and H2O
replaced by D2O) and purified by flash column chromatography (EtOAc: P.E. = 2:3 to 9:1) to
afford click product 15 as a white foam (495 mg, 81%). 1H NMR (CDCl3, 400 MHz): δ 1.43
(s, 9H), 1.45 (s, 18H), 1.61 – 1.78 (m, 4H), 2.46 – 2.55 (m, 2H), 2.62 – 2.73 (m, 2H), 2.97 (t,
2H, J 6.5), 3.09 – 3.38 (m, 12H), 4.41 (t, 2H, J 6.5), 7.29 – 7.35 (m, 1H), 7.38 – 7.45 (m, 2H),
7.80 – 7.85 (m, 2H). 13C NMR (CDCl3, 100 MHz): δ 26.5, 28.4, 28.5, 28.5, 28.8, 45.8, 46.8,
47.3, 47.7, 48.3, 52.3, 53.6, 55.1, 79.7, 79.8 120.1 (t, JC-D 25.9), 125.6, 128.1, 128.8, 130.6,
20
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147.4, 155.5, 155.6, 155.7. LRMS (ESI+): m/z 673.1 ([M+H]+, 62%), 695.1 ([M+Na]+,
+
100%). HRMS (ESI+): m/z Calcd. for C35H56DN7NaO6 [M+Na]+ 695.4322, found
695.4325. FTIR (ATR) νmax/cm-1: 2976, 2932, 1677, 1462, 1413, 1365, 1246, 1155, 1072,
1027, 911, 861, 775, 727, 695, 646.
1-(2-(4-Phenyl-1H-1,2,3-triazol-1-yl-5-d)ethyl)-1,4,8,11-tetraazacyclotetradecane 17
Protected ligand 15 (475 mg, 0.706 mmol) was deprotected using general synthetic procedure
C to afford Boc deprotected 17 as a hygroscopic beige solid (175 mg, 67%). 1H NMR (D2O,
500 MHz): δ 1.44 – 1.54 (m, 2H), 1.54 – 1.61 (m, 2H), 2.09 (t, 2H, J 6.4), 2.20 (t, 2H, J 5.6),
2.26 – 2.63 (m, 2H), 2.63 – 2.38 (m, 2H), 2.44 (t, 2H, J 5.8), 2.53 (t, 2H, J 5.8), 2.56 – 2.66
(m, 4H), 2.90 (t, 2H, J 5.5), J 4.52 (t, 2H, J 5.5), 7.43 – 7.48 (m, 1H), 7.51 – 7.56 (m, 2H),
2
13
7.80 – 7.84 (m, 2H). H NMR (H2O, 61 MHz): 8.47 (br s). C NMR (D2O, 125 MHz): δ
25.7, 26.6, 44.1, 44.9, 45.1, 45.6, 45.8, 46.3, 48.4, 50, 52.1, 52.5, 123.1, 125.7, 125.7, 129.2,
129.6, 129.6, 129.7, 147.3. LRMS (ESI+): m/z 373.3 ([M+H]+, 100%). HRMS (ESI+): m/z
+
Calcd. for C20H33DN7 [M+H]+ 373.2933, found 373.2937. FTIR (ATR) νmax/cm-1: 2944,
2821, 1462, 1360, 1286, 1224, 1123, 1073, 1049, 778, 728, 697.
Mercury perchlorate complex of 1-(2-(4-phenyl-1H-1,2,3-triazol-1-yl-5-d)ethyl)-1,4,8,11-
tetraazacyclotetradecane 19
Ligand 17 (55 mg, 0.15 mmol) was complexed with Hg(ClO4)2·4H2O (74 mg, 0.16 mmol) at
rt using general synthetic procedure E to afford mercury complex 19 as a hygroscopic white
2
solid (39 mg, 34%). H NMR (H2O, 61 MHz): 8.68 (br s). LRMS (ESI+): m/z 279.9 ([M–
2ClO4]2+, 100%), 658.9 ([M–ClO4]+, 16%). HRMS (ESI+): m/z Calcd. for
21
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+
C20H32DClHgN7O4 [M–ClO4]+ 673.2048, found 673.2046. FTIR (ATR) νmax/cm-1: 3264,
2872, 1453, 1081, 930, 884, 778, 730, 699, 622.
Tri-tert-butyl
11-((1-phenyl-1H-1,2,3-triazol-4-yl-5-d)methyl)-1,4,8,11-tetraazacyclotetra-
decane-1,4,8-tricarboxylate 16
Tri-Boc-propargylcyclam 11 (320 mg, 0.594 mmol) and phenyl azide (85 mg, 0.71 mmol)
were reacted using modified general synthetic procedure A (temperature = rt and H2O
replaced by D2O) and purified by flash column chromatography (EtOAc: P.E. = 3:2) to afford
1
click product 16 as an off-white foam (305 mg, 78%). H NMR (CDCl3, 400 MHz): δ 1.40
(s, 9H), 1.44 (s, 18H), 1.68 – 1.81 (m, 2H), 1.84 – 1.98 (m, 2H), 2.45 – 2.55 (m, 2H), 2.63 –
2.71 (m, 2H), 3.25 – 3.50 (m, 12H), 3.86 (s, 2H), 7.39 – 7.46 (m, 1H), 7.48 – 7.56 (m, 2H),
7.76 (d, 2H, J 7.8). 13C NMR (CDCl3, 100 MHz): δ 26.6, 27, 28.4, 28.5, 28.5, 45.5, 47.2,
48.9, 51.3, 52.1, 53, 79.6, 120.5, 128.6, 129.7, 137.1, 144.2, 155.5, 155.8. LRMS (ESI+): m/z
659.2 ([M+H]+, 96%), 681.1 ([M+Na]+, 100%). HRMS (ESI+): m/z Calcd. for
+
C34H55DN7O6 [M+H]+ 659.4349, found 695.4344. FTIR (ATR) νmax/cm-1: 2975, 2932,
1687, 1478, 1465, 1414, 1366, 1246, 1164, 984, 861, 761, 733, 688.
1-((1-Phenyl-1H-1,2,3-triazol-4-yl-5-d)methyl)-1,4,8,11-tetraazacyclotetradecane 18
Protected ligand 16 (290 mg, 0.440 mmol) was deprotected using general synthetic procedure
1
C to afford Boc deprotected 18 as a sticky off-white solid (158 mg, 100%). H NMR (D2O,
500 MHz): δ 1.75 – 1.83 (m, 2H), 1.98 – 2.06 (m, 2H), 2.72 – 2.77 (m, 2H), 2.79 (t, 2H, J
5.4), 2.87 – 2.96 (m, 4H), 2.98 – 3.07 (m, 6H), 3.08 (t, 2H, J 5.1), 3.90 (s, 2H), 7.57 – 7.63
(m, 1H), 7.57 – 7.69 (m, 2H), 7.76 – 7.81 (m, 2H). 13C NMR (D2O, 125 MHz): δ 23.7, 25.6,
45.6, 45.7, 46.4, 47.4, 48, 49.2, 49.5, 50, 52.7, 54.1, 121.4, 123.6, 129.9, 130.3, 136.6, 144.6.
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European Journal of Inorganic Chemistry
LRMS (ESI+): m/z 359.1 ([M+H]+, 100%). HRMS (ESI+): m/z Calcd. for C19H31DN7+
[M+H]+ 359.2777, found 359.2777. FTIR (ATR) νmax/cm-1: 2946, 2834, 1597, 1500, 1463,
1340, 1226, 1114, 1050, 982, 855, 761, 688, 516.
Mercury perchlorate complex of 1-((1-phenyl-1H-1,2,3-triazol-4-yl-5-d)methyl)-1,4,8,11-
tetraazacyclotetradecane 20
Ligand 18 (65 mg, 0.18 mmol) was complexed with Hg(ClO4)2·4H2O (94 mg, 0.20 mmol) at
rt using general synthetic procedure E to afford mercury complex 20 as a hygroscopic white
solid (43 mg, 31%). LRMS (ESI+): m/z 595.0 ([M–2ClO4+Cl]+, 100%). FTIR (ATR)
νmax/cm-1: 3549, 3272, 2863, 1597, 1500, 1463, 1428, 1068, 986, 931, 916, 863, 763, 688,
621, 517. HRMS (ESI+): m/z Calcd. for C19H30DClHgN7+ [M–2ClO4+Cl]+ 595.2095, found
595.2094.
NMR Titrations
To a solution of the substituted cyclam (0.5 mL, 2.5 mM in D2O) in an NMR tube were added
sequential aliquots of the salt/ competing ligand solution (250 mM in D2O) to deliver 1, 2, 5,
10, 20 and then 100 molar equivalents. Solutions were shaken for 2 minutes to ensure
complete mixing before spectra were recorded; spectra were calibrated to the residual proton
solvent peak in D2O (4.79 ppm).
Acknowledgements
We thank the Australian Research Council (DP120104035) and the National Health and
Medical Research Council (APP1084266) for funding. J. K.-H.Wong was supported by an
Australian Postgraduate Award (APA) from the Australian Government.
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