Rigid luminescent bis-zinc(II)-bis-cyclen complexes for the detection of phosphate anions and non-covalent protein labeling in aqueous solution

Article abstract of DOI:10.1002/ejoc.201100103

A series of water-soluble bis- and tetrakis-Zn^II-cyclen complexes with rigid structures was prepared to enhance the carboxylate and phosphate ion binding response in contrast to analogues with less-confined molecular structures. Boc-protected 6

Full text of DOI:10.1002/ejoc.201100103

FULL PAPER
DOI: 10.1002/ejoc.201100103
Rigid Luminescent Bis-Zinc(II)–Bis-Cyclen Complexes for the Detection of
Phosphate Anions and Non-Covalent Protein Labeling in Aqueous Solution
Mouchumi Bhuyan,[a] Evgeny Katayev,[a] Stefan Stadlbauer,[a] Hiroshi Nonaka,[b]
Akio Ojida,[b] Itaru Hamachi,[b] and Burkhard König*[a]
Keywords: Cross-coupling / Click chemistry / Palladium / Macrocycles / Luminescence / Molecular recognition
A series of water-soluble bis- and tetrakis-ZnII–cyclen com- nescent. They were studied by absorption and emission spec-
plexes with rigid structures was prepared to enhance the
carboxylate and phosphate ion binding response in contrast
to analogues with less-confined molecular structures. Boc-
protected 6-chloro-1,3,5-triazine–bis-cyclen was coupled to
different aryl and alkyl moieties in moderate to high yields
and subsequently converted into the corresponding bis- or
tetrakis-ZnII–cyclen complexes. The bis-ZnII–cyclen moiety is
known for its affinity to anions. Depending on the arene sub-
stituent some of the synthesized synthetic receptors are lumi-
troscopy for their response to the presence of phosphate
anions of biological relevance in buffered aqueous solution at
neutral pH and for their affinity to the genetically encodable
oligoaspartate and glutamate sequences (D4 and E4 tag) re-
cently introduced. The rigid structures of the compounds en-
hance the electronic coupling between the metal complex
binding site and the reporter dye. This leads to an increased
anion binding response in homogeneous aqueous solution.
We have recently reported the application of 1,3,5-tri-
azine-based bis-ZnII–cyclen complexes in phosphate ion re-
Introduction
The ubiquitous presence of phosphates in nature makes cognition.[11] However, the so-far prepared receptors have
their molecular recognition under physiological conditions a rather flexible molecular structure, and even though the
of immense interest.[1] Phosphates are vividly present in complexes bear luminescent labels, they did not respond to
RNA and DNA, in phosphorylated saccharides, and in the presence of phosphate anions in homogeneous solution.
phosphorylated proteins.[2] The nucleotide adenosine tri- As the rigidity of a molecule and the distance and connec-
phosphate (ATP), is the universal energy currency for me- tion between a binding site and a luminescent probe may
tabolism and is involved in intracellular energy transport be crucial to achieve high analyte binding response, we have
for various metabolic processes including biosynthetic reac- designed and synthesized modified bis-ZnII–cyclen recep-
tions, mobility, and cell division. The hydrolysis of ATP in tors, which are more rigid in their molecular structure.
cells produces pyrophosphate (PPi) along with adenosine
monophosphate (AMP) and it plays an important role in
intracellular signaling.[3] Hence, the development of artifi-
cial phosphate anion receptors is of great interest.
Transition metal complexes with vacant coordination
sites are well suited to serve as phosphate ion binding
sites.[4] Some widely used binding receptor moieties in phos-
phate recognition are zinc(II)–dipicolylamine (Dpa) com-
plexes as demonstrated by Hamachi,[5] Hong,[6] Koike,[7]
and Smith[8] and macrocyclic 1,4,7,10-tetraazacyclo-
dodecane (cyclen) transition metal complexes reported by
Kikuchi[9] and Kimura.[10]
Furthermore, the selective luminescent labeling of pro-
teins by markers is an important quest and an ongoing
challenge in molecular biology.[12] Covalent labeling uses re-
active dyes or genetic fusion of fluorescent proteins, for ex-
ample, green fluorescent proteins (GFPs).[13] Non-covalent
labeling strategies employ antibodies or snap tags.[14] Re-
cently, pairs of protein-fused peptide tags and complemen-
tary fluorescent chemical probes were reported for applica-
tions in protein labeling.[15]
The Hamachi group developed a new high affinity pep-
tide tag – artificial probe pair orthogonal to the His tag
– NiII–NTA pair, for protein labeling, employing coordina-
tion chemistry and multivalent interaction between a genet-
ically encodable oligoaspartate sequence (D4-tag) and a
corresponding oligonuclear ZnII–Dpa complex.[16] Because
artificial zinc dipicolylamine (ZnII–Dpa) receptors[17] and
zinc 1,4,7,10-tetraazacyclododecane (ZnII–cyclen) complex
derivatives[18] are both known for their application as phos-
phate binders under physiological conditions, we considered
using ZnII–cyclen derivatives as alternative artificial probes
[a] Institut für Organische Chemie, Universität Regensburg,
93040 Regensburg, Germany
Fax: +49-941-943-1717
E-mail: burkhard.koenig@chemie.uni-regensburg.de
[b] Department of Synthetic Chemistry and Biological Chemistry,
Graduate School of Engineering, Kyoto University,
Katsura campus, Kyoto 615-8510, Japan
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201100103.
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B. König et al.
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for the developed artificial probes for oligoaspartate (D4-
Complex 4c was characterized with the help of single-
tag) and oligoglutamate (E4-tag) tag sequences. Although crystal diffraction analysis. In order to obtain suitable crys-
the coordination geometry of ZnII–Dpa and ZnII–cyclen is tals we used a small amount of zinc(II) chloride present in
rather different, ZnII–cyclen derivatives show a comparable the water solution for crystallization. These conditions led
high binding affinity and fluorescence response and may to the formation of the complex [(4c-2Cl–)2+][ZnCl42–] (Fig-
therefore be used as an alternative artificial non-covalent ure 1). The cyclene moieties coordinate to zinc cations with
protein marker.
typical Zn–N distances of 2.1 Å to the three aliphatic nitro-
gen atoms and one longer Zn–N distance of 2.6 Å to the
triazine nitrogen. The anthracene ring is twisted from the
triazine plane by a torsion angle of 68°.
In attempts to include rigidity in the bis-ZnII–cyclen
complexes, apart from a Suzuki–Miyaura cross-coupling
strategy, two other different strategies, Sonogashira cross-
coupling and the click reaction, were also tried. These ap-
proaches are outlined in Schemes 2 and 3, but have not
been further developed due to the unsatisfactory chemical
yields of some of the reactions. Detailed experimental pro-
cedures and analytical data of all prepared compounds are
provided in the Experimental Section and in the Supporting
Information.
Results and Discussions
Syntheses of ZnII–Cyclen Complexes
The rigid bis-ZnII–cyclen based receptors are synthesized
mainly by using Suzuki–Miyaura cross-coupling reactions
as shown in Scheme 1. Threefold Boc-protected bis-cyclen
and compound 1 were synthesized following an earlier re-
ported procedure.[19] The protection of the cyclen azamac-
rocycle reduces polarity and prevents multi-N-substitution.
Trichlorotriazine reacts with two equivalents of threefold
Boc-protected cyclen to yield 1 in a clean twofold nucleo-
philic aromatic substitution. The remaining chloro substitu-
ent of triazine bis-cyclen 1 was then used for coupling with
UV/Vis and Luminescent Properties of ZnII–Cyclen
Complexes
Some of the synthesized complexes 4 bear fluorophores,
different boronic acids 2 by palladium-catalyzed Suzuki– and their UV/Vis and luminescent properties were investi-
Miyaura reaction. Synthesized ligands 3 are listed in Table 1 gated. The data are summarized in Table 2.
with their isolated yields. Obtained Boc-protected cyclen li-
The highest quantum yields were observed for com-
gands 3 were deprotected with trifluoroacetic acid to give pounds 4b–e. These compounds were studied in detail by
the corresponding ammonium salts in quantitative yields. using fluorescence titrations in 25 mm HEPES buffer,
Finally, complexation of the azamacrocyclic amines with pH 7.4 at 25 °C and binding affinities for carboxylates and
ZnCl2 gave ZnII–cyclen complexes 4.
phosphates were determined. The concentration of HEPES
Scheme 1. Synthesis of triazine bis-ZnII–cyclen complexes 4a–h.
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Detection of Phosphate Anions and Non-covalent Protein Labeling
Table 1. Structures and isolated yields of the synthesized bis- and tetrakis-Boc-protected cyclen ligands 3a–h obtained by Suzuki–Miyaura
cross coupling strategy.
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Scheme 2. Synthesis of modified triazine bis-cyclen ligand 5, for
potential metal complexation by using a Sonogashira cross-cou-
pling strategy.
Figure 1. ORTEP rendered view of the molecular structure of com-
2–
plex 4c. Hydrogen atoms, methanol, and the ZnCl4 anion are
Table 2. Absorption and emission data of synthesized complexes
4a–e and 4g. All compounds have similar emission maxima in
HEPES buffer. The emission quantum yields of the synthesized
complexes were measured in methanol as solvent with quinine sul-
fate as reference to explore variations in the photophysical proper-
ties.
omitted for clarity. Ellipsoids are shown at the 50% probability
level.
buffer was chosen to cover the range of binding affinities
which are characteristic for complexes 4b–e. We were inter-
ested to compare the selectivities of binding and emission
responses of rigid bis- and tetra-ZnII complexes arising
from the interaction with different anions. The studies per-
formed by the Hamachi group using ZnII–Dpa–Tyr and a
series of oligoaspartate peptides (Boc-Dn-NH2, n = 2–5)
and oligoglutamate peptides (Boc-En-NH2, n = 3, 4) re-
vealed that D4 tag (n = 4) has the highest affinity.[21] Based
on the reported results, we selected D4 and E4 tag sequences
for our investigations. The derived binding affinities of the
complexes with the carboxylates, pyrophosphate (PPi), and
O-phospho-l-serine are shown in Table 3.
Complex
Absorption[a]
λmax
Emission[b]
logε λexcitation [nm] λmax [nm] Φrel [%][c]
4a
4b
4c
4d
4e
4g
224
285
365
370
350
220
4.44
4.53
3.90
3.36
4.16
4.10
230
290
364
372
354
220
457
385
455
456
453
424
0.02
0.2
2.0
20
5.9
0.02
[a] Measured in HEPES buffer at
a concentration of c =
10–4 molL–1. [b] Measured in HEPES buffer at a concentration of
c = 10–5 molL–1. [c] Relative quantum yields were determined by
using quinine sulfate (Φquinine sulfate = 58%).[20]
Scheme 3. Synthesis of modified triazine bis-cyclen ligand 8, for potential metal complexation by using click chemistry.
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Detection of Phosphate Anions and Non-covalent Protein Labeling
Table 3. Binding characteristics of complexes 4b–e measured as 0.01–0.05 mm solutions in 25 mm HEPES buffer, pH 7.4, 25 °C.
Boc–E4–NH2
Boc–D4–NH2
PPi
[b]
O-Phospho-l-serine
logβ11 Ͻ 3
4d
logβ21 = 13.46(12)
logβ11 = 7.21(6)
1:1, 6.8
logβ21 = 12.24(8)
logβ11 = 6.74(3)
1:1, 2.2
logβ11 = 3.41(1)
1:1, 1.3
logβ11 Ͻ 3
logβ21 = 16.22(8)
logβ11 Ͼ 7
1:1, 4.8
logβ11 Ͼ 7
logβ12 = 13.38(8)
1:2, 1.3
logβ11 = 3.31(1)
1:1, 1.8
logβ11 Ͻ 3
[a]
H/G ratio, F/F0
1:2, 5.5
1:1, 1.21
[b]
[c]
4b
[a]
[a]
H/G ratio, F/F0
1:2, 0.1
[b]
[c]
[c]
4c
H/G ratio, F/F0
1:1, 3.4
logβ21 = 11.05(7)
logβ22 = 16.55(12)
1:1, 1.2
4e
[a]
H/G ratio, F/F0
1:1, 0.9
1:1, 0.9
[a] Stoichiometry of host (H)/guest (G) ratio was determined according to Job’s method; F/F0 = changes in fluorescence of zinc complex
upon addition of one equivalent of a guest. [b] Good fitting of the experimental curve was not possible; each stepwise binding constant
was in the range Ͼ106 m–1. [c] Changes in fluorescence response upon titration with guest were negligible.
Analysis of fluorescence responses (F/F0) in Table 3 re- crease.[a] The stoichiometry of the host (H)/guest (G) ratio
veals that in most cases the coordination of a guest to a was determined according to Job’s method; F/F0 = changes
zinc complex leads to an increase in emission. This is in in fluorescence of zinc complex upon addition of one equiv-
good agreement with reports on Zn2+–DPA complexes.[17d] alent of a guest.[b] Good fitting of the experimental curve
According to our ESI mass spectrometry investigations, at was not possible; each stepwise binding constant was in the
the concentration required for fluorescence measurement range Ͼ106 m–1.[c] Changes in fluorescence response upon
some di- and trinuclear zinc complexes are formed from the titration with guest were negligible.
parent tetranuclear zinc complexes. They are weakly fluo-
rescent due to quenching by photoinduced electron transfer
Coordination of Tetraaspartate (Boc–D4–NH2) and
(PET) from the uncomplexed aliphatic nitrogen atoms. The
Tetraglutamate (Boc–E4–NH2) Peptide Sequences
coordination of an anion induces complete Zn2+ cation co-
ordination to the nitrogen ligand and thus decreases PET
Coordination of D4 or E4 peptide sequences to bis-ZnII
quenching,[22] which results in a turn-on response. The pres- complexes 4c and 4e is rather weak and only small changes
ence of this mechanism was proved by addition of an excess in fluorescence are observed upon addition of ca. 10 equiv.
amount of zinc(II) chloride to the complexes, in this case a of a guest. The tetra-ZnII complexes have much higher
slight increase (1.1–1.4 fold) in fluorescence was observed. binding affinities for both E4 and D4 tags with stepwise
However, the coordination of the E4 and D4 tags to com- binding constants of ca. 107 m–1. Such a dramatic increase
plex 4d led to much larger increases in fluorescence – 6.8 is explained by the increased coulombic interaction in a
and 4.8, respectively. This data indicate that another turn- host–guest complex. The characteristic changes in emission
on mechanism is also present: The coordination of a guest and the binding isotherm for tetra-ZnII complex 4d are
rigidifies the structure, leading to a significant emission in- shown in Figure 2.
Figure 2. Changes in fluorescence of tetra-ZnII complex 4d upon addition of the E4 tag. The titrations were carried out in 25 mm HEPES
buffer, pH 7.4, 25 °C, λex = 320 nm, [4d]0 = 0.015 mm. The titration isotherm is shown in the right graph: rhombuses – experimental data;
dashed line – fitting curve.
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Binding isotherms were fitted to a model of stepwise 2:1,
In order to schematically depict the mode of interactions
1:1, and 1:2 binding. Aggregate formation with a 2:1 stoi- between the oligopeptide and the tetrazinc complexes we
chiometry appears only due to the addition of a mm solu- have conducted DFT (B3LYP, 6-31G*) calculations using
tion of the guest to the μm solution of a host. During first Spartan06 (Wavefunction Inc.). According to the calcula-
additions the complex is in large excess, thus several mole- tions, the scaffold of the two benzene-linked binuclear ZnII–
cules of the latter can coordinate to the tetracarboxylate. cyclen complexes (i.e., 4b) perfectly matches the oligopep-
The other binding stoichiometries were observed in solu- tide tags (D4, E4) structure and charge distribution. In a
tion and confirmed by ESI spectrometry. Comparison of sandwich-like aggregate of 4b and the tag protein, strong
the calculated and experimentally found isotope distribu- electrostatic interactions are likely, resulting in the observed
tion of the molecular ions is shown in Figure 3.
high apparent binding affinity. The electrostatic potential
surface is displayed as a wired mesh of the D4 tag in Fig-
ure 3. The red color indicates a high density of negative
charges at the carboxylate residues of the tag. The cationic
ZnII–cyclen favorably interacts with the anionic carboxylate
residues. The figure shows the formation of the sandwich-
like 1:2 receptor/peptide-tag aggregate of 4b and the D4 tag.
The energy-minimized structure of 4d shows that unlike 4b,
where the benzene and triazine moieties are in one plane,
the plane of anthracene is perpendicular to the plane of the
triazine. This creates steric hindrance, which may be respon-
sible for the observed 1:1 receptor to peptide binding
stoichiometry in 4d. The predicted twisted conformation of
the triazine anthracene moiety of complex 4d is in a good
agreement with the crystal structure of bis-ZnII–anthracene
complex 4c.
Coordination of Phosphate Anions
Bis-ZnII–cyclen complexes show high affinity for phos-
phate anions in aqueous solution, which is of interest for
potential applications in biological phosphate recognition.
However, the so-far developed bis-ZnII–cyclen based syn-
thetic receptors have flexible spacers between the receptor
moiety and the signaling unit. Due to this flexibility, the
luminescent group cannot respond effectively to the binding
event by a change in its emission properties. Figure 4 shows
one of our previously reported complexes with a flexible
linker between fluorophore and the receptor and its negligi-
ble change emission properties in the presence of analytes
in homogeneous solution.[23]
In the here-reported synthesized bis-ZnII–cyclen com-
plexes, the rigid molecular structure and the direct conjuga-
tion of the central triazine unit to the arenes allows trans-
mission of the anion binding event at the cyclen complex
to the signaling unit, which responds by changes in the
emission properties (Table 3). The affinities of the com-
plexes for monophosphate anions such as phenylphosphate
or O-phospho-l-serine are rather small, but they are excel-
lent for polyphosphates. The most efficient response was
observed for bis-ZnII–anthracene containing complex 4c,
whose fluorescence is 3.4-fold increased upon addition of
1 equiv. of pyrophosphate. The sensitivity for pyrophos-
phate is even higher for tetra-ZnII complexes: 5.5- and 0.1-
fold change in fluorescence for anthracene- (4d) and ben-
zene-containing (4b) complexes, respectively (Table 3).
Complexes 4b and 4d have opposite responses: the presence
of pyrophosphate quenches the fluorescence of 4b, but
strongly increases the fluorescence of 4d (Figure 5).
Figure 3. Comparison of the calculated (lower) and observed (up-
per) isotope distribution for ions: (a) m/z = 923 [4d – 8Cl– + D4 –
6H]2+ and (b) m/z = 1160 [4b – 8Cl– + 2D4 – 6H]2+; (c) schematic
geometry of the receptor–peptide tag aggregate of 4b with two mo-
lecules of D4 tag.
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Detection of Phosphate Anions and Non-covalent Protein Labeling
Figure 4. Dinuclear ZnII–cyclen dansyl complex with flexible linker between receptor and fluorophore (left); emission intensity changes
of binuclear ZnII–cyclen dansyl complex (80 μm in HEPES buffer, pH 7.4, λex = 330 nm, 25 °C) upon addition of various nucleotides and
phosphates (right).
Figure 5. Fluorescence response upon addition of sodium pyrophosphate to the solution of (a) complex 4b and solution of (b) complex
4d. Titrations were carried out in 25 mm HEPES buffer, pH 7.4, 25 °C.
Though the affinities of tetra-ZnII complexes for pyr- ure 6c). This could not be the case for the anthracene-con-
ophosphate were good, it was difficult to fit binding iso- taining tetra-ZnII complex, because it does not have a
therms with typical models. Job’s plot analysis showed that planar structure according to DFT calculations of 4d and
tetra-ZnII complexes bind pyrophosphate in a 1:2 stoichio- the X-ray structure of 4c, thus close interaction of the
metry. Using this model we were able to obtain a stepwise anthracene rings is not sterically favorable. To prove the
binding constant in the order 107 m–1 (Table 3). The binding dimerization of 4b, we conducted ESI measurements of
isotherm of bis-ZnII complex 4c was successfully fitted both complexes 4b and 4d in the presence of an excess
using a 2:1 and 2:2 stepwise binding model. Additional amount of pyrophosphate in aqueous solution. Though
proof for the formation of the complex with a 2:2 stoichi- the major peaks for both complexes corresponded to [4b –
ometry was obtained by ESI mass spectrometry, where the 8Cl– + 2PPi4– + 2Na+]2+ and [4d – 8Cl– + 2PPi4–
complex with the composition [(4c – 4Cl– + PPi4– 2Na+]2+, the presence of the dimer [(4b – 8Cl– + 2PPi4–
+
+
+
Na+)2]2+ was one of the major peaks (Supporting Infor- Na+)2]2+ was clearly observed, allowing the isotope distri-
mation, Figure S26). Thus, coordination of pyrophosphate bution to be compared with the predicted one (Figure 6).
to 4c induces dimerization of the complex. This observation For anthracene-containing tetra-ZnII complex 4d, the cor-
led us to suggest that strong quenching of the fluorescence responding dimer was also observed, but with much lower
of complex 4b upon interaction with pyrophosphate can intensity, which did not allow the isotopic distribution to
arise from a similar dimerization resulting in π–π stacking be resolved experimentally (see the Supporting Infor-
interactions of planar benzene–triazine moieties (Fig- mation).
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Figure 6. Isotope distribution for ions: (a) m/z = 923 [(4c – 4Cl– + PPi4– + Na+)2]2+, (b) m/z = 1036 [(4b – 8Cl– + 2PPi4– + Na+)2
+
Na+]3+, and (c) proposed structure of dimer [4b – 8Cl– + 2PPi4–]2.
the addition of aqueous Na2CO3 (2 m, 2 mL). The mixture was
heated at reflux for 48 h under a N2 atmosphere. After cooling, the
solvent was evaporated under reduced pressure to dryness. THF
was added, and the suspension was shortly placed in an ultra-
sonication bath. The mixture was then filtered, washed thoroughly
with THF, and the filtrate was evaporated under reduced pressure.
The residue was purified by column chromatography on silica gel
[ethyl acetate (EA)/petroleum ether (PE)] to afford pure products
3 as solids. Obtained ligand 3 was Boc-deprotected with trifluoro-
acetic acid (14 equiv. per Boc group) and passed through a basic
ion exchange column. The Boc-deprotected azamacrocycles, except
in the case of 4d, were dissolved in acetonitrile. To this solution
was slowly added anhydrous ZnCl2 (2.5 equiv. for 4a, 4c, 4e–h and
4.5 equiv. for 4b, 4d) dissolved in methanol, leading to the forma-
tion of a white precipitate. For the synthesis of 4d, Boc-deprotected
3d was dissolved in methanol/water (4:1), and to this solution was
slowly added anhydrous ZnCl2 (4.5 equiv.) dissolved in methanol.
The reaction mixture was heated at reflux overnight, which dis-
solved the precipitate. The hot solution is then decanted in a coni-
cal flask. Upon cooling a white precipitate was obtained, which
was filtered and analyzed by NMR spectroscopy.
Conclusions
We have obtained luminescent synthetic receptors based
on 1,3,5-triazine bis-ZnII–cyclen binding sites and arenes
by transition-metal-mediated cross-coupling reactions. The
synthesized complexes are rigid in structure and show excel-
lent analyte response in buffered aqueous solution. Com-
plexes 4b, 4c, and 4d have high affinities for pyrophosphate
and oligocarboxylate (Boc–D4–NH2, Boc–E4–NH2) anions
with changes in emission response reaching one order of
magnitude. Though the coordination geometry of ZnII–
cyclen is different in comparison to that of ZnII–Dpa, the
affinities for the target anions are comparable. Thus, our
complexes can be considered as alternative probes for
polyphosphates and polycarboxylates.
Experimental Section
General: All reagent-grade chemicals were used without purifica-
tion unless otherwise specified. Phenylboronic acid, benzene-1,4-
diboronic acid, phenyl acetylene, cyanuric chloride, ATP, ADP, O-
phospho-l-serine, and phenyl phosphate were obtained from Ald-
rich and used as received. UV/Vis absorption spectra were recorded
by using a Cary 50 Bio spectrophotometer, and emission spec-
troscopy was performed by using a Varian Cary Eclipse fluores-
cence spectrophotometer. Except for phenylboronic acid (2a) and
benzene-1,4-diboronic acid (2b), all other boronic acids (i.e., 2c–h)
were synthesized following reported procedures.[24,25]
Compound 4a: The Boc-protected ligand was obtained as a color-
less solid (53 mg, 50%). Rf = 0.68 (EA/PE, 50:50). It was then
Boc deprotected by using TFA to yield the free base (23 mg, 97%)
followed by zinc complexation (30 mg, 100%). Data for 3a: 1H
NMR (300 MHz, CDCl3): δ = 1.44 (s, 54 H, CH3-Boc), 3.41–3.91
(br. m, 32 H, CH2-cyclen), 8.441 (d, J = 6.9 Hz, 2 H), 7.469 (t, 1
H), 7.438 (t, 2 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 27.5 (+,
CH3-Boc), 49.1, 49.7, 50.2 (–, CH2-cyclen), 76.5, 78.9 (Cq, C-Boc),
156.4, 157.0 (Cq, C=O Boc), 168.6 (Cq, triazine-Caryl-N), 126.9,
127.430, 130.2, 135.5 (benzene) ppm. MS (ESI, DCM/MeOH +
10 mm NH4OAc): m/z (%) = 1094 (100) [M + H]+. Data for 4a: 1H
NMR (300 MHz, CDCl3): δ = 8.492 (t, 2 H), 7.512–7.623 (m, 3
H), 4.433–4.508 (m, 2 H), 4.149 (br. s, 2 H), 2.721–3.554 (m, 28
H) ppm. 13C NMR (75 MHz, CDCl3): δ = 42.4, 43.3, 44.5, 45.5,
General Procedure for the Synthesis of Complexes 4: To a mixture of
Boc-protected bis-cyclen chlorotriazene 1 (100 mg, 0.1 mmol) and
Pd(PPh3)4 (10 mg, 0.012 mmol, 12 mol-%) in DME was added re-
quired aryl boronic acid 2 (1.5 equiv. of monoboronic acid and
0.5 equiv. of diboronic acid), which was immediately followed by
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Detection of Phosphate Anions and Non-covalent Protein Labeling
45.6, 46.4, 47.6, 47.9 (–, CH2-cyclen), 128.1, 128.5, 132.2, 132.4, Compound 4e: The Boc-protected compound was obtained as a col-
135.1, 135.3, 170.3, 170.8 ppm. MS (ESI, DCM/MeOH + 10 mm
orless solid (62 mg, 54%). Rf = 0.68 (EA/PE, 50:50). It was then
Boc deprotected by using TFA (30 mg, 96%) followed by Zn com-
plexation (43 mg, 100%). Data for 3e: 1H NMR (300 MHz,
CDCl3): δ = 1.466 (s, 54 H, CH3-Boc), 3.150–3.896 (br. m, 32 H,
CH2-cyclen), 8.015–8.216 (m, 7 H), 8.571 (br. d, 1 H), 9.109 (br. s,
1 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 50.3, 51.0, 80.1, 124.3,
124.7, 125.2, 125.4, 125.9, 127.3, 127.5, 128.3, 130.71, 131.2 ppm.
MS (ESI, DCM/MeOH + 10 mm NH4OAc): m/z (%) = 1223 (100)
[M + H+], 1022.9 (20) [M + H+ – 2Boc]. Data for 4e: 1H NMR
(300 MHz, CDCl3): δ = 2.730–2.941 (br. m, 28 H), 3.472 (br. s, 2
H), 4.391 (br. s, 4 H), 4.935 (br. d, 4 H), 8.150 (t, 1 H), 8.290–8.421
(m, 6 H), 8.593 (d, 1 H), 9.042 (d, 1 H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 43.9, 45.0, 46.3, 47.3, 123.6, 123.9, 124.5, 125.4, 125.6,
126.0, 126.6, 127.3, 128.2, 128.7, 128.8, 130.1, 130.7, 132.2, 132.2,
169.3, 172.7 ppm. MS (ESI, H2O/MeOH + 10 mm NH4OAc): m/z
(%) = 434.8 (100) [M4+ + 2CH3COO–]2+. M.p. 285–287 °C. IR
NH4OAc): m/z (%) = 372.6 (100) [M4+ + 2CH3COO–]2+. M.p. 235–
238 °C. IR (ATR): ν = 3100, 2940, 1350, 1154, 968, 820 cm–1. UV
˜
(HEPES pH 7.4.25 mm): λmax (logε)
= 224 nm (4.4). MF:
[C25H43N11Zn2]4+4ClO4 . FW: 1026.25 gmol–1.
–
Compound 4b: The Boc-protected compound was obtained as a col-
orless solid (64 mg, 31%). Rf = 0.68 (EA/PE, 50:50). It was then
Boc-deprotected by using TFA (27 mg, 96%) followed by Zn com-
plexation (41 mg, 100%). Data for 3b: 1H NMR (300 MHz,
CDCl3): δ = 1.391 (s, 108 H, CH3-Boc), 3.370–3.594 (br. m, 64 H,
CH2-cyclen), 8.368 (s, 4 H) ppm. 13C NMR (300 MHz, CDCl3): δ
= 28.52 (+, CH3-Boc), 50.24 (–, CH2-cyclen), 79.91 (Cq, C-Boc),
128.11, 156.62, 169.46 ppm. MS (ESI, DCM/MeOH + 10 mm
NH4OAc) m/z (%) = 1060 (100) [M + 2H+]2+, 1010 (50) [M + 2H+ –
1
Boc] 2+, 2119.2 (10) [M + H+]. Data for 4b: H NMR (300 MHz,
[D6]DMSO): δ = 1.042–1.992 (br. m, 58 H), 2.925 (br., 6 H), 6.878
(s, 4 H) ppm. 13C NMR (75 MHz, [D6]DMSO): δ = 44.2, 46.3,
129.5, 140.5, 168.9, 171.4 ppm. MS (ESI, H2O/MeOH + 10 mm
NH4OAc): m/z (%) = 490.7 (100) [M8+ + 5CH3COO–]3+. M.p. 255–
(ATR): ν = 3125, 2890, 1650, 1545, 1175, 980, 880, 715 cm–1. UV
˜
(HEPES pH 7.4, 25 mm): λmax (logε) = 350 nm (4.16). MF:
–
[C35H47N11Zn2]4+Cl4 . FW: 894.39 gmol–1.
Compound 4f: The Boc-protected compound was obtained as a col-
orless solid (83 mg, 78%). Rf = 0.58 (EA/PE, 50:50). It was then
Boc deprotected by using TFA (37 mg, 97%) followed by Zn com-
plexation (72 mg, 100%). Data for 3f: 1H NMR (300 MHz,
CDCl3): δ = 1.478 (s, 54 H, CH3-Boc), 2.84 (t, 2 H, CH2), 3.12 (t,
2 H, CH2), 3.31–3.87 (br. m, 32 H, CH2-cyclen), 7.22–7.26 (m, 5
H, aromatic protons) ppm. 13C NMR (75 MHz, CDCl3): δ = 21.1
(CH2), 28.4, 28.5 2 (CH3-Boc), 41.1 (CH2), 50.1 (CH2-cyclen),
79.86 (Cq, C-Boc), 125.5, 128.2 (aromatic C), 144.2 (Cq, aromatic
C), 176.7 (Cq, triazine) ppm. MS (ESI, DCM/MeOH + 10 mm
NH4OAc): m/z (%) = 1126.7 (100) [M + H+], 1164.6 (40) [M +
257 °C. IR (ATR): ν = 3398, 2933, 1680, 1524, 1347, 1193, 1132,
˜
1087, 971, 813, 723 cm–1. UV (HEPES pH 7.4, 25 mm): λmax (logε)
= 285 nm (4.530). MF: [C44H92N22Zn4]8+Cl8. FW: 1474.50 gmol–1.
Compound 4c: The Boc-protected compound was obtained as a col-
orless solid (56 mg, 50%). Rf = 0.68 (EA/PE, 50:50). It was then
Boc deprotected by using TFA (25 mg, 95%) followed by Zn com-
plexation (36 mg, 100%). Data for 3c: 1H NMR (300 MHz,
CDCl3): δ = 1.451 (s, 54 H, CH3-Boc), 3.440–3.661 (br. m, 32 H,
CH2-cyclen), 7.316–7.439 (m, 4 H), 7.903 (br. d, 2 H), 8.003 (d, 2
H), 8.445 (s, 1 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 22.88,
26.30, 26.87, 27.48, 28.43, 48.95, 49.99, 79.05, 123.93, 124.28,
125.17, 126.12, 127.31, 127.82, 130.27, 133.22 ppm. MS (ESI,
DCM/MeOH + 10 mm NH4OAc): m/z (%) = 1023 (100) [M +
2H+]2+, 1199 (65) [M + H+], 1053 (35) [M + 2H+ – Boc]2+. Data
1
K+]. Data for 4f: H NMR (300 MHz, CDCl3): δ = 2.84 (t, 2 H,
CH2), 3.12 (t, 2 H, CH2), 3.29–3.55 (br. m, 32 H, CH2-cyclen),
7.22–7.26 (m, 5 H, aromatic protons) ppm. 13C NMR (75 MHz,
CDCl3): δ = 21.3 (CH2), 41.1 (CH2), 50.1 (CH2-cyclen), 79.8 (Cq,
C-Boc), 125.5, 128.2 (aromatic C), 144.2 (Cq, aromatic C), 176.7
(Cq, triazine) ppm. MS (ESI, H2O/MeOH + 10 mm NH4OAc): m/z
(%) = 386.5 (100) [M4+ + 2CH3COO–]2+. M.p. 290–293 °C. IR
1
for 4c: H NMR (300 MHz, [D6]DMSO): δ = 2.647–3.231 (br. m,
32 H), 4.249 (br. s, 2 H), 4.815 (br. s, 4 H), 7.445–7.573 (m, 4 H),
8.025 (d, 2 H), 8.168 (d, 2 H), 8.725 (s, 1 H) ppm. 13C NMR
(150 MHz, [D6]DMSO): δ = 30.6, 44.8, 46.8 (CH2-cyclen), 125.4,
126.2, 128.1, 130.7, 132.8, 133.1, (CH, aromatic), 158.0, 169.2,
172.5, 173.0 ppm. MS (ESI, H2O/MeOH + 10 mm NH4OAc): m/z
(%) = 423.6 (100) [M4+ + 2CH3COO–]2+. M.p. 274–282 °C. IR
(ATR): ν = 3290, 2942, 2971, 2863, 1645, 1470, 1385, 980, 824,
˜
–
728 cm–1. MF: [C27H47N11Zn2]4+Cl4 . FW: 798.3 gmol–1.
Compound 4g: The Boc-protected compound was obtained as a col-
orless solid (50 mg, 47%). Rf = 0.71 (EA/PE, 50:50). It was then
Boc deprotected by using TFA (23 mg, 96%) followed by Zn com-
plexation (40 mg, 100%). Data for 3g: 1H NMR (300 MHz,
CDCl3): δ = 1.445 (s, 54 H, CH3-Boc), 3.428–4.085 (br. m, 32 H,
CH2-cyclen), 6.839 (d, CH-ethylene, J = 15.9 Hz), 7.338–7.358 (m,
3 H, aromatic protons), 7.568 (d, J = 7.2 Hz, 2 H, aromatic pro-
tons), 7.959 (d, J = 15.9 Hz, 1 H, CH-ethylene) ppm. 13C NMR
(75 MHz, CDCl3): δ = 28.3, 28.6 (CH3-Boc), 49.1, (CH2-cyclen),
78.8 (Cq, C-Boc), 126.6, (C=C), 127.6, 127.9 (aromatic C), 135.0
(C=C), 168.9 (Cq, triazine) ppm. MS (ESI, DCM/MeOH + 10 mm
NH4OAc): m/z (%) = 1124.5 (100) [M + H+]. Data for 4g: 1H NMR
(300 MHz, CDCl3): δ = 2.67–3.76 (br. m, 38 H), 6.35 (d, CH-ethyl-
ene), 7.18–7.55 (m, aromatic protons), 7.79 (d, CH-ethylene) ppm.
13C NMR (75 MHz, CDCl3): δ = 43.9, 45.0, 46.3, 47.3, 123.6,
123.9, 124.4, 125.4, 125.6, 125.9, 126.6, 127.3, 128.2, 128.7, 128.8,
130.1, 130.7, 132.2, 132.2, 169.2, 172.7 ppm. MS (ESI, H2O/MeOH
(ATR): ν = 3090, 2943, 1345, 1087, 1130, 963, 825 cm–1. UV
˜
(HEPES pH 7.4, 25 mm): λmax (logε) = 365 nm (3.9). MF:
–
[C33H47N11Zn2]4+Cl4 . FW: 870.37 gmol–1.
Compound 4d: The Boc-protected compound was obtained as a col-
orless solid (65 mg, 30%). Rf = 0.68 (EA/PE, 50:50). It was then
Boc deprotected by using TFA (12 mg, 95%) followed by Zn com-
plexation (18 mg, 100%). Data for 3d: 1H NMR (300 MHz,
CDCl3): δ = 1.457 (s, 108 H, CH3-Boc), 3441–3.653 (br. m, 64 H,
CH2-cyclen), 7.283–7.317 (dd, 4 H), 7.834–7.868 (br., 4 H) ppm.
13C NMR (300 MHz, CDCl3): δ = 27.5 (+, CH3-Boc), 48.9 (–,
CH2-cyclen), 79.0 (Cq, C-Boc) ppm. MS (ESI, DCM/MeOH +
10 mm NH4OAc): m/z (%) = 1110.6 (100) [M + 2H+]2+, 2220 (6)
[M + H+]. Data for 4d: 1H NMR (300 MHz, D2O): δ = 2.667–
2.965 (m, 46 H), 2.987–3.011 (br., 3 H), 3.192–3.337 (br., 18 H),
7.4921–7.953 (dd, 4 H) ppm. 13C NMR (150 MHz, D2O): δ = 43.6,
44.8, 46.1, 47.6, 113.4, 115.3, 117.2, 119.2, 169.8, 173.9 ppm. MS
(ESI, H2O/MeCN): m/z (%) = 355.0 [M8++4Cl–]4+(100), 485.2 (50)
+ 10 mm NH4OAc): m/z (%) = 385.5 (100) [M4+ + 2CH3COO–]2+
.
IR (ATR): ν = 3385, 3083, 2966, 2896, 1680, 1644, 1525, 1465,
˜
1182, 965, 812, 720 cm–1. UV (HEPES pH 7.4, 25 mm): λmax (logε)
[M8++5Cl–]3+. M.p. 292–296 °C. IR (ATR): ν = 3025, 2893, 1640,
˜
= 220 nm (4.10). MF: [C27H45N11Zn2]4+Cl4 . FW: 796.29 gmol–1.
–
1585, 1125, 980, 720 cm–1. UV (HEPES pH 7.4, 25 mm): λmax
–
(logε)
=
370 nm (3.36). MF: [C52H84N22Zn4]8+Cl8 . FW:
Compound 4h: The Boc-protected compound was obtained as a col-
orless solid (56 mg, 52%). Rf = 0.68 (EA/PE, 50:50). It was then
1562.52 gmol–1.
Eur. J. Org. Chem. 2011, 2807–2817
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2815

B. König et al.
FULL PAPER
Boc deprotected by using TFA (24 mg, 92%) followed by Zn com-
plexation (48 mg, 100%). Data for 3h: 1H NMR (300 MHz,
CDCl3): δ = 0.87 (t, 3 H, CH3), 1.455 (s, 54 H, CH3-Boc), 1.576–
1.711 (m, 10 H, CH2), 2.458 (t, 2 H, CH2), 3.22–3.76 (br. m, 32 H,
CH2-cyclen) ppm. 13C NMR (75 MHz, CDCl3): δ = 28.5, 28.7
(CH3-Boc), 49.5 (CH2-cyclen), 78.8 (Cq, C-Boc) ppm. MS (ESI,
DCM/MeOH + 10 mm NH4OAc): m/z (%) = 1120.9 (100) [M +
1667, 1541, 1249, 1201, 1136, 901, 844 cm–1. MF: C27H44N14. FW:
564.39 gmol–1.
X-ray
C33H47Cl2N11Zn2·CH3OH·ZnCl4: M = 1038.73 gmol–1, mono-
clinic, P21/c, 13.33784(16) Å, 29.7188(4) Å, c =
Structure
Determination:
Crystal
data
for
a
=
b
=
10.88517(13) Å, α = 90°, β = 94.0245(11)°, γ = 90°, V =
4304.07(9) Å3, Z = 4, 18752 reflections measured, 8828 indepen-
dent (Rint = 0.0208), which were used in all calculations. The final
wR2 was 0.0972 (all data). Intensity data were collected with graph-
ite-monochromated Mo-Kα radiation (λ = 1.54184 Å) at 123 K
with a Goniometer Xcalibur, detector: Ruby (Gemini ultra Mo).
For data collection and structure solution and refinement the fol-
lowing programs were used: SHELXL,[27] R1 is calculated for ob-
served data and wR2 for all data.
1
H+]. Data for 4h: H NMR (300 MHz, [D6]DMSO): δ = 0.86 (t, 3
H, CH3-alkyl chain) 1.26 (br. s, 8 H), 1.75 (s, 7 H), 2.1 (s, 6 H),
2.89 (br. m, 16 H), 3.4 (s, 13 H) ppm. 13C NMR (75 MHz, [D6]-
DMSO): δ = 13.7 (CH3-alkyl chain), 17.8, 21.8, 22.1, 22.2, 26.2,
28.3 (CH2-alkyl chain), 30.9, 39.1 ppm. MS (ESI, H2O/MeOH +
10 mm NH4OAc): m/z (%) = 383.6 (100) [M4+ + 2CH3COO–]2+. IR
(ATR): ν = 2921, 2852, 1693, 1561, 1525, 1465, 1420, 1346, 1282,
˜
1085, 812 cm–1. MF: [C26H53N11Zn2]4+Cl4 . FW: 792.35 gmol–1.
–
CCDC-808617 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
Synthesis of Compound 5: In a two-necked flask, Boc-protected bis-
cyclen chlorotriazene 1 (0.2 g, 0.19 mmol), Pd/C (10%, Fluka),
PPh3, and CuI in a 1:0.04:0.16:0.04 molar ratio were placed under
a nitrogen atmosphere along with DIPEA (1.5 mL) as the suitable
base. After accurately purging with nitrogen, a solution of phenyl
acetylene (0.05 mL, 0.48 mmol) in CH3CN was added, the tem-
perature was raised, and the mixture was vigorously stirred over-
night. After cooling and filtering the mixture on a short plug of
Celite, the solution was evaporated under reduced pressure. The
crude reaction mixture was purified by column chromatography
(40% EA in PE) to get pure product 5 (51%). 1H NMR (300 MHz,
CDCl3): δ = 1.45 (s, 54 H, CH3-Boc), 3.24–4.00 (br. m, 32 H, CH2-
cyclen), 7.5–7.57 (dd, 2 H, benzene ring), 7.29–7.43 (br. m, 3 H,
benzene ring) ppm. 13C NMR (75 MHz, CDCl3): δ = 27.5, 48.9,
87.6, 120.8, 127.2, 128.3, 131.4, 158.0 ppm. MS (ESI, DCM/MeOH
+ 10 mm NH4OAc): m/z (%) = 1122.8 (100) [M + H+]. IR (ATR):
Supporting Information (see footnote on the first page of this arti-
cle): 1H and 13C NMR spectra, MS (ESI) characterization, and
fluorescence titration data including fitting curves; details of the
quantum chemical calculations.
Acknowledgments
M. B. thanks the Evonik Foundation and the Bavarian Research
Foundation for graduate fellowships. S. S. thanks the Japan Society
for the Promotion of Science for a fellowship. E. K. thanks the
Alexander von Humboldt Foundation for a fellowship. We thank
the University of Regensburg, the Fonds der Chemischen Industrie,
and the Deutsche Forschungsgemeinschaft for support of our
research.
ν = 3385, 3083, 2966, 2896, 2250, 1680, 1512, 1465, 1182, 965,
˜
812 cm–1. MF: C57H91N11O12. FW: 1122.4 gmol–1.
Synthesis of Compound 7: The monoazide derivative of cyanuric
chloride, 6 was synthesized following a reported procedure.[26] In a
round-bottomed flask, the monoazide derivative of trichlorotri-
azine (0.2 g, 1.08 mmol) and K2CO3 (0.6 g, 4.32 mmol) were sus-
pended in acetone (70 mL). To this suspension was slowly added a
solution of threefold Boc-protected cyclen (1.02 g, 2.16 mmol) in
acetone (30 mL). The reaction mixture was then heated under re-
flux for 12 h. The solvent was removed under reduced pressure, and
the crude mixture was purified by column chromatography (30%
EA in PE) to get pure product 7 (0.15 g, 20%). 1H NMR
(300 MHz, CDCl3): δ = 1.39 (s, 54 H, CH3-Boc), 3.04–3.75 (br. m,
32 H, CH2-cycle) ppm. 13C NMR (75 MHz, CDCl3): δ = 28.4, 31.7,
50.0, 53.8, 69.4, 79.9, 156.1, 168.2, 210.7 ppm. MS (ESI-Q1MS):
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˜
2974, 2928, 2865, 1694, 1540, 1361, 1169 cm–1. MF: C48H84N14O12.
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Synthesis of Compound 8: To a stirred solution of 7 (0.28 g,
0.29 mmol) in degassed dry THF (3 mL) was added phenylacetyl-
ene (0.05 mL, 0.48 mmol), CuI (6 mol-%), and TEA (0.1 mL,
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1
95%). H NMR (300 MHz, CDCl3): δ = 1.49 (s, 54 H, CH3-Boc),
3.27–3.85 (br. m, 32 H, CH2-cyclen), 7.29–7.52 (br. m, 5 H, benzene
ring), 7.97 (s, 1 H, triazole ring) ppm. 13C NMR (75 MHz, CDCl3):
δ = 28.5, 50.1, 80.1, 80.1, 80.2, 104.4, 126.0, 128.2, 128.7, 130.2,
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,
565.3 (40) [M + H+]. M.p. 95–98 °C. IR (ATR): ν = 2973, 2923,
˜
2816
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 2807–2817

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Received: January 24, 2011
Published Online: April 6, 2011
Eur. J. Org. Chem. 2011, 2807–2817
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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