Novel probes showing specific fluorescence enhancement on binding to a hexahistidine tag

Article abstract of DOI:10.1002/chem.200701896

The introduction of hexahistidine (His tag) is widely used as a tool for affinity purification of recombinant proteins, since the His tag binds selectively to nickel-nitrilotriacetic acid (Ni²⁺-NTA) complex. To develop efficient turn-on fluorescent probes for His-tagged proteins, we adopted a fluorophore displacement strategy, that is, we designed probes in which a hydroxycoumarin fluorophore is joined via a linker to a metal-NTA moiety, with which it forms a weak intramolecular complex, thereby quenching the fluorescence. In the presence of a His tag, with which the metal-NTA moiety binds strongly, the fluorophore is displaced, which results in a dramatic enhancement of fluorescence. We synthesized a series of hydroxycoumarins that were modified by various linkers with NTA (NTAC ligands), and investigated the chemical and photophysical properties of the free ligands and their metal complexes. From the viewpoint of fluorescence quenching, Ni ²⁺ and Co²⁺ were the best metals. Fluorescence spectroscopy revealed a 1:1 binding stoichiometry for the Ni²⁺ and Co²⁺ complexes of NTACs in pH 7.4 aqueous buffer. As anticipated, these complexes showed weak intrinsic fluorescence, but addition of a His-tagged peptide (H-(His)₆-Tyr-NH₂; Tyr was included to allow convenient concentration measurement) in pH 7.4 aqueous buffer resulted in up to a 22-fold increase in the fluorescence quantum yield. We found that the Co ²⁺ complexes showed superior properties. No fluorescence enhancement was seen in the presence of angiotensin I, which contains two nonadjacent histidine residues; this suggests that the probes are selective for the polyhistidine peptide.

Full text of DOI:10.1002/chem.200701896

DOI: 10.1002/chem.200701896
Novel Probes Showing Specific Fluorescence Enhancement on Binding to a
Hexahistidine Tag
Mie Kamoto, Naoki Umezawa,* Nobuki Kato, and Tsunehiko Higuchi*[a]
Abstract: The introduction of hexahis-
tidine (His tag) is widely used as a tool
for affinity purification of recombinant
proteins, since the His tag binds selec-
tively to nickel–nitrilotriacetic acid
(Ni2+–NTA) complex. To develop effi-
cient “turn-on” fluorescent probes for
His-tagged proteins, we adopted a fluo-
rophore displacement strategy, that is,
we designed probes in which a hydroxy-
coumarin fluorophore is joined via a
linker to a metal–NTA moiety, with
which it forms a weak intramolecular
complex, thereby quenching the fluo-
rescence. In the presence of a His tag,
with which the metal–NTA moiety
binds strongly, the fluorophore is dis-
placed, which results in a dramatic en-
hancement of fluorescence. We synthe-
sized a series of hydroxycoumarins that
were modified by various linkers with
NTA (NTAC ligands), and investigated
the chemical and photophysical proper-
ties of the free ligands and their metal
complexes. From the viewpoint of fluo-
rescence quenching, Ni2+ and Co2+
were the best metals. Fluorescence
spectroscopy revealed a 1:1 binding
stoichiometry for the Ni2+ and Co2+
complexes of NTACs in pH 7.4 aque-
ous buffer. As anticipated, these com-
plexes showed weak intrinsic fluores-
cence, but addition of a His-tagged
peptide (H-(His)6-Tyr-NH2; Tyr was in-
cluded to allow convenient concentra-
tion measurement) in pH 7.4 aqueous
buffer resulted in up to a 22-fold in-
crease in the fluorescence quantum
yield. We found that the Co2+ com-
plexes showed superior properties. No
fluorescence enhancement was seen in
the presence of angiotensin I, which
contains two nonadjacent histidine resi-
dues; this suggests that the probes are
selective for the polyhistidine peptide.
Keywords: cobalt
·
fluorescent
probes · molecular recognition ·
peptides · sensors
Introduction
nonspecific. The most commonly applied method for label-
ing a target protein selectively is by expressing the protein
as a fusion with a fluorescent protein, such as green fluores-
cent protein (GFP). This approach has been used to study
the dynamics of individual proteins in living cells.[2] Al-
though this technique offers absolute specificity, there
remain some limitations. The fluorescent protein requires a
period of up to several hours to mature into a soluble, fluo-
rescent protein, and this is too slow for some applications.
The folding of fluorescent proteins is known to be problem-
atic, especially at or above room temperature.[2] Also, the
fluorescence of GFP is sensitive to environmental factors,
such as pH, and can be difficult to distinguish from cellular
autofluorescence.[2] GFP variants have been produced
through protein engineering to overcome these limitations,
but the steric bulk of this protein (at least 220 aa) still has
the potential for significant perturbation of folding, traffick-
ing, and function of proteins to which it is fused.[3]
For proteomics research, huge numbers of proteins must be
characterized. The fluorescent labeling of proteins has
proven to be a powerful approach for following the dynamic
processes of protein synthesis and degradation, identifying
localization, and studying protein–protein interactions, both
in vitro and in vivo.[1] Many labeling techniques have been
developed that are based on the reaction of fluorescent dyes
bearing functional groups, such as succinimidyl ester or male-
imide, that react with primary amines or thiols exposed on
the protein surface. However, these techniques are typically
[a] M. Kamoto, Dr. N. Umezawa, Dr. N. Kato, Prof. Dr. T. Higuchi
Graduate School of Pharmaceutical Sciences
Nagoya City University
3-1, Tanabe-dori, Mizuho-ku
Nagoya, 467-8603 (Japan)
Fax : (+81)52-836-3435
Alternative protein-labeling strategies have emerged that
address the steric bulk and stability problems, as well as the
limited range of fluorescence characteristics of GFP and its
variants. Representative techniques involve the introduction
E-mail: umezawa@phar.nagoya-cu.ac.jp
higuchi@phar.nagoya-cu.ac.jp
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200701896.
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FULL PAPER
of a short peptide that is able to react/interact specifically
with a designed fluorescent molecule.[4,5] The combination
of a short peptide tag (a specific target for the fluorescent
probe) and a functional fluorescent molecule (a fluorescent
probe that recognizes the tag peptide) has great potential,
because technology is available for tagging proteins in a se-
lective, site-specific, and reversible manner.[6,7] This ap-
proach has several advantages, for example, it reduces per-
turbation that is caused by attachment of the fluorophore
and offers the possibility of using a wide variety of function-
al molecules, besides fluorophores, as labels.
Tsien and co-workers have developed a technique that
utilizes a tag peptide sequence containing four Cys residues
positioned to allow reaction with a biarsenical fluorogenic
compound.[6] The resulting biarsenical peptide adduct is
more fluorescent than the initial fluorogenic compound. The
tag peptide sequence necessary for reaction with the bi-
arsenical molecule is small (16 amino acid residues in a typi-
cal application); this implies minimal perturbation of the
cal properties of a series of hydroxycoumarins modified by
various linkers with NTA. We confirmed that the Co2+ com-
plexes of these probes showed substantial fluorescence
enhancement upon binding to hexahistidine, as expected.
Results and Discussion
Strategy for protein labeling with fluorescence enhance-
ment: As a specific interaction between tag peptide and flu-
orescent probe, we focused on the interaction of a hexahisti-
dine sequence with a metal complex. It is well-known that
the Ni2+–NTA or Co2+–NTA complex interacts selectively
with the hexahistidine sequence in aqueous media, by
means of coordination of the imidazoles of the histidine side
chains to the metal ion. Our strategy is summarized in
Scheme 1. To control the fluorogenic properties, we adopted
target protein, and presents
a minimal 4-Cys motif
(CCPGCC) that confers adequate specificity. The main dis-
advantage is the toxicity of the arsenic derivatives used. Al-
though Tsien has developed a protocol for the administra-
tion of antidotes that render the method broadly applicable,
alternative methods that do not rely on such toxic metals
would offer a distinct advantage.
Recently, several fluorescent derivatives of Ni2+–nitrilotri-
acetic acid complex (Ni2+–NTA), which targets the hexahis-
tidine tag ((His)6), have been reported.[8] The selective inter-
action between the His tag and Ni2+–NTA has been widely
used for the affinity purification of recombinant proteins,
and the His tag itself is thought to have little influence on
protein function.[9] However, most of the probes reported so
far show no significant change in fluorescence intensity
when the probe binds to the His tag, so detection requires
fluorescence anisotropy experiments,[8d,e] or the use of the
fluorescence resonance energy transfer technique with an-
other fluorophore, such as GFP.[8a] More accurate and sim-
pler monitoring of proteins would be possible if the probe
showed fluorescence enhancement upon binding to the
target protein. As far as we know, the only example of such
a probe so far is dansyl–NTA–Ni2+ complex,[10] which uses
the interaction between the field-sensitive dansyl group as a
fluorophore and an additional hydrophobic tritryptophan
motif coupled with the hexahistidine tag. This method is ele-
gantly designed, but the position of the hexahistidine tag is
limited to hydrophilic regions of the target protein and the
environment of the labeled protein affects the fluorescence.
In this paper, we describe the design and synthesis of a
series of His-tag-targeting fluorescent probes based on the
hydroxycoumarin fluorophore. The design employs a fluoro-
phore displacement strategy, that is, displacement of the flu-
orophore from a weak intramolecular complex with the His-
tag-targeting moiety in the presence of His tag results in a
large fluorescence enhancement. The tag sequence can be
incorporated at any position of the target protein. Here, we
describe the synthesis, chemical properties, and photophysi-
Scheme 1. Schematic illustration of our strategy for protein labeling.
an intramolecular fluorophore displacement strategy, as has
already been successfully applied to the fluorometric detec-
tion of nitric oxide,[11] pH,[12] and anions.[13] We hypothesized
that if an intramolecular fluorescent ligand coordinates to
the metal chelated by NTA with a relatively weak affinity, it
would be displaced in the presence of the hexahistidine pep-
tide sequence, resulting in enhanced fluorescence.
Design offluorescent probes : The structures of “hemilabile”
ligands consist of the fluorophore, a linker, and the metal–
NTA complex as the His-tag recognition site (Figure 1).
Several linkers were employed because the nature of the
linker is expected to influence the affinity of the fluoro-
phore coordination to the metal–NTA complex. Hydroxy-
coumarin derivatives, such as calcein blue, are known indica-
tors of various metal ions.[14] The fluorescence of such a mol-
ecule is significantly reduced upon coordination with a
metal ion, and so we considered that hydroxycoumarin
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T. Higuchi, N. Umezawa et al.
lated selectively at the 8-position with paraformaldehyde
and triethylamine.[15] The NTA units were synthesized from
3a–c. Compound 3a was synthesized by a Curtius reaction
starting from N-a-tert-butoxycarbonyl-l-glutamic acid g-
benzyl ester according to the reported procedure.[16] Com-
pounds 3b and 3c were readily synthesized from N-d-benzyl-
oxycarbonyl-l-ornithine
and
N-e-benzyloxycarbonyl-l-
lysine, respectively.[16,17] Bisalkylation of the a nitrogen of
3a–c with ethyl bromoacetate in the presence of potassium
carbonate provided the triesters 4a–c. Deprotection of the
benzyloxycarbonyl (Cbz) group by hydrogenolysis gave the
corresponding primary amines, and then condensation with
formylcoumarin 2, followed by reduction afforded 5a–c. Hy-
drolysis with lithium hydroxide provided the desired
NTACs, which were purified by reverse-phase HPLC. This
synthesis features reductive amination of an aldehyde and
amine, which may be compatible with various other fluoro-
phores and tag-peptide-recognizing motifs.
Figure 1. Structure of the designed fluorescent probes; M2+ is a metal ion
that can be chelated stably by NTA. (n=1–3).
would be a good candidate fluorophore for our purpose.
The hydroxyl group of coumarin and/or the secondary
amine may coordinate to the metal–NTA complex. To make
the probes switchable, metal–NTA should quench the fluo-
rescence of hydroxycoumarin efficiently. Therefore, we
screened various metal ions from the standpoint of fluores-
cence quenching, as described below.
Screening ofmetal ions : To find the appropriate metal ions
for NTACs, the fluorescence change was investigated upon
addition of various metal ions. Metal ions that can efficient-
ly quench the fluorescence of the hydroxycoumarin fluoro-
phore are required to develop “turn-on” fluorescent probes.
All the NTACs were highly fluorescent in Tris buffer
(50 mm, pH 7.4). Although the majority of the 21 metal ions
examined did not exhibit efficient quenching of hydroxycou-
marin fluorescence, several showed concentration-depen-
dent quenching. Representative results are shown in
Figure 2. Among the metal ions that we used, Ni2+ and
Co2+ were found to be the best from the standpoint of fluo-
rescence quenching, and they were selected for further
study because both Ni2+–NTA and Co2+–NTA are known
to interact selectively with hexahistidine sequences.
Synthesis ofligand molecules with lfuorophore : The de-
signed molecules were synthesized according to Scheme 2.
Commercially available 7-hydroxycoumarin (1) was formy-
A Jobꢁs plot analysis was performed to determine the
complexation stoichiometry of NTACs with Ni2+ or Co2+ in
neutral aqueous buffer. Eleven solutions containing NTAC
and Ni2+ or Co2+ in different ratios were prepared, and the
total concentration was kept at 10 mm. Fluorescence changes
(excitation wavelength: 365 nm, emission wavelength:
455 nm) of these samples were plotted as a function of the
molar fraction of NTACs. The plots showed a peak at 0.5
(Figure 3), which indicated the formation of a 1:1 complex.
This is consistent with our hypothesis that hydroxycoumarin
coordinates intramolecularly to the metal, which is chelated
by NTA.
Fluorescence response ofCo 2+–NTAC and Ni2+–NTAC
with hexahistidine peptide: We investigated the fluorescence
change of NTAC–metal complexes upon addition of a
model tag peptide. We first prepared a solution of Ni2+
–
NTAC or Co2+–NTAC complex by mixing NTAC solution
and NiCl2 or CoCl2 solution in Tris buffer (50 mm, pH 7.4).
A model hexahistidine tag peptide 6 (H-(His)6-Tyr-NH2; Tyr
was attached to allow convenient determination of concen-
tration) was added to the Ni2+–NTAC or Co2+–NTAC solu-
tion, and the fluorescence spectra were obtained. Represen-
Scheme 2. Synthesis of NTACs: a) paraformaldehyde, Et3N, CH3CN,
reflux, 10 h; 13%; b) ethyl bromoacetate, K2CO3, CH3CN, reflux, 20 h;
56% (4a), 91% (4b), 76% (4c); c) 10% Pd/C, MeOH, RT, 12 h;
d) 1) AcOH, THF or dichloroethane, RT, 2–6 h; 2) NaBH3CN, THF, RT,
12 h; 37% (5a), 55% (5b), 43% (5c); e) LiOH (1n), 30% aqMeOH,
08C, 2 h; 26% (NTAC-2), 45% (NTAC-3), 31% (NTAC-4).
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Binding to Hexahistidine
FULL PAPER
Figure 2. Relative fluorescence intensities of NTACs (5 mm) in the pres-
ence of various metal ions (Mn2+, Co2+, Ni2+, Cu2+, Ca2+, Mg2+) in Tris
buffer (50 mm, pH 7.4). a) NTAC-2, b) NTAC-3, c) NTAC-4. Excitation:
Figure 3. Jobꢁs plot of the fluorescence changes upon complexation of
NTACs with Ni2+ or Co2+ in Tris buffer (50 mm, pH 7.4) at 258C.
a) NTAC-2, b) NTAC-3, c) NTAC-4. [NTAC]+[metal ion]=10 mm. Exci-
2+
2+
2+
365 nm, emission: 455 nm. : Mn
,
: Co2+, &: Ni2+
,
: Cu
,
,
~
&
*
*
: Ca
”: Mg2+
.
2+
tation: 365 nm, emission: 455 nm. &: Ni2+
,
: Co .
~
tative results are shown in Figure 4a. The fluorescence in-
creased in a concentration-dependent manner. All NTAC–
metal complexes gave similar results. Most existing probes
that comprise a fluorophore and a metal-ion-chelating NTA
moiety contain Ni2+ as the metal ion;[8,10] however, Co2+
complexes showed larger fluorescence changes than Ni2+
–
NTAC complexes in our case. In addition, Ni2+–NTAC com-
plexes needed a much longer time for equilibration (more
than 3 h) compared with Co2+–NTAC complexes (~10 min).
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T. Higuchi, N. Umezawa et al.
peptide 6 in different ratios were prepared, and the total
concentration was kept at 10 mm. Jobꢁs plots showed a maxi-
mum at the molar ratio of 0.5, which indicated the forma-
tion of a 1:1 complex (Figure 5). The Jobꢁs plot for NTAC-4
at a lower ratio of peptide 6 showed nonlinearity. The
reason for this observation is currently unclear.
Figure 5. Jobꢁs plot of the fluorescence changes upon complexation of
Co2+–NTAC complexes with peptide 6 in 50 mm Tris buffer (pH 7.4) at
258C. [Co2+–NTAC complexes]+[peptide 6]=10 mm. Co2+–NTAC com-
plexes were prepared by mixing equimolar amounts of NTACs and
2+
2+
~
^
CoCl2. Excitation: 365 nm, emission: 455 nm. : Co –NTAC-2, : Co
–
NTAC-3, : Co2+–NTAC-4.
*
Fluorescence and chemical properties ofCo 2+–NTAC com-
plexes: The fluorescence and chemical properties of Co2+
–
–
NTAC complexes are summarized in Table 1. The Co2+
Figure 4. a) Emission spectra of Co2+–NTAC-4 complexes in the presence
of peptide 6 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20 mm) in Tris buffer
(50 mm, pH 7.4). Co2+–NTAC complex was prepared in situ by mixing
NTAC and CoCl2 (NTAC-4: 5 mm, CoCl2: 10 mm). All spectra were ob-
NTAC complexes were generated in situ by combining
NTAC with CoCl2 in a 1:10 ratio to suppress the NTAC
fluorescence completely. The fluorescence spectra were
measured in Tris buffer (50 mm, pH 7.4) in the absence or
presence of peptide 6. The fluorescence quantum yields
were 0.033–0.10 before addition of peptide 6, and were in-
creased 7.9–22-fold to 0.67–0.79 by the addition of peptide
6. Further, the binding affinity for peptide 6 was assessed
from the fluorescence intensity. In this case, Co2+–NTAC
complexes were generated by combining NTAC with CoCl2
in a 1:1 ratio, assuming that formation of NTAC–Co2+ com-
plex is complete, since the interaction of NTAC with Co2+ is
very strong. The apparent dissociation constants (Kd) of
Co2+–NTAC-2, Co2+–NTAC-3, and Co2+–NTAC-4 for pep-
tide 6 were calculated to be 2.2”10ꢀ6, 1.4”10ꢀ6, and 5.7”
tained with excitation at 365 nm. b) Fluorescence response of Co2+
–
NTAC complex (5 mm) in the presence of peptide 6 in Tris buffer (50 mm,
pH 7.4). Co2+–NTAC complex was prepared by mixing equimolar
amounts of NTAC and CoCl2 (NTAC: 5 mm, CoCl2: 5 mm). Excitation:
2+
2+
2+
~
^
*
365 nm, emission: 455 nm. : Co –NTAC-2, : Co –NTAC-3, : Co
–
NTAC-4.
Thus, for our present purpose, Co2+–NTAC complexes
are superior. The fluorescence increase of Co2+–NTACs was
dependent on the peptide concentration, as shown in Fig-
ure 4b. The intrinsic fluorescence of the Co2+–NTAC com-
plex was very weak, whereas strong blue fluorescence was
observed in the presence of peptide 6, as shown in Figure S1
(see the Supporting Information). The final fluorescence re-
covery upon binding of Co2+–NTAC complex to the peptide
6 reached >90% of the fluorescence intensity of the corre-
sponding metal-free dye.
Table 1. Chemical properties of Co2+–NTAC.[a]
Compound
Fluorescence quantum yield[b]
Kd [m][c]
Co2+–NTAC
Co2+–NTAC–peptide 6
NTAC-2
NTAC-3
NTAC-4
0.073
0.033
0.10
0.73
0.67
0.79
(2.2ꢁ0.4)”10ꢀ6
(1.4ꢁ0.4)”10ꢀ6
(5.7ꢁ2.7)”10ꢀ8
To determine the stoichiometry of the Co2+–NTAC and
hexahistidine peptide 6 complexes in solution, Jobꢁs plot
AHCTREUNG
AHCTREUNG
analyses of the fluorometric changes were made. The Co2+
–
[a] All data were measured in Tris buffer (50 mm, pH 7.4). [b] Quantum
yields were calculated by using that of quinine sulfate in H2SO4 (1.0n) as
a standard.[19] [c] Dissociation constants of Co2+–NTAC complexes for
peptide 6.
NTAC complexes were generated in situ by combining
NTAC with CoCl2 in a 1:1 ratio in Tris buffer (50 mm,
pH 7.4). Eleven solutions that contained Co2+–NTAC and
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Binding to Hexahistidine
FULL PAPER
10ꢀ8 m, respectively. These Kd values are comparable with
those of other Ni2+–NTA based probes (1-20 mm),[8a,b,d] and
suggest that Co2+–NTAC-4 would be the most efficient
among the molecules that we have developed. The long
spacer would favor easy displacement of the fluorophore by
the peptide.
with peptide 6 in pH 7.4 buffered aqueous solutions pro-
duced a selective increase in the fluorescence quantum yield
of up to 22-fold. No enhancement was observed upon addi-
tion of angiotensin I, which contains two nonadjacent histi-
dine residues. The strategy described here should be applica-
ble to various other fluorophores/functional compounds. Be-
cause the hexahistidine tag is the most frequently applied af-
finity tag in recombinant protein expression, numerous ap-
Selectivity ofCo 2+–NTAC-4: To confirm the selectivity of
the fluorescence response of Co2+–NTAC-4, the fluores-
cence spectra were measured in the presence of angioten-
sin I (H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-OH). Al-
though this peptide contains two histidines, they are not se-
quential. As shown in Figure 6, no fluorescence change of
plications
for
selective
fluorescent
labeling
or
functionalization of recombinant proteins can be envisioned.
Further work is in progress in our laboratory, especially on
protein labeling.
Experimental Section
General information: All reagents and solvents were of the highest com-
mercial quality and were used without further purification unless other-
wise noted. THF and diethyl ether (Et2O) were distilled from Na/benzo-
phenoneketyl immediately prior to use. CH2Cl2 was distilled from CaH2
immediately prior to use. MeOH and EtOH were distilled from Mg/I2.
TLC was performed on Merck precoated plates (silica gel 60 F254,
0.25 mm), and bands were visualized by fluorescence quenching. TLC
plates were also stained with potassium permanganate. 1H NMR spectra
were recorded on a JEOL GSX-400 or a JEOL JNM-LA 500 at 400 or
500 MHz, respectively. 13C NMR spectra were recorded on either
a
JEOL JNM-LA 500 or a Bruker AVANCE 600 at 125 or 150 MHz, re-
spectively. Chemical shifts are expressed as parts per million (ppm) by
using solvent as an internal standard. IR spectra were recorded on a
JASCO FT/IR-680 FTIR spectrophotometer. Fast atom bombardment
mass spectra (FABMS) were measured with a JEOL JMS-LCMATE
mass spectrometer, by using 3-nitrobenzyl alcohol as the matrix. Matrix-
assisted laser desorption ionization time of flight mass spectrometry
(MALDI-TOF MS) was done with a Shimadzu AXIMA-CFR-NC by
using a-cyano-4-hydroxycinnamic acid as the matrix. HRMS data were
obtained with a JEOL JMS-SX102A or a Bruker Daltonics APEX-III
mass spectrometer. Column chromatography was performed on BW-200
or -300 (Fuji Silysia Chemical, Aichi, Japan). Optical rotations were mea-
sured on a JASCO DIP-1000 polarimeter at room temperature, by using
the sodium D line.
Figure 6. Fluorescence response of Co2+–NTAC-4 complex (5 mm) in the
presence of angiotensin I (H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-
OH) or H6-angiotensin I (H-(His)6-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-
Leu-NH2) in Tris buffer (50 mm, pH 7.4). Co2+–NTAC complex was pre-
pared by mixing equimolar amounts of NTAC and CoCl2 (NTAC-4:
*
5 mm, CoCl2: 5 mm). Excitation: 365 nm, emission: 455 nm. : H6-angio-
*
tensin I, : angiotensin I.
8-Formyl-7-hydroxycoumarin (2): Paraformaldehyde (448 mg, 14 mmol)
and Et3N (1.1 mL, 8.0 mmol) were added to a solution of 7-hydroxycou-
marin (1; 324 mg, 2.0 mmol) in CH3CN (10 mL). The mixture was re-
fluxed for 10 h. The solution was cooled to room temperature and neu-
tralized with HCl (1n). The mixture was extracted with CH2Cl2 (10 mL”
3), washed with brine (10 mL”3), and dried over MgSO4. Purification by
flash column chromatography on silica gel (EtOAc/n-hexane, 1:3) afford-
ed 2 (50.5 mg, 13%) as a colorless solid. 1H NMR (400 MHz, CDCl3):
d=12.24 (s, 1H), 10.61 (s, 1H), 7.67 (d, J=9.6 Hz, 1H), 7.61 (d, J=
8.8 Hz, 1H), 6.90 (d, J=8.8 Hz, 1H), 6.34 ppm (d, J=9.6 Hz, 1H); MS
(FAB): m/z: 191 [M+H]+. These data were consistent with the literature
data for this compound.[15]
Co2+–NTAC-4 complex was observed upon addition of an-
giotensin I. On the other hand, an analogous peptide that in-
cluded the hexahistidine sequence, H6-angiotensin I (H-
(His)6-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-NH2),
showed strong fluorescence enhancement. These results sug-
gest that our molecules selectively recognize polyhisitidine
sequences.
General procedure for N-alkylation: Ethyl bromoacetate (10 equiv), fol-
lowed by finely ground K2CO3 (10–20 equiv) were added to a solution of
an appropriate side-chain-protected amino acid methyl ester (1 equiv) in
CH3CN. The reaction was heated at reflux for 20 h, cooled to room tem-
perature, and concentrated under reduced pressure. The crude product
was purified by flash column chromatography on silica gel.
Conclusions
We have developed novel “turn-on” fluorescent probes that
bind selectively to a hexahistidine tag peptide via a metal
ion–NTA moiety, with a substantial enhancement of their
fluorescence. The new probes consist of a hydroxycoumarin
fluorophore that is joined via a linker moiety to Co2+–NTA.
These complexes are intrinsically only weakly fluorescent,
probably due to the intramolecular coordination of the fluo-
rophore by the metal, but displacement of the fluorophore
Methyl (2S)-4-N-Benzyloxycarbonyl-2-N-bis(2-ethoxy-2-oxoethyl)-2,4-di-
aminobutanoate (4a): According to the general procedure, a solution of
3a[16] (3.97 g, 14.9 mmol) in CH3CN (60 mL) was treated with ethyl bro-
moacetate (16.5 mL, 150 mmol) and K2CO3 (20.6 g, 150 mmol). The
crude product was purified by column chromatography on silica gel
(EtOAc/n-hexane 1:4) to afford 4a (3.63 g, 56%) as a pale-yellow oil.
Rf =0.48 (EtOAc/n-hexane 2:3); [a]2D0 =ꢀ41.6 (c=1.05 in EtOH);
Chem. Eur. J. 2008, 14, 8004 – 8012
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8009

T. Higuchi, N. Umezawa et al.
1H NMR (400 MHz, CDCl3): d=7.38–7.29 (m, 5H), 6.44 (brs, 1H), 5.10
(m, 2H), 4.13 (q, J=7.2 Hz, 4H), 3.68 (s, 3H), 3.57 (m, 4H), 3.58–3.54 (s,
1H), 3.45–3.39 (m, 2H), 2.02–1.95 (m, 1H), 1.82–1.74 (m, 1H), 1.22 ppm
(t, J=7.2 Hz, 6H); 13C NMR (125 MHz, CDCl3): d=172.5, 171.1, 156.5,
136.9, 128.1, 127.6, 127.5, 66.0, 61.9, 60.6, 52.5, 51.3, 37.5, 29.1, 13.9 ppm;
IR (film): n˜ =3366, 2954, 1733, 1520 cmꢀ1; HRMS (EI;70 eV): m/z: calcd
for C21H30N2O8: 438.2002 [M]+; found: 438.2002.
(c=1.05 in EtOH); 1H NMR (400 MHz, CDCl3): d=7.69 (d, J=9.5 Hz,
1H), 7.38 (d, J=8.8 Hz, 1H), 6.99 (d, J=8.8 Hz, 1H), 6.16 (d, J=9.5 Hz,
1H), 4.54 (d, J=13.7 Hz, 1H), 4.44 (d, J=13.7 Hz, 1H), 4.15 (q, J=
7.1 Hz, 4H), 3.72 (s, 3H), 3.68–3.59 (m, 4H), 3.55–3.51 (m, 1H), 3.45–
3.14 (m, 2H), 2.06–2.00 (m, 3H), 1.77–1.75 (m, 1H), 1.25 ppm (t, J=
7.1 Hz, 6H); 13C NMR (100 MHz, CDCl3): d=171.8, 171.6, 161.9, 160.8,
153.7, 144.4, 130.0, 111.0, 110.9, 104.8, 64.5, 61.5, 53.8, 51.9, 47.2, 41.2,
27.7, 24.2, 14.0 ppm; IR (film): n˜ =2983, 1738, 1610, 1582 cmꢀ1; MS
(FAB): m/z: 493 [M+H]+; HRMS (ESI): m/z: calcd for C23H31N2O9:
493.2181 [M+H]+; found: 493.2180.
N-d-Benzyloxycarbonyl-N-a-bis(2-ethoxy-2-oxoethyl)-l-ornithine methyl
ester (4b): According to the general procedure, a solution of 3b[17]
(2.54 g, 9.08 mmol) in CH3CN (50 mL) was treated with ethyl bromoace-
tate (10.1 mL, 90.8 mmol) and K2CO3 (25.1 g, 181 mmol). The crude
product was purified by column chromatography on silica gel (EtOAc/n-
hexane 2:3) to afford 4b (3.72 g, 91%) as a pale-yellow oil. Rf =0.30
(EtOAc/n-hexane, 2:3); [a]2D0 =ꢀ21.3 (c=1.05 in EtOH); 1H NMR
(400 MHz, CDCl3): d=7.34–7.28 (m, 5H), 5.08 (brs, 2H), 4.12 (q, J=
7.3 Hz, 4H), 3.67 (s, 3H), 3.61 (m, 4H), 3.62 (s, 3H), 3.43 (m, 1H), 3.24–
3.23 (m, 2H), 1.74–1.68 (m, 4H), 1.33–1.22 ppm (m, 6H); 13C NMR
(125 MHz, CDCl3): d=172.3, 170.8, 156.0, 136.3, 127.9, 127.4, 127.4, 65.8,
63.9, 60.1, 52.2, 50.9, 40.1, 27.0, 25.7, 13.7 ppm; IR (film): n˜ =3383, 2953,
1730, 1537 cmꢀ1; HRMS (EI; 70 eV): m/z: calcd for C22H32N2O8: 452.2159
[M]+; found: 452.2151.
8-N-[5-(Bisethoxycarbonylmethylamino)-5-ethoxycarbonylpentyl]amino-
methyl-7-hydroxycoumarin (5c): According to the general procedure, 4c
(1.55 g, 4.65 mmol) was treated in CH3OH (500 mL) with 10% Pd/C
(150 mg) under H2. After workup, the resulting residue 4c’ was dissolved
in THF (10 mL) and a solution of 2 (590 mg, 3.10 mmol) and acetic acid
(355 mL, 6.20 mmol) in THF (10 mL) was added. The mixture was stirred
for 2 h, then sodium cyanoborohydride (214 mg, 4.65 mmol) was added.
The crude product was purified by flash column chromatography on
silica gel (MeOH/CH2Cl2 1:19) to afford 5c (667 mg, 43%) as a pale-
yellow oil. Rf =0.33 (MeOH/CH2Cl2 1:9); [a]2D0 =ꢀ25.3 (c=1.05 in
EtOH); 1H NMR (400 MHz, CDCl3): d=7.62 (d, J=9.5 Hz, 1H), 7.28 (d,
J=8.5 Hz, 1H), 6.80 (d, J=8.5 Hz, 1H), 6.16 (d, J=9.5 Hz, 1H), 4.35 (s,
2H), 4.14 (q, J=7.1 Hz, 4H), 3.69 (s, 3H), 3.63–3.59 (m, 4H), 3.46–3.36
(m, 1H), 2.84–2.79 (m, 2H), 1.76–1.45 (m, 6H), 1.25 ppm (t, J=7.1 Hz,
6H); 13C NMR (125 MHz, CDCl3): d=173.0, 172.1, 171.7, 163.6, 161.3,
153.2, 144.4, 128.5, 114.3, 111.1, 110.9, 107.6, 64.0, 60.9, 52.7, 52.6, 51.5,
48.3, 44.0, 29.6, 27.5, 22.9, 14.2 ppm; IR (film): n˜ =2952, 1733, 1605,
1589 cmꢀ1; MS (FAB): m/z: 507 [M+H]+; HRMS (ESI): m/z: calcd for
C25H35N2O8: 507.2337 [M+H]+; found: 507.2335.
N-e-Benzyloxycarbonyl-N-a-bis(2-ethoxy-2-oxoethyl)-l-lysine
methyl
ester (4c): This compound was synthesized according to the reported pro-
cedure (yield: 73%). The analytical data were consistent with the litera-
ture.[18]
General procedure for coupling to the hydroxycoumarin derivative: To a
solution of an appropriate triester (0.85–1.5 equiv) in CH3OH was added
10% Pd/C. This mixture was stirred vigorously under H2 (1 atm) for 12 h
at room temperature. The mixture was filtered through Celite, and the
filtrate was concentrated under reduced pressure. The resulting residue
was dissolved in THF or dichloroethane, and a solution of 2 (1 equiv)
and acetic acid (2 equiv) in THF or dichloroethane was added. The mix-
ture was stirred for 2–6 h at room temperature, then sodium cyanoboro-
hydride (1.5–2 equiv) was added. Stirring was continued for an additional
12 h at room temperature, the mixture was chilled to 08C, and water was
added with stirring. The solution was concentrated under reduced pres-
sure. The crude product was purified by flash column chromatography on
silica gel.
General procedure for hydrolysis: A solution of LiOH (1n, 6 equiv) was
added to
a solution of an appropriate coumarin-containing triester
(1 equiv) in 30% aqueous CH3OH at 08C. The reaction was allowed to
warm to room temperature, stirred for 2 h, neutralized with 30% acetic
acid, and concentrated under reduced pressure. The crude product was
purified by reverse-phase HPLC.
N-[3-(Biscarboxymethylamino)-3-carboxypropyl]-7-hydroxy-8-amino-
methylcoumarin (NTAC-2): According to the general procedure, LiOH
(1n, 1.4 mL, 1.4 mmol) was added to
a solution of 5a (113 mg,
N-[3-(Bisethoxycarbonylmethylamino)-3-ethoxycarbonylpropyl]-7-hy-
droxy-8-aminomethylcoumarin (5a): According to the general procedure,
4a (137 mg, 0.45 mmol) was treated in CH3OH (10 mL) with 10% Pd/C
(15 mg) under H2. After work-up, the resulting residue 4a’ was dissolved
in THF (2.0 mL) and a solution of 2 (100 mg, 0.53 mmol) and acetic acid
(60.2 mL, 1.05 mmol) in THF (2.0 mL) was added. The mixture was
stirred for 6 h, then sodium cyanoborohydride (66.1 mg, 1.05 mmol) was
added. The crude product was purified by column chromatography on
silica gel (MeOH/CH2Cl2 1:19) to afford 5a (79.7 mg, 37%) as a pale-
yellow oil. Rf =0.23 (MeOH/CH2Cl2 1:9); [a]2D0 =ꢀ10.22 (c=1.05 in
EtOH); 1H NMR (500 MHz, CDCl3): d=7.65 (d, J=9.5 Hz, 1H), 7.38 (d,
J=8.6 Hz, 1H), 6.99 (d, J=8.6 Hz, 1H), 6.21 (d, J=9.5 Hz, 1H), 4.71 (d,
J=12.7 Hz, 1H), 4.39 (d, J=12.7 Hz, 1H), 4.11(q, J=7.1 Hz, 4H), 3.74 (s,
3H), 3.70–3.56 (m, 4H), 3.49–3.38 (m, 2H), 3.28–3.24 (m, 1H), 2.52–2.42
(m, 1H), 2.09–2.04 (m, 1H), 1.21 ppm (t, J=7.1 Hz, 6H); 13C NMR
(125 MHz, CDCl3): d=171.8, 170.5, 160.9, 160.8, 154.1, 144.1, 129.9,
116.6, 112.2, 112.1, 108.4, 64.0, 61.8, 52.2, 47.7, 40.7, 24.5, 13.9 ppm; IR
(film): n˜ =2985, 1735, 1608, 1578 cmꢀ1; MS (FAB): m/z: 479 [M+H]+;
HRMS (ESI): m/z: calcd for C21H31N2O8: 479.2024 [M+H]+; found:
479.2027.
0.23 mmol) in 30% aqueous CH3OH (10 mL). The crude product was pu-
rified by reverse-phase HPLC by using a linear gradient system from
18:82 to 20:80 CH3CN/H2O (0.1% TFA) over 20 min, and the collected
fractions were lyophilized to afford NTAC-2 (32.6 mg, 26%) as a white
solid. The purity of this compound was determined as >97% by HPLC
analysis. 1H NMR (500 MHz, DMSO): d=7.90 (d, J=9.5 Hz, 1H), 7.51
(d, J=8.6 Hz, 1H), 6.91 (d, J=8.6 Hz, 1H), 6.26 (d, J=9.5 Hz, 1H), 4.96
(d, J=14.3 Hz, 1H), 4.83 (d, J=14.3 Hz, 1H), 4.11–4.07 (m, 1H), 4.04–
3.96 (m, 4H), 3.37–3.34 (m, 2H), 2.32–1.86 ppm (m, 2H); 13C NMR
(125 MHz, DMSO): d=173.0, 172.8, 160.0, 159.8, 153.9, 144.7, 129.0,
112.7, 111.3, 111.3, 108.8, 62.9, 53.2, 43.0, 34.8, 23.4 ppm; IR (KBr): n˜ =
3092, 1721, 1688, 1612, 1579 cmꢀ1; MS (MALDI): m/z: 409.5 [M+H]+;
HRMS (FAB): m/z: calcd for C18H17N2O8: 389.0985 [MꢀH2OꢀH]ꢀ;
found: 389.0994.
8-N-[4-(Biscarboxymethylamino)-4-carboxylbutyl]aminomethyl-7-hydrox-
ycoumarin (NTAC-3): According to the general procedure, LiOH (1n
1.2 mL, 1.2 mmol) was added to a solution of 5b (100 mg, 0.20 mmol) in
30% aqueous CH3OH (10 mL). The crude product was purified by re-
verse-phase HPLC by using a linear gradient system from 15:85 to 17:83
CH3CN/H2O (0.1% TFA) over 20 min, and the collected fractions were
lyophilized to dryness to afford NTAC-3 (49.0 mg, 45%) as a white solid.
The purity of this compound was determined to be >96% by HPLC
analysis. 1H NMR (500 MHz, CD3OD): d=7.90 (d, J=9.5 Hz, 1H), 7.51
(d, J=8.6 Hz, 1H), 6.91 (d, J=8.6 Hz, 1H), 6.26 (d, J=9.5 Hz, 1H), 4.96
(d, J=14.3 Hz, 1H), 4.83 (d, J=14.3 Hz, 1H), 4.04–3.96 (m, 4H), 4.11–
4.07 (m, 1H), 3.37–3.34 (m, 2H), 2.32–1.86 ppm (m, 4H); 13C NMR
(125 MHz, CD3OD): d=162.9, 161.7, 155.7, 146.3, 130.5, 114.6, 113.3,
112.5, 110.6, 65.6, 55.4, 48.2, 40.8, 24.2, 21.8 ppm; IR (KBr): n˜ =2952,
1717, 1651, 1608, 1578 cmꢀ1; MS (MALDI): m/z: 423.2 [M+H]+; HRMS
8-N-[4-(Bisethoxycarbonylmethylamino)-4-ethoxycarbonylbutyl]amino-
methyl-7-hydroxycoumarin (5b): According to the general procedure, 4b
(2.16 g, 6.78 mmol) was treated in CH3OH (100 mL) with 10% Pd/C
(200 mg) under H2. After workup, the resulting residue 4b’ was dissolved
in dichloroethane (20 mL) and a solution of 2 (860 mg, 4.52 mmol) and
acetic acid (518 mL, 9.04 mmol) in dichloroethane (20 mL) was added.
The mixture was stirred for 6 h, then sodium cyanoborohydride (426 mg,
6.78 mmol) was added. The crude product was purified by flash column
chromatography on silica gel (MeOH/CH2Cl2 1:19) to afford 5b (1.23 g,
55%) as a pale-yellow oil. Rf =0.37 (MeOH/CH2Cl2 1:9); [a]2D0 =ꢀ18.3
8010
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 8004 – 8012

Binding to Hexahistidine
FULL PAPER
(FAB): m/z: calcd for C19H19N2O8: 403.1141 [MꢀH2OꢀH]ꢀ; found:
Acknowledgements
403.1130.
This research was supported in part by a Grant-in-Aid for University and
Society Collaboration (12793009) to T.H., a Grant-in-Aid for Young Sci-
entists (B) (19790094) to N.U. from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT, Japan), and by a Grant-in-Aid
for Research in Nagoya City University to N.U. We thank Dr. T. Mori
and Dr. N. Soh (Kyushu University) for helpful comments, and Prof. H.
Sajiki and Dr. Y. Monguchi (Gifu Pharmaceutical University) for HRMS
(FAB) analysis.
8-N-[5-(Biscarboxymethylamino)-5-carboxylpentyl]aminomethyl-7-hy-
droxycoumarin (NTAC-4): According to the general procedure, LiOH
(1n, 1.2 mL, 1.2 mmol) was added to
a solution of 5c (100 mg,
0.20 mmol) in 30% aqueous CH3OH (10 mL). The residue was purified
by reverse-phase HPLC by using a linear gradient system from 13:87 to
15:85 CH3CN/H2O (0.1% TFA) over 20 min, and the collected fractions
were lyophilized to afford NTAC-4 (33.8 mg, 31%) as a white solid. The
purity of this compound was determined as >92% by HPLC analysis.
1H NMR (400 MHz, CD3OD): d=7.69 (d, J=9.2 Hz, 1H), 7.26 (d, J=
8.6 Hz, 1H), 6.57 (d, J=8.6 Hz, 1H), 5.90 (d, J=9.2 Hz, 1H), 4.21 (s,
2H), 3.53 (s, 4H), 3.39–3.35 (m, 1H), 2.94–2.90 (m, 2H), 1.68–1.33 ppm
(m, 6H); 13C NMR (150 MHz, DMSO): d=174.2, 173.9, 160.3, 159.7,
153.9, 144.7, 130.4, 112.5, 111.4, 111.1, 105.5, 64.8, 54.6, 46.9, 38.4, 29.2,
[1] a) G. T. Herrmanson, Bioconjugate Techniques, Academic Press, San
Diego, 1996; b) B. N. G. Giepmans, S. R. Adams, M. H. Ellismann,
R. Y. Tsien, Science 2006, 312, 217–224; c) A. Miyawaki, A.
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24.9 ppm; IR (KBr): n˜ =3030, 1717, 1684, 1612, 1580 cmꢀ1
; HRMS
(FAB): m/z: calcd for C20H23N2O9: 435.14033 [MꢀH]ꢀ; found: 435.14127.
Synthesis ofpeptides : Angiotensin I was purchased from the Peptide In-
stitute (Osaka, Japan). H-His6-Tyr-NH2 (6) and H6-angiotensin I were
synthesized on TentaGel S RAM resin (0.36 mmolg,ꢀ1 Watanabe Chemi-
cal Industries, Hiroshima, Japan). Four equivalents of N-9-fluorenylme-
thyloxycarbonyl (Fmoc) amino acid, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tet-
ramethyluronium hexafluorophosphate (HBTU), N-hydroxybenzotri-
azole hydrate (HOBt), and diisopropylethylamine (DIPEA) (8 equiv)
were used for each coupling. DMF was the solvent. For deprotection
steps, 20% piperidine in DMF was used. The completion of amino acid
couplings was monitored with the Kaiser test. The peptide was cleaved
and deprotected by shaking with TFA/triisopropylsilane/H2O (95:2.5:2.5
v/v/v) for 2 h, followed by precipitation into cold diethyl ether. The pre-
cipitate was collected by centrifugation/decantation prior to purification.
The purification was performed by reverse-phase HPLC by using a linear
gradient system from 11:89 to 13:87 CH3CN/H2O (0.1% TFA) over
20 min (peptide 6) and 20:80 to 22:78 CH3CN/H2O (0.1% TFA) over
20 min (H6-angiotensin I), and the collected fractions were lyophilized to
afford a white solid. The molecular weights of the peptides were con-
firmed by mass spectrometry. MS (MALDI): m/z: calcd for C45H55O20N8:
[3] J. Zhang, R. E. Campbell, A. Y. Ting, R. Y. Tsien, Nat. Rev. Mol.
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Ting, Nat. Methods 2005, 2, 99–104; f) K. M. Marks, P. D. Braun,
G. P. Nolan, Proc. Natl. Acad. Sci. USA 2004, 101, 9982–9987.
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L. A. Gross, B. R. Martin, G. K. Walkup, Y. Yao, J. Llopis, R. Y.
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1003.5 [M+H]+; found: 1003.5 (peptide 6); calcd for C98H133O36N19
2118.1 [M+H]+; found: 2118.0 (H6-angiotensin I).
:
Fluorometric analysis: Fluorescence spectra were measured with a Hita-
chi F4500. The slit width was 5.0 nm for excitation and emission. The
photomultiplier voltage was 700 V. Relative quantum yields of fluores-
cence were obtained by comparing the area under the corrected emission
spectrum of the test sample at 366 nm excitation with that of a solution
of quinine sulfate in H2SO4 (1.0n) (quantum yield: 0.55).[19] CoCl2
(10 equiv to NTACs) and peptide 1 (50 equiv to NTACs) were used for
the determination of quantum yields.
Determination ofapparent dissociation constants : Aliquots of a stock so-
lution of peptide 6 were added to a cuvette that contained a solution of
Co2+–NTAC complex (3.0 mL, 5 mm) in Tris buffer (50 mm, pH 7.4). To
determine the dissociation constant (Kd), the change of fluorescence in-
tensity with increasing peptide 6 concentration was measured. Excitation
and emission wavelengths were set at 355 and 455 nm, respectively. The
value of Kd was obtained by nonlinear least-squares fitting to Equa-
tion (1),[20] since a 1:1 complex is formed between Co2+–NTAC complex
and peptide 6 as shown from the results of a Jobꢁs plot. The Kd value was
calculated by using a Kaleida Graph (Synergy Software, Reading, PA,
USA).
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Roddam, C. Smith, H. R. Swenson, J. Chem. Soc. Perkin Trans. 1
1997, 2483–2493.
ðFꢀFmin
Þ
ꢀ
ꢁ
ðFmaxꢀFmin
Þ
2
1=2
¼
ð½MCŠ0þ½PŠ0þKdÞꢀfð½MCŠ0þ½PŠ0þKdÞ ꢀ4½MCŠ ½PŠ0g
0
½2 MCŠ
0
ð1Þ
[16] A. Nouvet, M. Binard, F. Lamaty, J. Martinez, R. Lazaro, Tetrahe-
dron 1999, 55, 4685–4698.
[17] A. Rosowsky, R. A. Forsch, H. Bader, J. H. Freisheim, J. Med.
Chem. 1991, 34, 1447–1454.
in which F is the observed fluorescence intensity, Fmin is the minimum
fluorescence intensity (in the absence of peptide 6), and Fmax is the maxi-
mum fluorescence intensity. [MC]0 and [P]0 are the total concentrations
of Co2+–NTAC complex and peptide 6, respectively.
[18] B. R. Hart, K. Shea, Macromolecules 2002, 35, 6192–6201.
Chem. Eur. J. 2008, 14, 8004 – 8012
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8011

T. Higuchi, N. Umezawa et al.
[19] J. N. Demas, G. A. Crosby, J. Phys. Chem. 1971, 75, 991–1024.
[20] a) K. A. Connors, Binding Constants, Wiley, New York, 1987,
pp. 339–362; b) J. R. Long, R. S. Drago, J. Chem. Educ. 1982, 59,
1037–1039; c) K. Hirose, J. Inclusion Phenom. Macrocyclic Chem.
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Received: December 1, 2007
Revised: June 13, 2008
Published online: July 21, 2008
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Chem. Eur. J. 2008, 14, 8004 – 8012