Stable Europium(III) Complexes with Short Linkers for Site-Specific Labeling of Biomolecules

Article abstract of DOI:10.1002/open.201700122

In this study, two new terpyridine-based Eu^III complexes were synthesized, the structures of which were optimized for luminescence resonance energy-transfer (LRET) experiments. The complexes showed high quantum yields (32 %); a single long life

Full text of DOI:10.1002/open.201700122

DOI: 10.1002/open.201700122
Stable Europium(III) Complexes with Short Linkers for
Site-Specific Labeling of Biomolecules
Felix Faschinger,[a] Martin Ertl,[b] Mirjam Zimmermann,[a] Andreas Horner,[a]
Markus Himmelsbach,[c] Wolfgang Schçfberger,[d] Gꢀnther Knçr,[b] and Hermann J. Gruber*[a]
In this study, two new terpyridine-based EuIII complexes were
synthesized, the structures of which were optimized for lumi-
nescence resonance energy-transfer (LRET) experiments. The
complexes showed high quantum yields (32%); a single long
lifetime (1.25 ms), which was not influenced by coupling to
protein; very high stability in the presence of chelators such as
ethylenediamine-N,N,N’,N’-tetraacetate and ethylene glycol-
bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid; and no inter-
action with cofactors such as adenosine triphosphate and gua-
nosine triphosphate. A special feature is the short length of
the linker between the EuIII ion and the maleimide or hydrazide
function, which allows for site-specific coupling of cysteine
mutants or unnatural keto amino acids. As a consequence, the
new complexes appear particularly suited for accurate distance
measurements in biomolecules by LRET.
1. Introduction
Lanthanide complexes have been successfully used in several
fields of bioanalytics. Examples are homogeneous time-re-
solved fluorescence (HTRF) assays;[1] dissociation-enhanced lan-
thanide fluorescent immunoassays (DELFIAs);[2] cell imaging;
and luminescence probes for oxygen, singlet oxygen, pH, or
other analytes.[3–5]
buffer components upon conformational changes or interac-
tions of biomolecules.[9]
For a number of reasons, LRET is better suited than FRET for
explicit distance determination.[7,8] 1) The origin of error from
the relative orientations of the donor and acceptor dyes is
minimized because of the isotropic nature of the donor.[10]
2) A wider range of distances can be analyzed if suitable
donor–acceptor pairs are selected.[7] 3) The narrow emission
lines of the lanthanide donor allow selective measurement of
the emission lifetime of the acceptor, as used in the “sensitized
emission” method.[7] 4) In LRET, the energy-transfer efficiency
(E=1ꢀtDA/tDonor) can be measured more accurately by compar-
ing the luminescence lifetime of the donor in the absence of
the acceptor (tDonor) versus in the presence of the acceptor
(tDA). Such a comparison of the lifetimes is much less suscepti-
ble to systematic errors than the comparison of emission in-
tensities used in FRET. FRET relies on organic dyes or fluores-
cent proteins, which require setups resolving lifetimes in the
nanosecond range, whereas Eu/Tb complexes used in LRET ex-
hibit lifetimes in the range of 1 to 2.5 ms.
A specialized biophysical application of lanthanide com-
plexes is the measurement of intra- or intermolecular distances
by luminescence resonance energy transfer (LRET),[6–8] which
may serve as an alternative to X-ray and NMR spectroscopy.
The latter methods can resolve very fine structural details, but
their material and time demands are very high. Very little
sample is required for fluorescence resonance energy transfer
(FRET), but it gives only crude distance estimations, because
the Fçrster distance can rarely be determined. For this reason,
FRET is mainly used to screen the influence of mutations or
[a] F. Faschinger, M. Zimmermann, Prof. A. Horner, Prof. Dr. H. J. Gruber
Institute of Biophysics, Johannes Kepler University Linz
Gruber Straße 40, 4040 Linz (Austria)
E-mail: hermann.gruber@jku.at
An essential requirement for energy-transfer measurements
by using lifetimes is the existence of a single lifetime of the
lanthanide donor in the absence of an acceptor, both before
and after coupling to the biomolecule of interest. The latter cri-
terion is fulfilled by some but not all lanthanide complexes
found in the literature.[5,11,12] Another requirement is high ther-
modynamic and kinetic stability to prevent loss of the lantha-
nide ion in the presence of chelators such as ethylene glycol-
bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA).
Several Eu/Tb complexes described in the literature fulfill
these criteria but still cannot be used for accurate distance
measurements, because they have unspecific coupling func-
tions such as N-hydroxysuccinimide esters or dichlorotriazinyl
groups that lead to statistical labeling of lysine residues (exem-
[b] M. Ertl, Prof. Dr. G. Knçr
Institute of Inorganic Chemistry, Johannes Kepler University Linz
Altenberger Straße 69, 4040 Linz (Austria)
[c] Prof. Dr. M. Himmelsbach
Institute for Analytical Chemistry, Johannes Kepler University Linz
Altenberger Straße 69, 4040 Linz (Austria)
[d] Prof. Dr. W. Schçfberger
Institute of Organic Chemistry, Johannes Kepler University Linz
Altenberger Straße 69, 4040 Linz (Austria)
Supporting Information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/open.201700122.
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This is an open access article under the terms of the Creative Commons
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general synthesis pathway was similar to that of chelate 9
(Figure 1):[12] thus, compound 5 (Scheme 1), which is a bis-bro-
momethyl derivative of terpyridine, and diethylenetriamine tri-
acetate (DTTA) derivative 11 (Scheme 2) were assembled into
cyclic chelate 12 (Scheme 3), and cyclic chelate 12 was further
modified to yield the final lanthanide complexes.
Figure 1. Example of known Eu complexes with linkers for protein coupling.
plified by DTBTA–Eu in Figure 1).[5,13] Other complexes actually
allow for site-specific labeling of a cysteine mutant by their
maleimide function; nevertheless, their use for distance meas-
urements is compromised because they have a long, flexible
linker between the lanthanide ion and the maleimide function
(see chelate 9 in Figure 1).[12]
The goal of our study was to design a lanthanide complex
that fulfills all requirements necessary for distance measure-
ments by LRET, including site-specific coupling and minimal
linker length. For the parent structure, we chose terpyridine-
based chelators that show high thermodynamic and kinetic
stability, due to the relatively rigid backbone and the high co-
ordination numbers.[4,5,14–16] In particular, we selected chelate 9
(Figure 1),[12] in which the terpyridine is cyclized with a diethy-
lene triamine skeleton. In this diethylene triamine unit, two ni-
trogen atoms are part of a glycine element and one nitrogen
atom is part of a lysine residue. Herein, we introduce a much
shorter linker between the complex and the maleimide func-
tion, and besides the maleimide moiety, we offer a hydrazide
for site-specific labeling of carbonyl functions.
Scheme 1. Synthesis of the dibromo terpyridine. Reagents and conditions:
a) mCPBA (5.8 equiv), CH2Cl2, RT, 24 h, 47%; b) (CH3)3SiCN (10 equiv), CH2Cl2,
RT, 20 min, PhCOCl (4 equiv), RT, 12 h, 70%; c) H2O/HOAc/concd H2SO4
(4:4:1), 90–1008C, 24 h, 100%; d) SOCl2 (10 equiv), MeOH, reflux, 18 h, 84%;
e) NaBH4 (5.7 equiv), EtOH, RT, 3 h, reflux, 1 h, 86%; f) LiBr (1.1 equiv), PBr3
(4.9 equiv), DMF, RT, 4 h, 86%.
One major difference from the published synthesis route of
chelate 9 was a different method for the preparation of bis-
bromomethyl terpyridine 5. Here, we used the methods re-
ported for the preparation of DTBTA–EuIII (Figure 1),[5] depicted
in Scheme 1.
The second deviation from the published synthesis of che-
late 9 was the use of protected diaminobutyric acid rather
than the longer lysine derivative as the starting material for
DTTA derivative 11. Except for this difference in the starting
material, which implied several additional synthesis steps, the
steps in Scheme 2 were analogous to those in the synthesis of
chelate 9.
The coupling of the bis-bromomethyl terpyridine 5 with
DTTA derivative 11 (Scheme 3, step a), removal of the protect-
ing group (Scheme 3, step b), and lanthanide insertion
(Scheme 3, step c) were performed as described for chelate
9.[12] However, the primary amino group was then directly con-
2. Results and Discussion
The goal of this study was to synthesize analogues of chelate
9 (Figure 1) that carry either a maleimide or a hydrazide func-
tion on a much shorter linker than that found in chelate 9. The
Scheme 2. Synthesis of the 2,4-diamonobutyric acid derivative of DTTA. Reagents and conditions: a) TBTA (2 equiv), BF3·OEt2 (0.18 equiv), CH2Cl2, cyclohexane,
RT, 12 h, 93%; b) NH4HCO2 (10 equiv), Pd/C (0.08 equiv), 1-propanol/MeOH (1:1), RT, 18 h, 96%; c) tert-butyl bromoacetate (1 equiv), DIPEA (1 equiv), DMF, 0–
258C, 12 h, 100%; d) PPh3 (1.18 equiv), NBS (1.17 equiv), CH2Cl2, 0–258C, 4 h, 45%; e) 7 (1 equiv), 9 (2 equiv), K2CO3 (10 equiv), CH3CN, reflux, 18 h, 71%;
f) NH4HCO2 (42 equiv), Pd/C (0.25, equiv), 1-propanol/MeOH (1:1), RT, 18 h, 84%.
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Scheme 3. Synthesis of lanthanide complexes for site-specific labeling. Reagents and conditions: a) 11 (1 equiv), 5 (1 equiv), Na2CO3 (10 equiv), CH3CN, 12 h,
50%; b) TFA/CH2Cl2 (1:1), RT, 12 h, 96%; c) 2 mm 13/17 in H2O, LnCl3 (1 equiv), pH 6.5, RT, 12 h, 100%; d) N-methoxycarbonylmaleimide (54 equiv), H2O/sat.
NaHCO3 (1:11), 08C, 15 min, RT, 30 min, 60–89%; e) 15 (2.0 equiv), DIPEA (16 equiv), DMF, RT, 12 h, 93%; f) TFA/CH2Cl2 (1:1), RT, 12 h, 100%.
verted into a maleimide group (Scheme 3, 14-Eu and 14-
Tb),[17,18] which thereby avoided the additional increase in the
length of the linker associated with attachment of the maleimi-
dopropionyl group in chelate 9 (Figure 1).
The synthesis of carbazide derivatives 16, 17, and 17-Eu was
essentially new. The hydrazide function in 17 is known for its
chemoselective reaction with aldehydes and ketones in aque-
ous media, as used for site-specific labeling of N-terminal
serine/threonine or oligosaccharide side chains after periodate
treatment[19,20] or for coupling to site-specifically incorporated
unnatural amino acids containing a ketone function.[21]
heating at reflux with thionyl chloride in methanol. After re-
crystallization from toluene, pure 3 was obtained in 84% yield.
The methyl ester was reduced by using sodium borohydride in
ethanol, and important was the careful workup with saturated
NaHCO3 to obtain pure 4 in 86% yield. Bromide substitution of
the hydroxy group was achieved with an approximately 4.9-
fold excess amount of PBr3 and LiBr in dry DMF. The dropwise
addition of PBr3 at 08C was crucial, and the solution was red as
long as active PBr3 was present in the solution. Due to the
high excess amount of PBr3, the solution had to remain col-
ored as long as unreacted alcohol 4 was present, for which re-
action control was performed by TLC. After solvent removal
and basic extraction, pure 5 was isolated in 86% yield.
2.1. Synthesis of 6,6’’-Dibromomethyl-2,2’:6’,2’’-terpyridine 5
The synthesis of 6,6’’-dibromomethyl-2,2’:6’,2’’-terpyridine 5
was performed in a manner similar to the strategy of Nishioka
et al., starting with the terpyridine because the biphenyl side
chain with the aromatic amine function was not required
here.[5,13] In the first step, bis-N-oxide 1 was synthesized ac-
cording to Nishioka et al.[5] by using a 5.8-fold excess amount
of meta-chloroperoxybenzoic acid (mCPBA). The yield of 47%
was moderate and can probably be improved by using a small-
er amount of mCPBA and by washing during the workup with
acetone rather than acetonitrile.[22] The introduction of carboni-
trile groups in the 6- and 6’’-positions was conducted by using
(CH3)3SiCN and benzoyl chloride in a modified Reissert–Henze
reaction.[23] The 70% yield in the reaction for 2 is rather high
for the regioselective reaction. The carbonitrile groups were
hydrolyzed in acetic acid and concentrated sulfuric acid at 90–
1008C, and subsequently, the methyl ester was obtained by
2.2. Synthesis of Diethylenetriamine Triacetate Derivative 11
The synthesis of DTTA derivative 11 was based on the strat-
egies reported by Deslandes et al. and Bechara et al.[12,14,15] The
side-chain amino group of 2,4-diaminobutyric acid (Dab) was
designed to carry the coupling function (e.g., maleimide) for
site-specific labeling. Commercially available Z-Dab(Boc)-
OH·DCHA (Z=Cbz=benzyloxycarbonyl, Boc=tert-butoxycar-
bonyl, DCHA=dicyclohexylammonium) was first transformed
into the free acid by suspending it in ethyl acetate and adding
1 m phosphoric acid, whereupon everything dissolved in the
two-phase system. The tert-butyl ester was introduced with
tert-butyl 2,2,2-trichloroacetimidate (TBTA) by using BF3 as a
catalyst in a mixture of cyclohexane and dichloromethane.[24]
Upon completion of the reaction and removal of the solvent,
the residue was suspended in 10% Na2CO3 for 2 h; otherwise,
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trichloroacetimidate derivative contaminants could not be re-
moved (the contaminants were observed as two broad singlets
the pH was raised to 6. The appearing solid byproducts were
filtered off, and the product-containing solution was adjusted
with water to an appropriate volume for purification of 14-Eu/
Tb by HPLC. Reverse-phase HPLC was performed by applying a
gradient from water to acetonitrile by using 0.1% acetic acid
as an additive. Purification resulted in 60% yield of 14-Eu and
89% yield in the case of 14-Tb. The site-specific labeling reac-
tion of 14-Eu with a cysteine-containing peptide or protein is
illustrated in Scheme 4 at the top.
1
at d=5.8 and 6.6 ppm in the H NMR spectrum). Esterification
attempts with di-tert-butyl dicarbonate and 4-(N,N-dimethyla-
mino)pyridine (DMAP) as the catalyst were unsuccessful,
whereas the method mentioned above gave 6 in 93% yield.
The benzyloxycarbonyl protecting group was removed by
using ammonium formate as the hydrogen source with Pd/C
as the catalyst.[25,26] Cleavage of the Cbz group of 6 was ach-
ieved in 96% yield in a mixture of 1-propanol and MeOH,
which ensured complete solvation of 6 and ammonium for-
mate. The primary amino group of N-benzylethanolamine was
alkylated in DMF with tert-butyl bromoacetate to give 8 in
quantitative yield. In the next step, alcohol 8 was converted
into corresponding bromide 9 by an Appel reaction by using
N-bromosuccinimide (NBS) and triphenylphosphine. For this
purpose, the reactants (except for NBS) were dried carefully by
evaporation of dry toluene, and NBS was added in portions at
08C. The order of adding the reactants as well as the use of
strictly dry CH2Cl2 were very important to obtain a product
with proper quality. During purification by silica-gel chroma-
tography some product loss was unavoidable because the tri-
phenylphosphine oxide fraction partially overlapped with the
product fraction, and this is the reason behind the moderate
yield of 45%. The a-amino group of 7 was alkylated with
2 equivalents of bromide 9 in dry refluxing acetonitrile with
solid potassium carbonate as the base.[12] Extraction and purifi-
cation by column chromatography gave pure 10 in 71% yield.
The method for cleavage of the benzyl group was related to
the method described above for cleavage of the Cbz group.
Thus, diamine 11 was obtained in 84% yield by using ammoni-
um formate in MeOH/1-propanol, yet by using a higher
amount of the Pd catalyst than that used for cleavage of the
Cbz group.[25,26]
Scheme 4. Site-specific labeling reaction of a cysteine-containing peptide or
protein with 14-Eu (top) and of a periodate-treated N-terminal serine (or
threonine) with 17-Eu (bottom). Reagents and conditions: a) Buffer pH 7.5,
tris(2-carboxyethyl)phosphine, 1 mm EDTA;[27] b) sodium periodate, pH 6.5;[20]
c) acetate buffer pH 4.5.[20]
2.4. Synthesis of a Carbonyl-Reactive Lanthanide Complex
The site-specific labeling of glycoproteins or N-terminal ser-
ines/threonines is achieved by periodate oxidation and subse-
quent reaction with a hydrazide linker (a model reaction of N-
terminal serine with 17-Eu is presented in Scheme 4 at the
bottom).[19,20] Amine-reactive nitrophenyl ester 15 was synthe-
sized from Boc-hydrazine and 4-nitrophenyl chloroformate in
dry THF according to the protocol of Gante and Weitzel in
96% yield (Scheme 5).[28] Coupling of 15 with 13 was achieved
in dry DMF with N,N-diisopropylethylamine (DIPEA) (Scheme 3,
step e). Purification was performed by HPLC (method B) with
an aqueous acetic acid system, and elution of the pure prod-
uct (93% yield) was achieved by applying a gradient of in-
creasing acetonitrile content. The Boc group of 16 was quanti-
tatively cleaved in TFA/CH2Cl2 (1:1), and metalation of 17 was
performed by using the procedure described for 13. Final
product 17-Eu was isolated by evaporation of water. Thereby,
2.3. Synthesis of a Sulfhydryl-Reactive Lanthanide Complex
Activated terpyridine 5 was treated with a stoichiometric
amount of DTTA derivative 11 in refluxing acetonitrile with
sodium carbonate as the base. The crude product was extract-
ed with brine and not with ethylenediamine-N,N,N’,N’-tetraace-
tate (EDTA) solution, as described by Deslandes et al.[12] Pure
complex precursor 12 was isolated after column chromatogra-
phy in a yield comparable (50%) to that given in their
report.[12] Cleavage of the Boc and tert-butyl ester groups was
performed in trifluoroacetic acid (TFA)/CH2Cl2 (1:1) at a concen-
tration of 12.05 mm 12 to obtain 13 in 96% yield.
To synthesize a sulfhydryl-reactive probe, the lanthanide ion
was introduced into chelate 13. Metalation was achieved by
combining aqueous solutions of lanthanide(III) chloride and a
stoichiometric amount of 13, whereas the pH was repeatedly
adjusted to 6.5 with dilute NaOH. Finally, the primary amino
group of 13-Eu/Tb was directly converted into a maleimide
group with N-methoxycarbonylmaleimide in saturated NaHCO3
by using a method described originally by Keller and Ruding-
er.[17] Management of the temperature and time was very im-
portant during the reaction, and to quickly stop the reaction,
Scheme 5. Synthesis of Boc-aza-Gly 4-nitrophenyl ester 15: Reagents and
conditions: a) Boc-hydrazine (2.0 equiv), 4-nitrophenyl chloroformate
(1.0 equiv), THF, RT, 12 h, 96%.
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some sodium chloride was formed as a byproduct, which origi-
nated from adjustment of the pH. It was not removed, because
it did not influence further steps.
2.5. Luminescent BSA Conjugate
Bovine serum albumin (BSA) was chosen for labeling with sulf-
hydryl-reactive complex 14-Eu, because on average every
second BSA molecule possesses one accessible sulfhydryl
group.[29] The coupling was performed overnight in 2-[4-(2-hy-
droxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES)-buffered
saline (HBS) 7.3 containing 1 mm EDTA under an argon atmos-
phere. In contrast to the general procedure shown in
Scheme 4, the reducing agent, tris(2-carboxyethyl)phosphine
(TCEP), was omitted for two reasons: 1) TCEP cleaves important
disulfides in BSA, which causes destabilization and the appear-
ance of additional thiol groups. 2) The endogenous free cys-
teine of BSA is known to resist oxidation for a very long
time.[29] The labeled BSA was isolated by size-exclusion chroma-
tography, and a labeling efficiency of 91% was estimated by
comparing the extinction coefficients of BSA[30] and 14-Eu (for
the spectra, see Figure 2; also see calculations in the Support-
ing Information).
Figure 2. Absorption spectra of BSA labeled with 14-Eu, 14-Eu, 14-Tb, and
17-Eu in HBS 7.3. The values were renormalized to the appropriate extinc-
tion coefficients at l=335 nm (11900, 11580, and 10420 mꢀ1 cmꢀ1 for 14-
Eu, 14-Tb, and 17-Eu, respectively) to estimate the labeling efficiency of BSA
with 14-Eu.
2.6. Photophysical Properties
The most important photophysical data are summarized in
Table 1. The absorption spectra of chelates 14-Eu, 14-Tb, and
17-Eu and that of 14-Eu-labeled BSA are shown in Figure 2.
The characteristic luminescence spectra of 14-Eu, 17-Eu, and
14-Tb in HBS 7.3 are presented in Figure 3. The europium
7
emission bands arise from 5D0! FJ (J=0–4) transitions, and
the strongest emission is at l=611 nm (J=2). The terbium
5
7
bands result from D4! FJ (J=6–3) transitions, and the stron-
gest emission is at l=541 nm (J=5).
Figure 3. Emission spectra of 14-Eu, 14-Tb, and 17-Eu in HBS 7.3. The excita-
tion was performed at l=335 nm with excitation and emission slits of 1 nm.
The spectrum of 17-Eu was measured with a spectrometer with poorer sen-
sitivity in the long-wavelength region.
The luminescent quantum yield was determined by a rela-
tive method by using rhodamine 6G as a standard.[31,32] The
data were collected in aerated HBS 7.3 and were corrected for
changes in refractive indices (see Figure S1 in the Supporting
Information). The quantum yield determined for 14-Eu [F=
(32.2ꢁ2.0)%] is comparable to the highest values measured
for Eu complexes.[34] Coupling 14-Eu to BSA affected the quan-
tum yield only marginally [F=(28.8ꢁ2.3)%], which led to the
conclusion that conjugation to proteins has no adverse effect.
Tb-complex 14-Tb had a rather poor quantum yield [F=
(18.4ꢁ2.4)%] relative to those of other known Tb complexes.
Analogous observations were also reported by Bechara
et al.[14,15] A similar distinction between Eu and Tb was seen
upon comparing the lifetimes: 14-Eu and 17-Eu showed slow
Table 1. Spectroscopic properties of 14-Eu, 14-Tb, 17-Eu, and 14-Eu-labeled BSA.
Compound
lmax [nm]
e [mꢀ1 cmꢀ1
]
F
[a] [%]
tDonor [ms]
tD [b] [ms]
q[c]
2O
14-Eu
14-Eu on BSA
14-Tb
236
227
236
14700
107100 (278 nm)
14300
32.3ꢁ2.0
28.8ꢁ2.3
18.4ꢁ2.4
1197ꢁ5
1254ꢁ7
961ꢁ8
1767ꢁ8
1765ꢁ13
–
0.02ꢁ0.01
ꢀ0.02ꢁ0.01
–
205ꢁ19
1199ꢁ6
17-Eu
235
13900
–
–
–
[a] Quantum yield was determined by the relative method by using rhodamine 6G as a standard.[31,32] The measurement was performed in aerated HBS 7.3.
Excitation was performed at l=335 nm by using a 1 nm slit for emission and excitation. The data were corrected for changes in the refractive indices.
[b] Measured in deuterated water for 14-Eu and deuterated HBS 7.3 for 14-Eu-labeled BSA. [c] The hydration state q was calculated by using the equation
q=1.2(1000/tDonorꢀ1000/tD Oꢀ0.25), in which tDonor is the lifetime in nondeuterated buffer and tD is the lifetime in deuterated buffer.[33]
2 O
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single-exponential decays with t=(1197ꢁ5) and (1199ꢁ6) ms,
respectively. These long lifetimes are comparable to those of
other Eu complexes with good luminescence properties. In
contrast to the Eu complexes, 14-Tb showed two lifetimes, one
with t=(961ꢁ8) ms and the other one with t=(205ꢁ19) ms.
The banana-shaped curve presented in Figure 4 (red dashed
line) is a clear indicator of this multiexponential decay. The fact
that 14-Tb possesses a low quantum yield and two lifetimes,
both of which are shorter than expected for a good Tb com-
plex, demonstrates that 14-Tb is not suited for LRET
experiments.
decrease in signal intensity was observed over the whole 24 h
time period (see Figure S3), and the characteristic emission
spectrum of 17-Eu was fully conserved (data not shown),
which implies that EuIII stays tightly bound to the chelate.
Apart from signal intensity, the lifetime of 14-Eu-labeled BSA
listed in Table 2 also remained unchanged in the presence of
Table 2. Lifetimes of 14-Eu-labeled BSA in HBS 7.3 (3 mm) under different
conditions (see curves in Figure S5).
Additive[a]
Buffer
t
[b] [ms]
–
–
HBS 7.3
50 mm phosphate
HBS 7.3
HBS 7.3
HBS 7.3
HBS 7.3
HBS 7.3
HBS 7.3
1254ꢁ7
1227ꢁ11
1250ꢁ5
1248ꢁ13
1232ꢁ8
1244ꢁ6
1256ꢁ5
1255ꢁ3
2 mm ATP
2 mm AMP
2 mm adenosine
2 mm GTP
2 mm EDTA
2 mm EGTA
[a] The pH of the nucleotide, adenosine, EGTA, or EDTA stock solution
was adjusted to pH 7.3 before it was used as an additive in the experi-
ments. [b] All lifetimes exhibited a monoexponential decay and were
fitted to the equation I=I0 +I1·eꢀ(t/t), in which I is the total luminescence
intensity, I0 is time-independent background light, I1 is the luminescence
intensity from the Eu emission, t is the time and t is the luminescence
lifetime of the Eu emission.
2 mm EDTA or EGTA. Numerous proteins require metal ions
such as zinc(II) or copper(II) for their proper function. It is
known that some lanthanide complexes possess low stability
against zinc(II) and copper(II), which cause transmetalation
with the lanthanide.[36] For this reason, the stability of 17-Eu
(3 mm) in the presence of 10 mm ZnII (physiological concentra-
tion[37]) and 200 mm ZnII was examined by monitoring the lumi-
nescence intensity for more than 24 h (Figure S3). No change
in luminescence intensity was observed, which indicates that
the Eu complex could be used in the presence of physiological
zinc(II) concentrations without any concerns. Analogous meas-
urements were also performed with 20 mm (physiological con-
centration[38]) and 200 mm copper(II) sulfate. In both cases, the
luminescence was also constant for 24 h, but the total lumines-
cence in 200 mm copper(II) sulfate was already at the begin-
ning of the experiment 1.5 times lower than that in absence of
CuII (data not shown). This was attributed to the colloidal
nature of the 200 mm copper(II) sulfate solution at pH 7.3.
Lanthanide ions bound to lanthanide-binding tags and
some lanthanide complexes were reported to be sensitive to
inorganic phosphate, so that physiological phosphate buffers
could not be used.[6,39] No such sensitivity to phosphate was
seen in BSA-bound 14-Eu during 15 min exposure to 50 mm
phosphate buffer (Table 2).
Figure 4. Lifetimes of 14-Eu-labeled BSA, 17-Eu, 14-Eu, and 14-Tb in HBS 7.3
at a concentration of 3 mm. Chelates 14-Eu and 17-Eu show single exponen-
tial decays, which were undisturbed by protein coupling, whereas 14-Tb ex-
hibits biexponential behavior with two shorter lifetimes. The inset illustrates
the parallel time courses for all Eu complexes, which reflects equal lifetimes.
The lifetimes of free 14-Eu and BSA-bound 14-Eu were mea-
sured in nondeuterated and deuterated buffer (Figure S2 and
Table 1). A general reason for the shorter lifetimes in nondeu-
terated buffer is the presence of water in the outer hydration
shell of the lanthanide complexes.[33] In deuterated buffer, free
14-Eu and BSA-bound 14-Eu exhibited the same lifetimes
(Table 1). In nondeuterated buffer, however, BSA-bound 14-Eu
showed a small but significant increase in the lifetime from t=
(1197ꢁ5) to (1254ꢁ7) ms. Using the method of Beeby et al.,[33]
the q values for free 14-Eu and BSA-bound 14-Eu were calcu-
lated as 0.02ꢁ0.01 and ꢀ0.02ꢁ0.01, respectively. This q value
is interpreted as the number of water molecules in the inner
coordination sphere.[33] We cannot be fully sure whether the
measured effect is truly significant, but if so, then the data sug-
gest reduced access of water to EuIII in the BSA-bound state.
Chelates 14-Eu and 17-Eu were intended for labeling of pro-
teins and for measuring distances within proteins or between
interacting proteins. Many proteins are CaII dependent and re-
quire EGTA for adjustment of defined CaII concentrations. In
such cases, it is important that EuIII cannot be extracted by
EGTA but that it remains bound. The luminescence intensity of
17-Eu was monitored over a period of at least 24 h with 2 mm
EDTA, EGTA, or Ca-EGTA (2 mm EGTA and 2.1 mm CaCl2 result-
ing in 100 mm free CaII ion concentration)[35] in the buffer. No
Weitz et al. reported quenching of a luminescent Tb com-
plex by millimolar concentrations of adenosine 5’-monophos-
phate (AMP), adenosine 5’-diphosphate (ADP), and especially
adenosine 5’-triphosphate (ATP), which was explained by pho-
toelectron transfer caused by stacking of the nucleobases with
the antenna molecule.[40] Fortunately, no indication of interac-
tions with nucleotides was found with BSA-bound 14-Eu. Slow
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gradual titration with up to 25 mm ATP, ADP, and AMP caused
minor changes in the emission intensity of BSA-bound 14-Eu
(Figure 5), and prolonged incubation at the highest concentra-
tion showed completely stable emission (Figure S4). The lumi-
nescence lifetime of 14-Eu-labeled BSA remained constant
manner (Figure 5) but not the luminescence lifetime (Table 2
and Figure S5). Fortunately, only nucleotides (e.g., ATP, ADP,
AMP, and GTP) but not nucleosides (e.g., adenosine) are typi-
cally required as important cofactors for in vitro studies, for
which distances are measured by LRET. In conclusion, new
complex 14-Eu appears to be unperturbed by most buffer
components that are to be expected.
At a given concentration of GTP, the long light path inside a
fluorimeter cuvette causes a much larger reduction in the exci-
tation intensity than the short light path of an inverted micro-
scope, for which the imaged volume is located immediately
above the surface of the glass slide. Unfortunately, the Nd:YAG
laser used in our inverted microscope had an excitation wave-
length of only 266 nm, which is very close to the absorption
maximum of the nucleotides. To compensate for signal loss
due to the filter effect caused by the nucleotides, the laser
power was increased, and this resulted in decay curves with
slightly increased background signals. This becomes clear
upon comparing the curves with and without nucleotides in
Figure S5. In spite of this increased background, the fitted life-
times summarized in Table 2 were not noticeably influenced
by the higher background and gave the same results within
experimental uncertainty.
Figure 5. The emission of BSA-linked 14-Eu in HBS 7.3 (3 mm) was monitored
at l=615 nm (2.5 nm slit) with excitation at l=335 nm (10 nm slit), while ti-
trating with different nucleotides or adenosine. a) Emission intensities were
corrected for the dilution caused by the addition of the 100 mm nucleotide
stock solution (the stock solution of adenosine was 10 mm). b) Data were in
addition to panel A corrected for the filter effect caused by the nucleotide
solutions. The error bars are shown to one side only to minimize overlap-
ping of the data.
3. Conclusions
New terpyridine-based Eu complexes were designed and syn-
thesized for site-specific labeling of thiols and carbonyls in pro-
teins, peptides, and nucleic acids. The Eu complexes were
found to possess high thermodynamic and kinetical stability,
as previously observed for similar complexes. The high quan-
tum yield and the long lifetimes were not affected by coupling
to proteins or by typical buffer components such as ethylene-
diamine-N,N,N’,N’-tetraacetate, ethylene glycol-bis(2-aminoe-
thylether)-N,N,N’,N’-tetraacetic acid, phosphate, calcium(II),
zinc(II), copper(II), and nucleotides. These findings show that
the new Eu complexes fulfill the crucial requirements for dis-
tance measurements by luminescence resonance energy trans-
fer (LRET). Unlike the Eu complex, the Tb complex of the same
chelator was not suitable for LRET, as it exhibited two lifetimes,
both of which were shorter than that of the Eu complex.
The most significant new feature of the new Eu complexes
was the fact that the linkers were much shorter than those in
comparable published lanthanide chelates. The 2,4-diaminobu-
tyric acid linker module is shorter than the lysine linker in a
comparable complex, and the terminal amine of this linker was
not extended with an additional heterobifunctional linker; in-
stead, it was directly converted into a maleimide or a hydra-
zide function, with minimal length extension. The maleimide
and hydrazide derivatives could be used for site-specific label-
ing of biomolecules by relying on simple well-established cou-
pling protocols. The short length of the linker is expected to
minimize systematic errors upon measuring intra- and intermo-
lecular distances by LRET. Presently, we are examining this cru-
cial aspect in appropriate test systems.
after the addition of 2 mm AMP or ATP (see Table 2 and Fig-
ure S5). Guanosine 5’-triphosphate (GTP) caused a stronger
concentration dependence of the emission intensity of 14-Eu-
bound BSA (see Figure 5a), but this was not due to quenching,
as explained in the following. The reason for the concentration
dependence of the emission intensity is the redshifted absorp-
tion spectrum of GTP (or of a contamination commonly pres-
ent in commercial GTP), which caused absorption of the excita-
tion beam used to excite 14-Eu (for the absorption spectra of
the nucleotide stock-solutions, see Figure S6) in the fluores-
cence cuvette. This “filter effect” caused a GTP-concentration-
dependent decrease in the emission intensity of the Eu com-
plex (Figure 5a), which disappeared once we corrected the
data for the absorption caused by the GTP component (Fig-
ure 5b). Moreover, the emission intensity was not dependent
on the time of GTP incubation (Figure S4), and the lifetime of
14-Eu-labeled BSA was also unaffected by GTP (Table 2, Fig-
ure S5). Only adenosine caused a strong concentration-depen-
dent reduction in 14-Eu emission, which was clearly not just
due to a filter effect, because the absorption of adenosine at
l=335 nm was too small for this purpose. By analogy to the
findings of Weitz et al., stacking of adenosine with the antenna
of the chelate could be responsible for the intensity loss.[40]
The higher tendency of adenosine (relative to ATP, ADP, and
AMP) to form stacks with 14-Eu seems plausible in light of its
much lower solubility, due to the lack of a phosphate group.
Interestingly, adenosine influenced only the luminescence in-
tensity of 14-Eu-labeled BSA in a concentration-dependent
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0.5 mLminꢀ1, and the absorbance was monitored at l=280 and
335 nm.
Experimental Section
General Methods
Analytical-grade solvents and reagents were used as long as they
were commercially available. 2,2’:6’,2“-Terpyridine, N-bromosuccini-
mide (NBS), benzoyl chloride (PhCOCl), lithium bromide (ultradry,
99.998%), phosphorus tribromide, Na-benzyloxycarbonyl-Ng-tert-bu-
tyloxycarbonyl-l-2,4-diaminobutyric acid dicyclohexylammonium
salt [Z-Dab(Boc)-OH·DCHA], europium(III) chloride hexahydrate
(99.99%), terbium(III) chloride hexahydrate (99.99%), trimethylsilyl
cyanide [(CH3)3SiCN], trifluoroacetic acid, and adenosine were pur-
chased from Alfa Aesar. tert-Butyl trichloracetimidate (TBTA), N-ben-
zylethanolamine, boron trifluoride diethyl ether (BF3·OEt2), tert-
butyl carbazate, meta-chloroperoxybenzoic acid (mCPBA), adeno-
sine 5’-triphosphate (ATP) disodium salt hydrate, guanosine 5’-tri-
phosphate (GTP) sodium salt hydrate, adenosine 5’-monophos-
phate (AMP) sodium salt hydrate, ethylene glycol-bis(2-aminoethy-
lether)-N,N,N’,N’-tetraacetic acid (EGTA), and disodium dihydrogen
ethylenediamine-N,N,N’,N’-tetraacetate (EDTA) were ordered from
Sigma–Aldrich. Sodium borohydride and N-methoxycarbonylmalei-
mide were purchased from Fluka. Dry acetonitrile, dry N,N-dime-
thylformamide (DMF), and 4-nitrophenyl chloroformate were ob-
tained from Acros Organics, whereas dry ethanol, triphenylphos-
phine (PPh3), and dry N,N-diisopropylethylamine (DIPEA) were ob-
tained from Merck Millipore. HBS 7.3 [150 mm NaCl, 5 mm 2-[4-(2-
hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) adjusted
with NaOH to pH 7.3] and 50 mm phosphate buffer (sodium dihy-
drogen phosphate monohydrate was adjusted with NaOH to
pH 7.5) were used in the experiments. Toluene and dichlorome-
thane were dried and stored over 4 ꢂ molecular sieves (Merck)
under an argon atmosphere. Dry peroxide-free tetrahydrofuran
(THF) was prepared by passing twice through a bed of basic alumi-
na (60, Merck) of approximately the same volume as the THF and
was used immediately. Reactions requiring an inert atmosphere
were performed under argon 5.0. Thin-layer chromatography (TLC)
was performed on Merck Millipore 60 ꢂ silica sheets without a fluo-
rescence indicator. TLC staining was performed in a chamber with
sublimating iodine. For selective staining of primary or Boc-protect-
ed amines, a solution of 0.1% ninhydrin in acetic acid/1-butanol
mixture (1:50, v/v) was sprayed onto the TLC plates, and subse-
quently, the plates were heated to 120–1508C until violet spots
became visible. Column chromatography was performed on silica
gel (Davisil, 70–200 mm, porosity 60 ꢂ). Reverse-phase (RP) HPLC
was performed by using a Dionex P680 HPLC pump with a Dionex
PDA 100 detector and a polymer based reverse phase column
(Hamilton PRP-3, 10 mm, 300 ꢂ, 305ꢃ7.0 mm). The flow rate was
7 mLminꢀ1, and the absorbance was monitored at l=210, 230,
280, 335, and 359 nm. Method A: The solvents were 3% acetoni-
trile in water (solvent A), acetonitrile (solvent B), and 1% acetic
acid in water (solvent C). The compounds were separated by apply-
ing a gradient from solvent A to solvent B, while keeping the frac-
tion of solvent C constant at 10%. The solvent gradient started
with 0% solvent B for 10 min, and solvent B was then linearly in-
creased to 22% from 10 to 45 min and then kept at 22% for
10 min. Finally, the column was regenerated with 90% solvent B
for 15 min. Method B: Solvents A, B, and C were the same as in
method A, and the fraction of solvent C was also kept constant at
10%. The fraction of solvent B was kept constant during the initial
10 min and was then raised from 0 to 90% within 60 min, and fi-
nally kept constant at 90% for another 15 min. Size-exclusion chro-
matography was performed in HBS 7.3 at 48C with a GE Healthcare
(Little Chalfont, UK) ꢄKTA pure system equipped with a Super-
dex 200 increase column (10 mmꢃ300 mm, GL). The flow rate was
Physical Measurements
1H NMR and 13C NMR spectra were recorded with a Bruker Avance
300 MHz spectrometer, but in some cases, a Bruker Avance-III
700 MHz spectrometer was used. Chemical shifts are given in parts
per million according to the solvent signal. Electrospray (ES) mass
spectra were obtained with an Agilent MSD SL ion-trap mass spec-
trometer (Agilent, Waldbronn, Germany), and high-resolution mass
spectrometry (HRMS) was performed with a LTQ Orbitrap XL from
Thermo Fisher Scientific (Waltham, MA, USA), operating in positive-
ion mode by using electrospray ionization. Absorption measure-
ments were performed with a Hitachi U-3010 or an Agilent
Cary 300. Emission spectra were measured with a Hitachi F-4500 or
a Horiba Fluorolog-3–22. The quantum yields were determined in
aerated HBS 7.3 by the relative method[41] by using rhodamine 6G
(F=94%)[31,32] as a standard. The quantum yield determinations
were performed with a Cary 300 UV/Vis spectrophotometer and a
Fluorolog-3–22 fluorimeter, whereby the data were corrected for
changes in refractive indices. Excitation of reference standard and
samples was performed at l=335 nm, and the concentration was
held below an absorbance value of 0.1. During all the measure-
ments, the same quartz glass cuvette was used, and the orienta-
tion of the cuvette in the spectrometers was always identical.
Stability of the Eu Complexes
The stability was assessed with a Hitachi F-4500 by using a quartz
glass cuvette with dimensions of 8.5 mmꢃ5 mmꢃ2 mm (wꢃhꢃd;
the small window 5 mmꢃ2 mm was used for excitation, and the
large window 8.5 mmꢃ5 mm was used for emission detection).
For the measurements, excitation was at l=335 nm (10 nm slit),
emission was observed at l=615 nm (2.5 nm slit), and the photo-
multiplier voltage was set to 700 V. To a solution of 14-Eu-labeled
BSA in HBS 7.3 (3 mm, 120 mL) increasing amounts of a 100 mm
stock solution of nucleotides (adjusted to pH 7.3) were added at
3 min intervals, until a concentration of approximately 25 mm was
reached (the measurements were performed in triplicate). In the
case of adenosine, a 10 mm stock solution (adjusted to pH 7.3) was
used, and it was added up to a final concentration of about
2.5 mm. Solutions with the highest nucleotide concentrations were
monitored at 1 min intervals for at least 10 min to examine
changes due to prolonged incubation. The luminescence intensity
of 17-Eu in HBS 7.3 (3 mm) in the presence and absence of 2 mm
EDTA, 2 mm EGTA, 2 mm Ca-EGTA buffer, 10 mm zinc(II) chloride,
and 200 mm zinc(II) chloride buffer was monitored over more than
24 h to assess the stability of the complex against competitors.
The EDTA (350 mm) and the EGTA (100 mm) stock solutions were
adjusted to pH 7.3 prior to the preparation of the final dilutions.
For the Ca-EGTA buffer, the EGTA concentration was 2 mm and the
CaCl2 concentration was 2.1 mm, which resulted in a free CaII ion
concentration of 100 mm according to Schoenmakers et al.[35] Con-
trol experiments were performed by measuring solutions contain-
ing EuCl3 (3 mm) and EDTA (2 mm) or EGTA (2 mm) in HBS 7.3.
Lifetime Measurements
The time-resolved measurements were performed with a home-
built setup, and the exact measurement conditions are described
in the Supporting Information.
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suspended in dry acetonitrile (27 mL) and heated at reflux for 1 h.
In the meantime, 9 (524 mg, 1.60 mmol, 2 equiv) was dissolved in
dry acetonitrile (5.4 mL) and added to the stirred suspension. The
mixture was stirred overnight at reflux temperature, and the turn-
over was checked by TLC (silica gel, CHCl3/MeOH/acetic acid=
9:1:0.1, Rf =0.83). After complete conversion, the mixture was fil-
tered, and the solid was washed with acetonitrile. The combined
acetonitrile layer was taken to dryness under reduced pressure,
and the obtained residue was dissolved in CHCl3 and washed with
10% Na2CO3 (2ꢃ) and brine (1ꢃ). The organic layer was dried with
Na2SO4, and the solvent was removed under vacuum to obtain a
crude product (507 mg). Purification was done by chromatography
(silica gel, gradient elution starting with CHCl3/n-heptane=3:2,
and n-heptane was stepwise decreased, followed by pure chloro-
form and finally increasing the MeOH content stepwise to CHCl3/
MeOH=95:5) to give 10 (433 mg, 563 mmol, 71%). 1H NMR
(300 MHz, CDCl3, 258C, Me4Si): d=1.38–1.46 (m, 36H, C(CH3)3),
1.60–1.83 (m, 2H, b-CH2), 2.57–2.86 (m, 8H, N-CH2-CH2-N), 3.01–
3.15 (m, 1H, g-CHI2), 3.22 (s, 4H, CH2-COOC4H9), 3.26–3.41 (m, 2H,
Syntheses
The syntheses of compounds 1–5,[5] 8,[14,15] 9,[14,15] and Z-Dab(Boc)-
OH (according to the manufacturer’s procedure) were performed
in analogy to published procedures and are described in the Sup-
porting Information.
tert-Butyl Na-Benzyloxycarbonyl-Ng-tert-butyloxycarbonyl-l-2,4-dia-
minobutyrate (6): Z-Dab(Boc)-OH (805 mg, 2.29 mmol) was dis-
solved in dry CH2Cl2 (5 mL), and a solution of TBTA (818 mL,
4.6 mmol, 2.0 equiv) in dry cyclohexane (10 mL) was added under
an argon atmosphere. BF3·OEt2 (50.8 mL, 411 mmol, 0.18 equiv) was
added as a catalyst, and the mixture was stirred overnight. The
precipitate was filtered off and washed with CH2Cl2. The combined
CH2Cl2 layer was taken to dryness under reduced pressure. Saturat-
ed Na2CO3 was added to the oily residue, and the mixture was
stirred for 2 h. CHCl3 was added to the suspension, the phases
were separated, and the organic solution was washed with 10%
Na2CO3 (2ꢃ) and 0.1m NaH2PO4 (2ꢃ). The organic solution was
dried with Na2SO4, and the solvent was removed under reduced
3
g-CHII2 and a-CH), 3.70–3.83 (m, 4H, CH2-C6H5), 6.02 (t, JH,H =5.4 Hz,
1
1H, NH), 7.21–7.32 ppm (m, 10H, C6H5); 13C NMR (75 MHz, CDCl3,
258C, Me4Si): d=28.35 (3C, C(CH3)3), 28.39 (3C, C(CH3)3), 28.62 (3C,
C(CH3)3), 29.2 (1C, b-CH2), 37.4 (CH2, g-CH2), 49.3 (2C, N-CH2-CH2-N-
CH2-CH2-N), 52.4 (2C, N-CH2-CH2-N-CH2-CH2-N), 55.2 (2C, CH2-C6H5
or CH2-COOC4H9), 58.2 (2C, CH2-C6H5 or CH2-COOC4H9), 60.4 (1C, a-
CH), 78.7 (1C, C(CH3)3), 80.9 (1C, C(CH3)3), 81.0 (1C, C(CH3)3), 127.3
(2C, para-CH of C6H5), 128.4 (4C, meta-CH of C6H5), 129.4 (4C,
ortho-CH of C6H5), 138.7 (2C, ipso-C of C6H5), 156.2 (1C, C carbonyl
of Boc), 170,7 (2C, C carbonyl of t-butyl ester), 172,3 ppm (1C, C
carbonyl of t-butyl ester); HRMS (ESI+): m/z (%): 769.5098 [M+H]+
(100), 791.4913 [M+Na]+ (10) (C43H68N4O8 requires 769.5110 [M+
H]+, 791.4929 [M+Na]+).
pressure to give 6 (870.3 mg, 2.13 mmol, 93%). H NMR (300 MHz,
CDCl3, 258C, Me4Si): d=1.44 (s, 9H, C(CH3)3), 1.45 (s, 9H, C(CH3)3),
1.60–1.72 (m, 1H, b-CHI2), 1.99–2.10 (m, 1H, b-CHII2), 2.94–3.05 (m,
1H, g-CHI2), 3.37–3.45 (m, 1H, g-CHII2), 4.30 (dt, JH,H =8.7 Hz, JH,H
3
3
=
4.2 Hz, 1H, a-CH), 5.11 (s, 2H, CH2-C6H5), 5.14 (brs, 1H, NH), 5.53 (d,
3JH,H =7.6 Hz, 1H, NH), 7.32–7.37 ppm (m, 5H, aromatic CH of
C6H5); 13C NMR (75 MHz, CDCl3, 258C, Me4Si): d=28.1 (3C, C(CH3)3),
28.6 (3C, C(CH3)3), 33.8 (1C, b-CH2), 36.8 (1C, g-CH2), 52.3 (1C, a-
CH), 67.2 (1C, CH2-C6H5), 79.4 (1C, C(CH3)3), 82.6 (1C, C(CH3)3), 128.2
(2C, ortho-CH of C6H5), 128.3 (1CH, para-CH of C6H5), 128.7 (2C,
meta-CH of C6H5), 136.4 (1C, ipso-C of C6H5), 156.1 (1C, C carbonyl),
156.5 (1C, C carbonyl), 171.5 ppm (1C, C carbonyl); HRMS (ESI+):
m/z (%): 409.2334 [M+H]+ (100), 426.2598 [M+NH4]+ (40),
431.2151 [M+Na]+ (10), 817.4587 [2M+H]+ (15), 839.4406 [2M+
Na]+ (30) (C21H32N2O6 requires 409.2333 [M+H]+, 426.2599 [M+
NH4]+, 431.2153 [M+Na]+, 817.4594 [2M+H]+, 839.4413 [2M+
Na]+).
tert-Butyl
Na,Na-Bis({N-[(2-tert-butoxy)-2-oxoethyl]}-2-aminoethyl)
Ng-tert-Butyloxycarbonyl-2,4-diaminobutyrate (11): Compound 10
(420 mg, 546 mmol) was dissolved in 1-propanol (4.5 mL), and 10%
Pd/C (143 mg, 134 mmol, 0.25 equiv) was added. Ammonium for-
mate (1.43 g, 22.6 mmol, 42 equiv) was separately dissolved in
MeOH (4.5 mL) and was added under an argon atmosphere. The
suspension was stirred at RT overnight. The suspension was filtered
through Celite and washed with 1-propanol, and the solvent was
evaporated under vacuum. The crude product was dissolved in
CHCl3 and washed with 10% Na2CO3 (2ꢃ). The organic solution
was dried with Na2SO4, and the solvent was removed under re-
duced pressure to give 11 (270.1 mg, 459 mmol, 84%). 1H NMR
(300 MHz, CDCl3, 258C, Me4Si): d=1.437 (s, 9H, C(CH3)3), 1.444 (s,
9H, C(CH3)3), 1.465 (s, 18H, C(CH3)3), 1.65–1.80 (m, 1H, b-CHI2),
tert-Butyl Ng-tert-Butyloxycarbonyl-l-2,4-diaminobutyrate (7): Com-
pound 6 (165 mg, 404 mmol) was dissolved in 1-propanol (1.7 mL),
and 10% Pd/C (33 mg, 31 mmol, 0.08 equiv) was added. Ammoni-
um formate (264 mg, 4.2 mmol, 10 equiv) was separately dissolved
in MeOH (1.7 mL) and added under an argon atmosphere. The sus-
pension was stirred at RT overnight. The suspension was filtered
through Celite and washed with 1-propanol, and the solvent was
evaporated under vacuum. The crude product was dissolved in
CHCl3 and washed with saturated NaHCO3 (2ꢃ). The organic solu-
tion was dried with Na2SO4, and the solvent was removed under
reduced pressure to give 7 (106.8 mg, 389 mmol, 96%). 1H NMR
(300 MHz, CDCl3, 258C, Me4Si): d=1.44 (s, 9H, C(CH3)3), 1.47 (s, 9H,
1.86–1.93 (m, 1H, b-CHII ), 2.55–2.90 (m, 8H, N-CH2-CH2-N), 3.09–
2
3
3.25 (m, 1H, g-CHI2), 3.32 (d, JH,H =2.5 Hz, 4H, CH2-COOC4H9), 3.26–
3
3.45 (m, 2H, g-CHII2 and a-CH), 6.72 ppm (t, JH,H =4.9 Hz, 1H, NH);
C(CH3)3), 1.59–1.70 (m, 1H, b-CHI2), 1.89–1.99 (m, 1H, b-CHII2), 3.19–
3.29 (m, 1H, g-CHI2), 3.31–3.37 (m, 1H, g-CHII2), 3.39 (dd, JH,H
13C NMR (75 MHz, CDCl3, 258C, Me4Si): d=28.3 (6C, C(CH3)3), 28.4
(3C, C(CH3)3), 28.7 (3C, C(CH3)3), 29.2 (1C, b-CH2), 37.2 (1C, g-CH2),
47.6 (2C, NH-CH2-CH2-N-CH2-CH2-NH), 50.6 (2C, CH2-COOC4H9), 51.5
(2C, NH-CH2-CH2-N-CH2-CH2-NH), 60.4 (1C, a-CH), 78.5 (1C, C(CH3)3),
81.1 (1C, C(CH3)3), 81.2 (2C, C(CH3)3), 156.5 (1C, COOC4H9), 172.0
(2C, COOC4H9), 172.2 ppm (1C, COOC4H9); HRMS (ESI+): m/z (%):
589.4164 [M+H]+ (100), 611.3974 [M+Na]+ (15) (C29H56N4O8 re-
quires 589.4171 [M+H]+, 611.3990 [M+Na]+).
3
=
8.7 Hz, 3JH,H =4.5 Hz, 1H, a-CH), 5.23 ppm (brs, 1H, NH); 13C NMR
(75 MHz, CDCl3, 258C, Me4Si): d=28.2 (3C, C(CH3)3), 28.6 (3C,
C(CH3)3), 34.6 (1C, b-CH2), 38.2 (1C, g-CH2), 53.9 (1C, a-CH), 79,2
(1C, C(CH3)3), 81.4 (1C, C(CH3)3), 156.1 (1C, C carbonyl of Boc),
175.2 ppm (1C, COOC(CH3)3); HRMS (ESI+): m/z (%): 275.1963 [M+
H]+ (100), 297.1780 [M+Na]+ (10), 549.3850 [2M+H]+ (35),
571.3667 [2M+Na]+ (10) (C13H26N2O4 requires 275.1965 [M+H]+,
297.1785 [M+Na]+, 549.3858 [2M+H]+). 571.3677 [2M+Na]+).
Di-tert-butyl 2,2’-(8-{1-(tert-Butoxy)-4-[(tert-butoxycarbonyl)amino]-
1-oxobutan-2-yl}-5,8,11-triaza-1,2,3(2,6)-tripyridinacyclododeca-
phane-5,11-diyl)diacetate (12): Compound 11 (175 mg, 297 mmol)
and 5 (125 mg, 297 mmol, 1 equiv) were dried by azeotropic distil-
lation (3ꢃ) with dry toluene under reduced pressure and dissolved
tert-Butyl Na,Na-Bis({N-benzyl-N-[(2-tert-butoxy)-2-oxoethyl]}-2-ami-
noethyl) Ng-tert-Butyloxycarbonyl-2,4-diaminobutyrate (10): Com-
pound 7 (219 mg, 798 mmol, dried by evaporating three times with
dry toluene) and K2CO3 (1.1 g, 8.0 mmol, 10 equiv) were dissolved/
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in dry acetonitrile (110 mL). Na2CO3 (315 mg, 2.97 mmol, 10 equiv)
was added, and the suspension was heated at reflux overnight
under an argon atmosphere (with an argon bubbler connected to
the outlet of the reflux condenser). The suspension was filtered
and washed with acetonitrile and CHCl3, and the solvent was
evaporated under vacuum. The crude product was dissolved in
CHCl3 and washed with brine (2ꢃ). The organic solution was dried
with Na2SO4, and the solvent was removed under reduced pres-
sure. The crude mixture was purified by chromatography (silica gel,
gradient elution starting with CHCl3/n-heptane=98:2, followed by
CHCl3, increasing the MeOH content stepwise and ending with
CHCl3/MeOH=9:1) to give 12 (126 mg, 149 mmol, 50%). 1H NMR
(300 MHz, CDCl3, 258C, Me4Si): d=1.17 (s, 27H, C(CH3)3), 1.36 (s,
General Procedure for Lanthanide Complex Formation: 2 mm Euro-
pium(III) chloride hexahydrate [or terbium(III) chloride hexahydrate]
in water was added in portions to a 2 mm solution of the ligand in
water. During the addition, the pH was repeatedly adjusted to 6.5
with dilute sodium hydroxide (150 mm). The solution was stirred
overnight under an argon atmosphere at RT. Water was removed
under reduced pressure to give the lanthanide complex in quanti-
tative yield together with some sodium chloride.
Europium(III)
2,2’-[8-(3-Amino-1-carboxylatopropyl)-5,8,11-triaza-
1,2,3(2,6)-tripyridinacyclododecaphane-5,11-diyl]diacetate (13-Eu):
See the general procedure for preparation. 1H NMR (700 MHz,
[D4]methanol, 258C): d=49.20, 35.45, 29.15, 22.85, 20.08 18.23,
16.89, 14.93, 14.37, 11.18, 10.54, 7.27, 6.99, 5.60, 3.75, 3.42, 3.30,
2.66, 0.93, 0.74, 0.08, ꢀ0.90, ꢀ7.81, ꢀ14.01, ꢀ15.07, ꢀ17.86,
ꢀ18.68, ꢀ21.20 ppm; 13C NMR (176 MHz, [D4]methanol, 258C): d=
125.4, 164.9, 119.1, 107.3, 111.8, 7.1, 93.1, 147.7, 111.8, 7.1, 141.4,
48.5, 35.4, 35.4, 62.3, 98.2, 71.6, 90.7, 71.6, 62.3, 42.6, 42.6, 29.5,
67.8, 132.7, 136.8, 142.1, 161.7, 165.0, 167.5, 174.2, 177.5, 189.9,
213.7 ppm; UV/Vis (H2O): lem (e)=235 (14400), 283 (8300), 292
(11500), 335 nm (10900 molꢀ1 dm3 cmꢀ1); HRMS (ESI+): m/z (%):
726.1681 [M+H]+ (85), 728.1695 [M+H]+ (100), 748.1498 [M+
Na]+ (10), 750.1512 [M+Na]+ (12) (C29H32N7O6 requires 726.1685
[M+H]+, 728.1699 [M+H]+, 748.1505 [M+Na]+, 750.1519 [M+
Na]+).
9H, C(CH3)3), 1.75–1.89 (m, 2H, b-CH2), 2.63–3.00 (m, 8H, N-CH2-
3
CH2-N), 3.06–3.19 (m, 6H, CH2-COOC4H9 and g-CH2), 3.65 (d, JH,H
=
6.7 Hz, 1H, a-CH), 3.96 (d, 2JH,H =12.9 Hz, 2H, N-CHI2(meso)-C-N),
4.19 (d, 2JH,H =12.8 Hz, 2H, N-CHII2(meso)-C-N), 5.00 (brs, 1H, NH),
7.35 (d, 3JH,H =7.5 Hz, 2H, CH of 5 and 5’’ in terpyridine), 7.92 (t,
3
3JH,H =7.7 Hz, 2H, CH of 4 and 4’’ in terpyridine), 8.01 (d, JH,H
=
7.6 Hz, 2H, CH of 3 and 3’’ in terpyridine), 8.11 ppm (s, 3H, CH of
3’, 4’ and 5’ in terpyridine); 13C NMR (75 MHz, CDCl3, 258C, Me4Si):
d=26.4 (1C, b-CH2), 27.9 (9C, C(CH3)3), 28.5 (3C, C(CH3)3), 38.7 (1C,
g-CH2), 49.5 (2C, N-CH2-CH2-N-CH2-CH2-N), 53.2 (2C, N-CH2-CH2-N-
CH2-CH2-N), 57.1 (2C, CH2-COOC4H9), 57.8 (2C, N-CH2(meso)-C-N),
60.9 (1C, a-CH), 79.2 (1C, C(CH3)3), 82.0 (2C, C(CH3)3), 82.6 (1C,
C(CH3)3), 120.5 (2C, CH of 3 and 3’’ in terpyridine), 121.7 (2C, CH of
3’ and 5’ in terpyridine), 124.0 (2C, CH of 5 and 5’’ in terpyridine),
138.5 (2C, CH of 4 and 4’’ in terpyridine), 139.2 (1C, CH of 4’ in ter-
pyridine), 155.0 (2C, C of 2, 2’’ or 2’, 6’ in terpyridine), 155.3 (2C, C
of 2, 2’’ or 2’, 6’ in terpyridine), 156.1 (1C, C of COOC4H9), 158.1
(2C, C of 6 and 6’’ in terpyridine), 171.8 (2C, C of CH2-COOC4H9),
173.2 ppm (1C, C of COOC4H9); HRMS (ESI+): m/z (%): 846.5128
[M+H]+ (90), 868.4941 [M+Na]+ (100) (C46H67N7O8 requires
846.5124 [M+H]+, 868.4943 [M+Na]+).
Europium(III) 2,2’-{8-[3-(N-maleimido)-1-carboxylatopropyl]-5,8,11-
triaza-1,2,3(2,6)-tripyridinacyclododecaphane-5,11-diyl}diacetate
(14-Eu): Compound 13-Eu (8.8 mg, 12.1 mmol) was dissolved in
water (280 mL), and saturated NaHCO3 (3.1 mL) was added under
an argon atmosphere. The solution was stirred at 08C, and N-me-
thoxycarbonylmaleimide (101 mg, 654 mmol, 54 equiv) was added
in one portion. After 15 min, the solution was warmed to RT and
stirred for another 30 min. Subsequently, the pH of the solution
was adjusted to pH 6 with 6m HCl. The solution was concentrated
under vacuum, and the solid was dissolved in a mixture of water
(4 mL) and MeOH (0.6 mL). The solution was filter through cotton
and was concentrated under vacuum to a volume of about 200 mL.
The product was diluted with water to a total volume of 900 mL
and was loaded on an HPLC column. The collected fractions were
evaporated under vacuum to dryness. HPLC (method A): tR =
19.3 min. Pure product 14-Eu (7.9 mg, 9.8 mmol, 60%) was isolated.
UV/Vis (HBS 7.3): lmax (e)=236 (14700), 283 (8900), 292 (12500),
2,2’-[8-(3-Amino-1-carboxypropyl)-5,8,11-triaza-1,2,3(2,6)-tripyridina-
cyclododecaphane-5,11-diyl]diacetic acid (13): Compound 12
(20.4 mg, 24.1 mmol) was dissolved in dry CH2Cl2 (1 mL) and TFA
(1 mL) to give a concentration of 12.05 mm for 12. The mixture
was stirred overnight at RT. Toluene was added to the solution,
and the solvent was removed under reduced pressure. The result-
ing product was evaporated from toluene (1ꢃ), MeOH (3ꢃ), and
water (1ꢃ) to give 13 (24 mg, 23.3 mol, 96%). 1H NMR (300 MHz,
335 nm (11900 molꢀ1 dm3 cmꢀ1); luminescence (HBS 7.3, lexc
=
3
CDCl3, 258C, Me4Si): d=1.97–2.18 (m, 2H, b-CH2), 3.00 (t, JH,H
=
335 nm): lem (%)=578 (22), 586 (26), 595 (38), 611 (100), 615 (81),
618 (58), 622 (27), 649 (9), 681 (48), 684 (27), 690 (17), 695 (17), 700
(31), 704 nm (32); luminescence quantum yield (rhodamine 6G
standard): F=0.32; HRMS (ESI+): m/z (%): 806.1583 [M+H]+ (85),
808.1598 [M+H]+ (100), 828.1401 [M+Na]+ (17), 830.1415 [M+
Na]+ (20), 838.1843 [M+MeOH+H]+ (32), 840.1858 [M+MeOH+
H]+ (35), 862.1678 [M+MeOH+K]+ (10) (C33H32N7O8Eu requires
806.1584 [M+H]+, 808.1597 [M+H]+, 828.1403 [M+Na]+,
830.1417 [M+Na]+, 838.1846 [M+MeOH+H]+, 840.1860 [M+
MeOH+H]+, 862.1679 [M+MeOH+H]+).
6.3 Hz, 2H, g-CH2), 3.13–3.21 (m, 2H, N-CH2-CH2-N-CH2-CH2-N),
3.35–3.42 (m, 2H, N-CH2-CH2-N-CH2-CH2-N), 3.48–3.51 (m, 1H, a-
CH), 3.62–3.70 (m, 4H, N-CH2-CH2-N-CH2-CH2-N), 3.74 (s, 4H, CH2-
COOH), 4.72 (m, 4H, N-CH2(meso)-C-N shown in the HSQC), 7.80 (d,
3
3JH,H =7.7 Hz, 2H, CH of 5 and 5’’ in terpyridine), 8.27 (t, JH,H
=
3
7.8 Hz, 2H, CH of 4 and 4’’ in terpyridine), 8.43 (d, JH,H =8.0 Hz, 2H,
CH of 3 and 3’’ in terpyridine), 8.61–8.72 ppm (m, 3H, CH of 3’, 4’
and 5’ in terpyridine); 13C NMR (75 MHz, CDCl3, 258C, Me4Si): d=
23.3 (1C, b-CH2), 37.3 (1C, g-CH2), 47.0 (2C, N-CH2-CH2-N-CH2-CH2-
N), 53.1 (2C, N-CH2-CH2-N-CH2-CH2-N), 55.4 (2C, CH2-COOH), 57.3
(2C, N-CH2(meso)-C-N), 61.2 (1C, a-CH), 116.3 (1C, 1JC,F =291.7 Hz,
CF3-COOH), 124.3 (2C, CH of 3 and 3’’ in terpyridine), 125.6 (2C, CH
of 3’ and 5’ in terpyridine), 128.4 (2C, CH of 5 and 5’’ in terpyri-
dine), 141.9 (2C, CH of 4 and 4’’ in terpyridine), 147.16 (1 or 2C, C
of 2, 2’’ or CH of 4’), 147.19 (1 or 2C, C of 2, 2’’ or CH of 4’), 148.1
(2C, CH of 2’ and 6’ in terpyridine), 150.7 (2C, CH of 6 and 6’’ in
terpyridine), 162.9 (1C, 2JC,F =35.5 Hz, CF3-COOH), 170.4 (2C, CH2-
COOH), 173.6 ppm (1C, CH-COOH); HRMS (ESI+): m/z (%):
578.2721 [M+H]+ (100), 600.2536 [M+Na]+ (15) (C29H35N7O6 re-
quires 578.2722 [M+H]+, 600.2541 [M+Na]+).
Terbium(III)
2,2’-[8-(3-Amino-1-carboxylatopropyl)-5,8,11-triaza-
1,2,3(2,6)-tripyridinacyclododecaphane-5,11-diyl]diacetate (13-Tb):
See the general procedure for the preparation. UV/Vis (H2O): lmax
(e)=235
(14100),
283
(8100),
292
(11300),
335 nm
(10700 molꢀ1 dm3 cmꢀ1); MS (ESI+): m/z (%): 734.2 [M+H]+ (100),
756.2 [M+Na]+ (50), 772.1 [M+K]+ (30).
Terbium(III)
2,2’-{8-[3-(N-Maleimido)-1-carboxylatopropyl]-5,8,11-
triaza-1,2,3(2,6)-tripyridinacyclododecaphane-5,11-diyl}diacetate
(14-Tb): Maleimide 14-Tb was prepared according to the procedure
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outlined for 14-Eu with 13-Tb (1.4 mg, 1.9 mmol). HPLC (method A):
tR =19.0 min. Pure product 14-Tb (1.4 mg, 1.7 mmol, 89%) was iso-
lated. UV/Vis (HBS 7.3): lmax (e)=236 (14300), 283 (8600), 292
(12100), 335 nm (11500 molꢀ1 dm3 cmꢀ1); luminescence (HBS 7.3,
lexc =335 nm): lem (%)=486 (28), 501 (10), 541 (100), 546 (78), 549
(51), 553 (28), 583 (29), 588 (18), 620 (15), 624 nm (12); lumines-
cence quantum yield (rhodamine 6G standard): F=0.18; HRMS
(ESI+): m/z (%): 814.1636 [M+H]+ (70), 836.1458 [M+Na]+ (15),
846.1897 [M+MeOH+H]+ (100), 868.1716 [M+MeOH+Na]+ (25)
(C33H32N7O8Tb requires 814.1639 [M+H]+, 836.1458 [M+Na]+,
846.1901 [M+CH3OH+H]+, 868.1720 [M+CH3OH+Na]+).
1H NMR (300 MHz, D2O, 258C): d=1.84–2.00 (m, 2H, b-CH2), 3.05–
3.12 (m, 2H, g-CH2), 3.17–3.33 (m, 4H, N-CH2-CH2-N-CH2-CH2-N),
3.34–3.38 (m, 1H, a-CH), 3.61–3.77 (m, 8H, N-CH2-CH2-N-CH2-CH2-N
3
and N-CH2-COOH), 4.76 (brs, 4H, N-CH2(meso)-C-N), 7.80 (d, JH,H
=
3
7.7 Hz, 2H, CH of 5 and 5’’ in terpyridine), 8.26 (t, JH,H =7.9 Hz, 2H,
CH of 4 and 4’’ in terpyridine), 8.41 (d, JH,H =8.0 Hz, 2H, CH of 3
and 3’’ in terpyridine), 8.63 (d, JH,H =7.6 Hz, 2H, CH of 3’ and 5’ in
terpyridine), 8.73 ppm (t, JH,H =8.0 Hz, 1H, CH of 4’ in terpyridine);
3
3
3
13C NMR (75 MHz, D2O, 258C): d=27.0 (1C, b-CH2), 37.5 (1C, g-CH2),
47.3 (2C, N-CH2-CH2-N-CH2-CH2-N), 53.4 (2C, N-CH2-CH2-N-CH2-CH2-
N), 56.1 (2C, N-CH2-COOH), 57.2 (2C, N-CH2(meso)-C-N), 61.5 (1C, a-
CH), 124.2 (2C, CH of 3 and 3’’ in terpyridine), 125.5 (2C, CH of 3’
and 5’ in terpyridine), 128.1 (2C, CH of 5 and 5’’ in terpyridine),
141.4 (2C, CH of 4 and 4’’ in terpyridine), 147.0 ppm (1C, CH of 4’
in terpyridine); MS (ESI+): m/z (%):636.2 [M+H]+ (80), 658.3 [M+
Na]+ (100), 674.2 [M+K]+ (30), 690.3 [M+MeOH+Na]+ (35), 712.4
[MꢀH+2K]+ (35).
Europium(III) 2,2’-{8-[1-Carboxylato-3-(hydrazinecarboxamido)prop-
yl]-5,8,11-triaza-1,2,3(2,6)-tripyridinacyclododecaphane-5,11-diyl}dia-
cetate (17-Eu): See the general procedure for lanthanide complex
formation for the preparation. UV/Vis (HBS 7.3): lmax (e)=235
(13900), 283 (7560), 292 (10430), 336 nm (10430 molꢀ1 dm3 cmꢀ1);
luminescence (HBS 7.3, lexc =335 nm): lem =578, 586, 594, 610, 615,
618, 622, 647, 679, 688, 693, 698, 702 nm; HRMS (ESI+): m/z (%):
784.1850 [M+H]+ (90), 786.1864 [M+H]+ (100), 806.1669 [M+
Na]+ (21), 808.1682 [M+Na]+ (23), 822.1407 [M+K]+ (10),
824.1420 [M+K]+ (11) (C30H34N9O7Eu requires 784.1852 [M+H]+,
786.1866 [M+H]+, 806.1672 [M+Na]+, 808.1686 [M+Na]+,
822.1411 [M+K]+, 824.1425 [M+K]+).
tert-Butyloxycarbonyl-aza-glycine 4-Nitrophenyl Ester (15): Boc-hy-
drazine (251 mg, 1.9 mmol) was dissolved in dry THF (2.0 mL) and
nitrophenyl chloroformate (191 mg, 0.95 mmol, 0.5 equiv) was
added in portions. The solution was stirred overnight, and the pre-
cipitate was filtered off. The filtrate was dissolved in CHCl3 and ex-
tracted with 100 mm phosphate buffer pH 6.0 (2ꢃ) and 100 mm
phosphate buffer pH 7.5 (sat. with NaCl) for as often as needed
until the buffer phase no longer turned yellow. The organic solu-
tion was dried with Na2SO4 and concentrated under vacuum to
1
give 15 (320 mg, 908 mmol, 96%). H NMR (300 MHz, CDCl3, 258C,
Me4Si): d=1.50 (s, 9H, C(CH3)3), 6.51 (brs, 1H, NH), 7.11 (brs, 1H,
3
NH), 7.36 (d, 3JH,H =8.9 Hz, 2H, ortho CH), 8.26 ppm (d, JH,H =9.0 Hz,
2H, meta CH).
2,2’-(8-{3-[2-(tert-Butoxycarbonyl)hydrazine-1-carboxamido]-1-car-
boxypropyl}-5,8,11-triaza-1,2,3(2,6)-tripyridinacyclododecaphane-
5,11-diyl)diacetic acid (16): A solution of 15 (3.4 mg, 10 mmol,
2 equiv) in dry DMF (120 mL) was added to dry 13 (5.0 mg,
4.8 mmol, dried by evaporation with dry toluene, 3ꢃ). DIPEA
(13.5 mL, 77 mmol, 16 equiv) was added, and the solution was
stirred under an argon atmosphere at RT overnight. Subsequently,
the mixture was frozen in liquid nitrogen and attached to a liquid-
nitrogen-cooled cold trap, and the solvent was removed with stir-
ring at 10–100 Pa. The residue was dissolved in 0.1% acetic acid/
5% MeOH in water (1 mL). This solution was loaded onto the HPLC
column, and the collected fractions were evaporated under
vacuum to dryness. HPLC (method B): tR =15.5 min. Pure product
16 (4.1 mg, 4.5 mmol, 93%) was isolated. 1H NMR (300 MHz,
[D4]methanol, 258C, Me4Si): d=1.46 (s, 9H, C(CH3)3), 1.56–1.73 (m,
1H, b-CHI2), 1.74–1.89 (m, 1H, b-CHII2), 2.91–3.14 (m, 2H, g-CH2),
3.14–3.45 (m, 4H, N-CH2-CH2-N-CH2-CH2-N), 3.45–3.54(m, 1H, a-CH),
3.54–3.70 (m, 4H, N-CH2-CH2-N-CH2-CH2-N), 3.76 (brs, 4H, N-CH2-
BSA labeling: A solution of 14-Eu (0.1 mg, 124 nmol) in MeOH/H2O
(1:1, 200 mL) was transferred to a 1.5 mL vial commonly used in au-
tosamplers and taken to dryness under reduced pressure. BSA
(13.2 mg, 100 nmol, 0.81 equiv) was separately dissolved in HBS 7.3
containing 1 mm EDTA (0.5 mL), and foam formation was avoided
by stirring the solution slowly until complete solvation. The BSA
solution was added to the vial containing dry 14-Eu, and subse-
quently, the air was displaced by argon and the vial was closed
with a crimp cap. The vial was cautiously swirled from time to time
and was left to stand overnight at room temperature. Labeled BSA
was purified by size-exclusion chromatography in HBS 7.3 with a
Superdex 200 increase column (10 mmꢃ300 mm, GE Healthcare,
flow rate=0.5 mLminꢀ1), while collecting 0.5 mL fractions. The
product was detected by its absorbance at l=280 and 330 nm
(for the chromatogram see Figure S69).
3
COOH), 4.73 (brs, 4H, N-CH2(meso)-C-N), 7.61 (d, JH,H =6.9 Hz, 2H,
CH of 5 and 5’’ in terpyridine), 8.07–8.21 ppm (m, 7H, CH of 3, 4,
3’, 4’, 5’, 3’’ and 4’’ in terpyridine); 13C NMR (75 MHz, [D4]methanol,
258C, Me4Si): d=27.1 (3C, C(CH3)3), 29.4 (1C, b-CH2), 36.1 (1C, g-
CH2), 46.8 (2C, CH2, N-CH2-CH2-N-CH2-CH2-N), 53.3 (2C, N-CH2-CH2-
N-CH2-CH2-N), 56.7 (2C, N-CH2-COOH), 58.0 (2C, N-CH2(meso)-C-N),
59.9 (1C, a-CH), 122.4 (1C, CH of 3, 4, 3’, 4’, 5’, 3’’ or 4’’ in terpyri-
dine), 122.6 (1C, CH of 3, 4, 3’, 4’, 5’, 3’’ or 4’’ in terpyridine), 123.8
(2C, CH of 5 and 5’’ in terpyridine), 138.8 (C, CH of 3, 4, 3’, 4’, 5’, 3’’
or 4’’ in terpyridine), 139.1 ppm (C, CH of 3, 4, 3’, 4’, 5’, 3’’ or 4’’ in
terpyridine); MS (ESI+): m/z (%): 736.5 [M+H]+ (100), 758.4 [M+
Na]+ (40), 774.3 [M+K]+ (5), 734.3 [MꢀH]ꢀ (100), 756.1 [M+
Nꢀ2H]ꢀ (10).
2,2’-{8-[1-Carboxy-3-(hydrazinecarboxamido)propyl]-5,8,11-triaza-
1,2,3(2,6)-tripyridinacyclododecaphane-5,11-diyl}diacetic acid (17):
Dry 16 (4.1 mg) was treated overnight in dry CH2Cl2 (190 mL) and
TFA (190 mL) under an argon atmosphere to remove the Boc
group. After the reaction was finished, toluene was added to the
solution. The residue was evaporated from toluene (3ꢃ), MeOH
(3ꢃ), and Milli-Q water (1ꢃ) to give 17 (3.9 mg, 4.5 mmol, 100%).
Acknowledgements
We gratefully acknowledge financial support of the Austrian Sci-
ence Funds (FWF, project FWFW1250). The NMR spectrometers
were acquired in collaboration with the University of South Bohe-
mia (CZ) with financial support from the European Union
through the EFRE INTERREG IV ETC-AT-CZ program (project
M00146, “RERI-uasb”). We would further like to thank Klemens
Winkler for support and fruitful discussions regarding the LRET
setup and lifetime measurements, Nicole Ollinger for help in pro-
tein purification, and Maria Wiesauer for helpful discussions con-
cerning chemical syntheses and characterization.
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11
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&
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[20] S. D. Mikolajczyk, D. L. Meyer, J. J. Starling, K. L. Law, K. Rose, B. Dufour,
R. E. Offord, Bioconjugate Chem. 1994, 5, 636.
Conflict of Interest
[21] Z. Zhang, B. A. C. Smith, L. Wang, A. Brock, C. Cho, P. G. Schultz, Bio-
chemistry 2003, 42, 6735.
The authors declare no conflict of interest.
[22] R. P. Thummel, Y. Jahng, J. Org. Chem. 1985, 50, 3635.
[23] W. K. Fife, J. Org. Chem. 1983, 48, 1375.
Keywords: energy transfer · fluorescent probes · lanthanides ·
luminescence · macrocyclic ligands
[24] M. J. O’Donnell, J. T. Cooper, M. M. Mader, J. Am. Chem. Soc. 2003, 125,
2370.
[25] D. C. Gowda, B. Rajesh, S. Gowda, Indian J. Chem. 2000, 504.
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Received: July 5, 2017
Version of record online && &&, 2017
&
ChemistryOpen 2017, 00, 0 – 0
www.chemistryopen.org
12
ꢁ 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

FULL PAPERS
F. Faschinger, M. Ertl, M. Zimmermann,
A. Horner, M. Himmelsbach,
W. Schçfberger, G. Knçr, H. J. Gruber*
&& – &&
Stable Europium(III) Complexes with
Short Linkers for Site-Specific Labeling
of Biomolecules
The missing link: Highly stable Eu com-
plexes possessing long emission life-
times and high quantum yields are pre-
pared. A unique feature of these com-
plexes is the combination of a short
linker with bioorthogonal coupling
functions, which brings the EuIII probe
very close to the attachment site on the
biomolecule and reduces systematic
errors upon measuring intra- and inter-
molecular distances by luminescence
resonance energy transfer.
ChemistryOpen 2017, 00, 0 – 0
www.chemistryopen.org
13
ꢁ 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ