Design and synthesis of fluorescent probe for polyhistidine tag using macrocyclic nickel(II) complex and fluorescein conjugate

Article abstract of DOI:10.1246/bcsj.20100288

We report a newly designed polyhistidine tag (His-tag) targeting fluorescent probe, NiL^ODCF, which we synthesized based on the fluorophore displacement mechanism. A macrocyclic nickel(II) complex (NiL ^O) was employed as a novel binding site for a His-tag motif, and we chose dichlorofluorescein (DCF) (=2′,7′-dichloro-3′,6′- dihydroxyspiro[iso-benzofuran-1(3H),9′-(9H)xanthen]-3-one) as the fluorophore. A hypochromic shift of NiLODCF from the metal-unbound form (L ^ODCF) in the absorption spectrum suggested that the phenolic oxygen atom of DCF interacted directly with the NiL^O complex, resulting in efficient fluorescence quenching (Φ = 0.084) in a neutral aqueous solution. When a model peptide having a hexahistidine sequence (H6Y1: YHHHHHH) was added to the solution of NiL^ODCF, a significant fluorescence enhancement in the emission (Φ = 0.60) was observed. The stoichiometry of the ternary complex between NiL^ODCF and H6Y1 was 1:1. The fluorescence intensity increased as the concentration of H6Y1 increased, and the dissociation constant (K_d) was determined to be 24 ± 1 μM, consistent with that for the Ni-NTA complex and His6-fused proteins (K_d = 1-20 μM). These results indicate that macrocyclic NiL^O can serve as a novel binding site for the polyhistidine sequence and that NiL^ODCF would be applicable to a switchable fluorescent probe for such His-tagged proteins.

Full text of DOI:10.1246/bcsj.20100288

386
Bull. Chem. Soc. Jpn. Vol. 84, No. 4, 386–394 (2011)
© 2011 The Chemical Society of Japan
Design and Synthesis of Fluorescent Probe for Polyhistidine Tag
Using Macrocyclic Nickel(II) Complex and Fluorescein Conjugate
Masayasu Taki,*1,2 Fumiyoshi Asahi,1 Tasuku Hirayama,1 and Yukio Yamamoto1
1Graduate School of Human and Environmental Studies, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501
2Graduate School of Global Environmental Studies, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501
Received October 6, 2010; E-mail: taki@chem.mbox.media.kyoto-u.ac.jp
We report a newly designed polyhistidine tag (His-tag) targeting fluorescent probe, NiLODCF, which we synthesized
based on the fluorophore displacement mechanism. A macrocyclic nickel(II) complex (NiLO) was employed as a novel
binding site for a His-tag motif, and we chose dichlorofluorescein (DCF) (=2¤,7¤-dichloro-3¤,6¤-dihydroxyspiro[iso-
benzofuran-1(3H),9¤-(9H)xanthen]-3-one) as the fluorophore. A hypochromic shift of NiLODCF from the metal-unbound
form (LODCF) in the absorption spectrum suggested that the phenolic oxygen atom of DCF interacted directly with the
NiLO complex, resulting in efficient fluorescence quenching (Φ = 0.084) in a neutral aqueous solution. When a model
peptide having a hexahistidine sequence (H6Y1: YHHHHHH) was added to the solution of NiLODCF, a significant
fluorescence enhancement in the emission (Φ = 0.60) was observed. The stoichiometry of the ternary complex between
NiLODCF and H6Y1 was 1:1. The fluorescence intensity increased as the concentration of H6Y1 increased, and the
dissociation constant (Kd) was determined to be 24 « 1 ¯M, consistent with that for the Ni-NTA complex and His6-fused
proteins (Kd = 1-20 ¯M). These results indicate that macrocyclic NiLO can serve as a novel binding site for the
polyhistidine sequence and that NiLODCF would be applicable to a switchable fluorescent probe for such His-tagged
proteins.
Fluorescent labeling of proteins has been an indispensable
NTA (M = CoII or NiII) complexes,24,25 designed on the basis
of a fluorophore displacement strategy,27,28 which show a turn-
on response in the emission upon binding to a His-tag-fused
protein. In this system, fluorescence is largely quenched due to
a labile interaction with a paramagnetic metal center (CoII
or NiII) supported by an NTA ligand. When a His-tag-fused
protein is present, dissociation of the fluorophore from the
coordination sphere of the metal ion occurs, increasing the
strength of the fluorescence. However, the hydroxycoumarin
fluorophore requires a short excitation wavelength (µ360 nm),
which can cause serious damage to cells. Although fluorescein
and boron-dipyrromethene (BODIPY) derivatives are the most
commonly used as a visible light excitable fluorescent
molecule, electron-donating ability of the amino group intro-
duced into these fluorophores usually causes fluorescence
quenching through a photoinduced electron-transfer pro-
cess,29-32 which may result in decreasing the fluorescence
intensity of the fluorophore-displaced form. Anionic functional
groups such as carboxylate and phenolate are other candidates
for the coordinative donors to the metal ions. However, the
affinities of Ni-NTA complexes for these functional groups are
much weaker compared with those for aromatic and aliphatic
amines.14,17,33 Indeed, Lippard et al. have reported that
fluorophore emission of the NTA-fluorescein construction
remained almost unchanged upon complexation with NiII,23
indicating no interaction between the Ni-NTA complex and the
anionic fluorescein dye. For all these reasons, the choice of
an appropriate fluorophore is quite limited when a Ni-NTA
complex is utilized as the His-tag binding site of the fluorescent
probe based on the fluorophore displacement mechanism.
technique for studying protein dynamics both in vitro and in
vivo.1,2 Such studies are mostly performed by expressing
fluorescent proteins (FPs) at the specific site of the target
protein.3,4 Although FPs can be easily incorporated into
proteins through genetic fusion, the relatively large size of
FPs (ca. 27 kDa) can sterically interfere with functions,
trafficking, and locations of the original proteins.5,6 One
strategy for overcoming these problems with FPs is to utilize
a pair of an artificial short peptide (tag) and a small synthetic
organic fluorescent probe that has sites capable of recognizing
the tag sequence.7-9 A variety of tag-probe pairs have been
developed for analyzing protein expression and protein-protein
interactions, as well as for fluorescent imaging of proteins in
living systems.10-19 Among them, polyhistidine tag (His-tag)
and nickel(II)-2,2¤,2¤¤-nitrilotriacetato complex (Ni-NTA) are
the most well-known complementary pair and various Ni-NTA
derivatives with an appended fluorophore have been developed
as the probe.20-26 However, only a few fluorescent Ni-NTA
complexes, for which the labeling event can be monitored
by means of a detectable fluorescent signal, have been
reported.24-26 Therefore, in most cases, removal of the non-
labeled probe molecule is required to reduce the fluorescence
background prior to analyzing protein dynamics and the optical
imaging experiment.
Ni-NTA complexes are known to bind not only with
nitrogen-containing aromatic heterocyclic compounds but also
with aliphatic amines. By taking advantage of such coordina-
tive ability of Ni-NTA complexes, Higuchi, Umezawa, et al.
recently developed hydroxycoumarin-based fluorescent M-
Published on the web April 7, 2011; doi:10.1246/bcsj.20100288

M. Taki et al.
Bull. Chem. Soc. Jpn. Vol. 84, No. 4 (2011)
387
Diethyl 4-[3-(N-Benzyloxycarbonyl)aminopropoxy]-2,6-
pyridinedicarboxylate (1). To a solution of diethyl
4-hydroxy-2,6-pyridinedicarboxylate35 (2.5 g, 10 mmol) in
CH3CN (10 mL) were added a solution of benzyl 3-bromopro-
pylcarbamate (2.8 g, 10 mmol) in CH3CN (10 mL) and K2CO3
(2.1 g, 0.016 mmol). The solution was refluxed overnight and
the filtrate was evaporated. The residue was purified by flash
chromatography (hexane-EtOAc, 1:1) to give 1 as a white solid
1
(2.8 g, 62%). H NMR (270 MHz, CDCl3): ¤ 1.46 (t, 6H, J =
7.3 Hz), 2.08 (m, 2H), 3.43 (m, 2H), 4.20 (t, 2H, J = 5.9 Hz),
4.47 (q, 4H, J = 7.3 Hz), 4.92 (m, 1H), 5.10 (s, 2H), 7.30-7.40
(m, 5H), 7.77 (s, 2H). 13C NMR (68 MHz, CDCl3): ¤ 14.2,
29.1, 37.9, 62.4, 66.3, 66.8, 114.2, 128.1, 128.5, 136.3, 150.2,
156.4, 164.6, 166.6. ESI-TOF (pos.): calcd for C22H27N2O7
[M + H]+ 431.2, found 431.3.
Figure 1. Structure of
a designed fluorescent probe
NiLODCF consisting of a macrocyclic NiII complex
(NiLO), dichlorofluorescein (DCF), and a flexible linker.
4-[3-(N-Benzyloxycarbonyl)aminopropoxy]-2,6-pyridine-
dimethanol (2). To a solution of 1 (2.2 g, 5.0 mmol) in a
mixture of EtOH (10 mL) and THF (15 mL) was added NaBH4
(0.84 g, 22.2 mmol) in portions over 5 min at 0 °C. After
stirring for 1 h at room temperature, the reaction mixture was
evaporated. The residue was dissolved in water (100 mL) and
extracted with EtOAc (3 © 100 mL). The combined organic
layer was dried over MgSO4 and evaporated to give 2 as a
white solid (1.6 g, 93%). This compound was used for the next
Therefore, it has been challenging to develop alternatives to
Ni-NTA that can both quench the fluorescence via a labile
interaction with anionic functional groups of the fluorophore
and bind with the His-tag motif of proteins.15,19
In this paper, we report a newly designed polyhistidine tag
(His-tag) targeting fluorescent probe, NiLODCF (Figure 1),
which we synthesized based on the fluorophore displacement
mechanism. This probe consists of two units, macrocyclic
nickel(II) complex (NiLO)34 as a novel binding site for a
His-tag motif and dichlorofluorescein (DCF) (=2¤,7¤-dichloro-
3¤,6¤-dihydroxyspiro[isobenzofuran-1(3H),9¤-(9H)xanthen]-3-
one) as a visible light excitable fluorophore, and these
components are linked by a flexible chain. We found that
NiLODCF exhibits an increase in fluorescence in the presence
of the His-tag and that NiLO can act as the His-tag binding
site with a comparable binding affinity to the Ni-NTA/His6
pair.
1
step without further purification. H NMR (270 MHz, CDCl3):
¤ 2.01 (m, 2H), 3.38 (m, 2H), 4.07 (t, 2H, J = 5.8 Hz), 4.67
(s, 4H), 5.01 (brs, 1H), 5.09 (s, 2H), 6.69 (s, 2H), 7.29-7.39
(m, 5H). 13C NMR (68 MHz, CDCl3): ¤ 29.2, 38.0, 64.4, 65.6,
66.7, 105.5, 128.0, 128.1, 128.5, 136.4, 156.6, 160.7, 166.3.
ESI-TOF (pos.): calcd for C18H23N2O5 [M + H]+ 347.2, found
347.3.
4-[3-(N-Benzyloxycarbonyl)aminopropoxy]-2,6-pyridine-
dimethyl Ditosylate (3). A solution of tosyl chloride (1.4 g,
7.1 mmol) in THF (3 mL) was added to an ice-cooled solution
of 2 (1.2 g, 3.6 mmol) in THF (5 mL), and then 5 mL of 2.4 M
aqueous NaOH solution was added to the mixture. After
stirring for 4 h at room temperature, the reaction mixture was
evaporated. The residue was diluted with water (30 mL) and
extracted with CHCl3 (3 © 30 mL). The combined organic
layer was dried over MgSO4 and evaporated. The residue was
purified by flash chromatography (hexane-EtOAc, 1:1) to give
Experimental
All chemicals used in this study were commercial products
of the highest available purity and were further purified by
standard methods, if necessary. A macrocyclic nickel complex
[Ni(LO)(CH3CN)2](ClO4)2,34 diethyl 4-hydroxypyridine-2,6-di-
carboxylate,35 and 6-carboxy-2¤,7¤-dichloro-3¤,6¤-bis(pivaloyl-
oxy)spiro[isobenzofuran-1(3H),9¤-(9H)xanthen]-3-one,36 were
prepared according to published procedures. A model peptide
of histidine-tag was synthesized by solid-phase synthesis
(Fmoc-method) as previously reported.37 NMR spectra were
recorded on a JEOL JNM-EX-270 (at 270 MHz to 1H, 68 MHz
to 13C) or a JEOL ¡-500 (at 500 MHz to 1H, 126 MHz to 13C).
Chemical shifts are given in ppm relative to tetramethylsilane
(TMS). Electrospray ionization mass spectra (ESI-MS) were
recorded with micrOTOF II focus (Bruker Daltonics). Matrix-
assisted laser desorption/ionization time-of-flight mass spec-
trometry (MALDI-TOF) was performed on autoflex III (Bruker
Daltonics). High-resolution mass spectra (HRMS) were mea-
sured with a JMS-DX-300 (JEOL). Elemental analysis was
done at the Elementary Analysis Center in the Graduate School
of Pharmaceutical Science, Kyoto University. TLC analyses
were performed on Silica gel 60-F254 (Merck). Flash chroma-
tography was performed on silica gel (Merck Silica Gel 60).
Reverse phase HPLC was performed on Waters Delta 600
system (10 © 250 mm2 XBridgeTMC18).
1
3 as a white solid (1.8 g, 77%). H NMR (270 MHz, CDCl3):
¤ 2.02 (m, 2H), 2.44 (s, 6H), 3.39 (m, 2H), 4.04 (t, 2H,
J = 5.6 Hz), 4.97 (s, 4H), 5.11 (s, 2H), 6.81 (s, 2H), 7.30-7.40
(m, 9H), 7.80 (d, 4H, J = 8.1 Hz). 13C NMR (68 MHz, CDCl3):
¤ 21.57, 29.06, 37.91, 65.80, 66.67, 71.06, 107.6, 128.0, 128.0,
128.1, 128.5, 129.9, 132.5, 136.4, 145.1, 155.1, 156.4, 166.4.
ESI-TOF (pos.): calcd for C32H35N2O9S2 [M + H]+ 655.2,
found 655.2.
4-[3-(N-Benzyloxycarbonyl)aminopropoxy]-2,6-bis(bromo-
methyl)pyridine (4). A solution of 3 (2.9 g, 4.4 mmol) and
LiBr (3.8 g, 44 mmol) in acetone (35 mL) was refluxed for 8 h.
After the reaction mixture was evaporated, the residue was
dissolved in EtOAc (100 mL) and the organic layer was washed
with water (3 © 100 mL), dried over MgSO4, and evaporated
to give the product 4 as a white solid (2.2 g, 100%). This
compound was used for the next step without further
purification. 1H NMR (270 MHz, CDCl3): ¤ 2.05 (m, 2H), 3.41
(m, 2H), 4.10 (t, 2H, J = 5.9 Hz), 4.47 (s, 4H), 4.93 (brs, 1H),

388
Bull. Chem. Soc. Jpn. Vol. 84, No. 4 (2011)
Fluorescent Probe for Polyhistidine Tag
5.11 (s, 2H), 6.87 (s, 2H), 7.30-7.40 (m, 5H). 13C NMR
(68 MHz, CDCl3): ¤ 29.2, 33.4, 38.0, 65.8, 66.7, 109.2, 128.1,
128.2, 128.5, 136.4, 156.4, 158.2, 166.3. ESI-TOF (pos.): calcd
for C18H20N2O379Br179Br1Na1 [M + Na]+ 492.97, found
492.97; calcd for C18H20N2O379Br181Br1Na1 [M + Na]+
494.97, found 494.97; calcd for C18H20N2O381Br181Br1Na1
[M + Na]+ 496.97, found 496.97.
to give amine 7 as a yellow oil (190 mg, 100%). The purity was
1
confirmed by H NMR and compound 7 was used for the next
1
step without further purification. H NMR (270 MHz, CDCl3):
¤ 1.66 (m, 4H), 1.94 (m, 2H), 2.92 (t, 2H, J = 6.3 Hz), 3.22
(t, 4H, J = 5.3 Hz), 3.32 (t, 4H, J = 7.4 Hz), 4.06 (t, 2H, J =
5.9 Hz), 4.52 (s, 4H), 6.93 (s, 2H), 7.65-7.75 (m, 6H), 8.05 (d,
2H, J = 9.4 Hz).
Bis[3-(2-nitrophenylsulfonylamino)propyl] Ether (5).
A
2¤,7¤-Dichloro-3-oxo-3¤,6¤-bis(pivaloyloxy)spiro[isobenzo-
furan-1(3H),9¤-(9H)xanthene]-6-carboxylic Acid 2,5-Dioxo-
solution of 2-nitrophenylsulfonyl chloride (2.06 g, 20 mmol) in
THF (50 mL) was added to a solution of bis(3-aminopropyl)
ether (1.03 g, 7.8 mmol) and NaHCO3 (2.62 g, 31 mmol) in
THF (50 mL) at 0 °C. The mixture was stirred overnight at
room temperature and evaporated in vacuo. The residue was
dissolved in saturated NaHCO3 aqueous solution (100 mL) and
extracted with CH2Cl2 (3 © 100 mL). The combined organic
layers were dried over MgSO4 and evaporated to give
dinosylamide 5 as a white solid (3.98 g, 100%). The purity
1-pyrrolidinyl Ester (8).
A solution of 6-carboxy-2¤,7¤-
dichloro-3¤,6¤-bis(pivaloyloxy)spiro[isobenzofuran-1(3H),9¤-
(9H)xanthen]-3-one36 (330mg, 0.53mmol), 1-ethyl-3-(3-dimeth-
ylaminopropyl)carbodiimide hydrochloride (EDC) (140 mg,
0.74 mmol), and N-hydroxysuccinimide (67 mg, 0.58 mmol)
in dry CH2Cl2 (10 mL) were stirred for 4 h at room temperature
under argon atmosphere in the dark. EDC (20 mg, 0.11 mmol)
was further added to the reaction mixture and stirring was
continued for an additional 3 h. After removal of the solvent,
the residue was dissolved in EtOAc (50 mL) and the organic
layer was washed with brine (3 © 50 mL), dried over MgSO4,
and evaporated. The residue was purified by flash chromatog-
raphy (CHCl3) to give 8 as a pale yellow solid (250 mg, 67%).
1H NMR (270 MHz, CDCl3): ¤ 1.40 (s, 18H), 2.90 (s, 4H),
6.85 (s, 2H), 7.17 (s, 2H), 7.91 (s, 1H), 8.22 (d, 1H, J = 8.1
Hz), 8.44 (d, 1H, J = 8.1 Hz). ESI-TOF (pos.): calcd for
C35H30N1O11Cl2 [M + H]+ 710.12, found 710.12.
Compound 9. A solution of 7 (240 mg, 0.35 mmol) and 8
(260 mg, 0.36 mmol) in dry DMF (5 mL) was stirred overnight
at room temperature under argon atmosphere. After evapora-
tion, the residue was dissolved in CHCl3 (20 mL) and the
organic layer was washed with brine (20 mL). The aqueous
layer was further extracted with CHCl3 (2 © 20 mL). The
combined organic layer was dried over MgSO4 and evaporated.
The residue was purified by flash chromatography (hexane-
EtOAc, 1:2) to give 9 as a pale yellow solid (220 mg, 50%).
1H NMR (500 MHz, CDCl3): ¤ 1.39 (s, 18H), 1.67 (m, 4H),
2.12 (m, 2H), 3.20 (t, 4H, J = 6.5 Hz), 3.30 (t, 4H, J = 6.3 Hz),
3.62 (m, 2H), 4.09 (t, 2H, J = 6.0 Hz), 4.52 (s, 4H), 6.59 (brs,
1H), 6.87 (s, 2H), 6.93 (s, 2H), 7.12 (s, 2H), 7.56 (s, 1H), 7.66
(dd, 2H, J = 7.5, 2.0 Hz), 7.69-7.75 (m, 4H), 8.04 (dd, 2H,
J = 7.5, 2.0 Hz), 8.08 (dd, 1H, J = 7.9, 1.4 Hz), 8.16 (d, 1H,
J = 7.9 Hz). 13C NMR (126 MHz, CDCl3): ¤ 27.1, 28.1, 28.6,
29.7, 37.8, 39.4, 44.5, 53.5, 66.2, 67.1, 109.5, 112.8, 116.7,
122.6, 122.9, 124.3, 126.3, 127.7, 128.9, 129.5, 130.7, 131.7,
133.1, 133.7, 141.7, 148.2, 149.0, 149.6, 152.6, 157.1, 165.7,
166.5, 167.7, 175.6. HRMS (FAB): m/z calcd for C59H59-
N6O1835Cl2S2 [M + H]+ 1273.2704, found 1273.2716; calcd
for C59H59N6O1835Cl137Cl1S2 [M + H]+ 1275.2675, found
1275.2670.
1
was sufficient for the following reaction. H NMR (270 MHz,
CDCl3): ¤ 1.82 (m, 4H), 3.22 (m, 4H), 3.50 (t, 4H, J = 5.5 Hz),
5.78 (t, 2H, J = 5.8 Hz), 7.70-7.77 (m, 4H), 7.80 (d, 2H,
J = 9.2 Hz), 8.14 (d, 2H, J = 9.7 Hz). 13C NMR (68 MHz,
CDCl3): ¤ 29.1, 42.0, 69.2, 125.3, 131.0, 132.9, 133.4, 133.5,
147.9. ESI-TOF (pos.): calcd for C18H22N4O9S2Na1 [M + Na]+
525.07, found 525.15.
15-[3-(N-Benzyloxycarbonyl)aminopropoxy]-3,11-bis(2-
nitrobenzenesulfonyl)-7-oxa-3,11,17-triazabicyclo[11.3.1]-
heptadeca-1(17),13,15-triene (6). A mixture of 5 (0.97 g, 2.1
mmol) and Na2CO3 (1.1 g, 10 mmol) in dry DMF (80 mL) was
stirred for 2 h at 100 °C under argon atmosphere. After cooling
to room temperature, a solution of 4 (1.0 g, 2.0 mmol) in dry
DMF (80 mL) was added dropwise to the mixture over 5 h by
using a dropping funnel. After stirring overnight at room
temperature, the reaction mixture was evaporated. The residue
was dissolved in CH2Cl2 (100 mL) and washed with 0.1 M
aqueous NaOH solution (3 © 100 mL). The combined organic
layer was dried over MgSO4 and evaporated. The crude product
was purified by flash chromatography (hexane-EtOAc, 1:3) to
give 6 as a white solid (0.86 g, 52%). 1H NMR (270 MHz,
CDCl3): ¤ 1.67 (m, 4H), 2.00 (m, 2H), 3.21 (t, J = 5.3 Hz, 4H),
3.31 (t, 4H, J = 7.3 Hz), 3.38 (m, 2H), 4.00 (t, 2H, J = 5.6 Hz),
4.52 (s, 4H), 4.93 (brs, 1H), 5.12 (s, 2H), 6.90 (s, 2H), 7.26-
7.38 (m, 5H), 7.63-7.72 (m, 6H), 8.06 (d, 2H, J = 8.9 Hz).
13C NMR (68 MHz, CDCl3): ¤ 27.8, 28.9, 37.8, 44.2, 53.3,
65.4, 66.5, 66.8, 109.3, 124.1, 128.0, 128.4, 130.4, 131.7,
132.8, 133.7, 136.4, 148.0, 156.4, 156.8, 166.5. ESI-TOF
(pos.): calcd for C36H41N6O12S2 [M + H]+ 813.22, found
813.26; calcd for C36H40N6O12S2Na1 [M + Na]+ 835.20, found
835.23.
15-(3-Aminopropoxy)-3,11-bis(2-nitrobenzenesulfonyl)-
7-oxa-3,11,17-triazabicyclo[11.3.1]heptadeca-1(17),13,15-
triene (7). A mixture of 6 (220 mg, 0.28 mmol) in 25% HBr-
acetic acid solution (2 mL) was stirred for 1 h at room
temperature. The reaction mixture was poured into Et2O
(100 mL) and the resulting precipitation was collected by
filtration. The deliquescent material thus obtained was dis-
solved in MeOH and the solvent was again evaporated to
dryness. The residue was dissolved in saturated NaHCO3
solution (20 mL) and extracted with CHCl3 (3 © 20 mL). The
combined organic layer was dried over MgSO4 and evaporated
LODCF. A solution of KOH (670 mg, 12 mmol) in H2O
(2 mL) was added to a solution of PhSH (125 mL, 1.23 mmol)
in CH3CN (5 mL) at 0 °C under argon atmosphere. After
stirring for 30 min, a solution of 9 (520 mg, 0.41 mmol) in
CH3CN (5 mL) was added to the reaction mixture and the
resulting solution was further stirred overnight at 50 °C. The
mixture was neutralized with 1 M HCl and the solvent was
removed by evaporation. The residue was purified by reversed-
phase chromatography on an ODS column (H2O/CH3CN
containing 0.1% TFA 70:30 ¼ 10:90) to give a TFA salt of

M. Taki et al.
Bull. Chem. Soc. Jpn. Vol. 84, No. 4 (2011)
389
LODCF as an orange solid (270 mg, 81%). 1H NMR (500 MHz,
CD3OD): ¤ 1.96-2.03 (m, 6H), 3.26 (t, 4H, J = 5.8 Hz), 3.41
(t, 2H, J = 6.4 Hz), 3.70 (t, 4H, J = 5.5 Hz), 4.06 (t, 2H, J =
6.1 Hz), 4.31 (s, 4H), 6.59 (s, 2H), 6.81 (s, 2H), 6.91 (s, 2H),
7.58 (s, 1H), 8.04 (d, 1H, J = 7.9 Hz), 8.10 (d, 1H, J = 7.9 Hz);
13C NMR (126 MHz, CD3OD): ¤ 18.3, 27.3, 29.7, 37.9, 47.0,
50.9, 58.3, 67.8, 69.2, 105.0, 110.2, 112.1, 119.0, 124.3, 127.0,
127.0, 129.3, 129.6, 130.1, 130.9, 131.5, 142.5, 152.6, 153.3,
168.2, 168.7, 169.7; HRMS (FAB): m/z calcd for C37H37N4-
O835Cl137Cl1 [M + H]+ 737.1959, found 737.1965.
crystals (104 mg, 92%). Anal. Calcd for C23H29N5O9Cl2Ni:
C, 42.56; H, 4.50; N, 10.79%. Found: C, 42.34; H, 4.32; N,
10.89%.
Steady-State Absorption and Fluorescence Spectroscopy.
The UVabsorption spectra were recorded on a Hewlett-Packard
8453 spectrometer. Fluorescence spectra were recorded using a
Hitachi F-2500 spectrometer with a slit width of 2.5 nm. The
photomultiplier voltage was 400 V. To reduce the fluctuation in
the excitation intensity during measurement, the lamp was kept
on for 1 h prior to the experiment. The path length was 1 cm
with a cell volume of 3.0 mL. Quantum yields were determined
by using fluorescein in 0.1 M NaOH (Φ = 0.95) as the
fluorescence standard.38
Determination of Apparent Dissociation Constant. The
dissociation constant (Kd) of the ternary complex between
NiLODCF and H6Y1 was determined by fluorescence spec-
troscopy. A series of HEPES-buffered solutions (50mM,
pH 7.20) of 1.0 ¯M NiLODCF containing various concentrations
of H6Y1 peptide ([H6Y1] = 0, 1.4, 4.1, 6.9, 9.6, 13.7, 20.5, 27.2,
40.4, 53.3, and 126.1¯M) were prepared. The sample solutions
were stirred for 3h at room temperature to reach equilibrium and
the fluorescence spectra were measured. The subtracted fluores-
cent intensities at 528nm (¦F528 = F ¹ F0, where F0 means the
initial fluorescence intensity) were plotted against the concen-
trations of H6Y1 and the experimental data were analyzed by
nonlinear least square curve fitting using the following equation:
NiLODCF.
To a mixture of ligand LODCF (45.3 mg,
62 ¯mol) and Et3N (28 ¯L, 0.2 mmol) in MeOH (8 mL) was
added Ni(ClO4)2¢6H2O (25.7 mg, 70 ¯mol) in MeOH (2 mL).
After removal of the solvent, the residue was purified by
reversed-phase chromatography on an ODS column (H2O/
MeOH 50:50 ¼ 10:90) to give NiLODCF as a red-orange solid
(40 mg, 81%). MALDI-TOF (pos.): m/z calcd for C37H35N4O8-
Cl2Ni1 [M + H]+ 791.12, found 791.17; HRMS (FAB): m/z
calcd for C37H35N4O835Cl2Ni1 [M + H]+ 791.1185, found
791.1196.
[Ni(LO)(bpy)](ClO4)2. A solution of [Ni(LO)(CH3CN)2]-
34
(ClO4)2 (101 mg, 0.18 mmol) and 2,2¤-bipyridyl (42 mg, 0.27
mmol) in CH3CN (1 mL) was stirred for 15 min at room
temperature, the mixture was poured into 50 mL of Et2O. The
precipitation thus generated was collected and recrystallized
from CH3CN/Et2O to give [Ni(LO)(bpy)](ClO4)2 as blue
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
½Niꢁ0 þ ½H6Y1ꢁ þ Kd ꢂ ð½Niꢁ0 þ ½H6Y1ꢁ þ KdÞ ꢂ 4½Niꢁ0½H6Y1ꢁ
2½Niꢁ0
ꢀF528 ¼ ꢀF1 ꢀ
ð1Þ
in which ¦F¨ represents the difference between the initial (F0)
and final (F¨) fluorescence intensities, and [Ni]0 means the
total concentration of NiLODCF, that is 1.0 ¯M.
character of the Ni-NTA complex itself. If a DCF fluorophore,
which has a net charge of ¹2 at pH 7, is utilized for the probe
based on the fluorophore displacement mechanism, then the
corresponding nickel(II) complex requires the following
features: (1) a cationic character, which allows the dye to
interact with the nickel(II) center, resulting in efficient fluores-
cence quenching, (2) less steric hindrance around the metal
center for facile accessibility of DCF and the His-tag sequence,
and (3) comparable binding affinity for the His-tag motif as
Ni-NTA complexes.
X-ray Diffraction Data Collection and Determination of
Structure. A single crystal was mounted on a CryoLoop
(Hamptom Research Co.). Data of X-ray diffraction were
collected by a Rigaku RAXIS-RAPID imaging plate two-
dimensional area detector with graphite monochromated
Mo K¡ radiation (- = 0.71075 ¡) to 2ªmax of 55.0°. All data
were corrected for Lorentz and polarization effects. All the
crystallographic calculations were performed by using a
crystal structure software package of the Rigaku Corporation
(CrystalStructure version 3.8.2).39 The crystal structure was
solved by direct methods and refined by full-matrix least
squares using SIR-92.40 All hydrogen atoms were refined using
the riding model. Crystallographic data have been deposited
with Cambridge Crystallographic Data Centre: Deposition
number for [Ni(LO)(bpy)](ClO4)2 is CCDC-795728. Copies of
the data can be obtained free of charge via http://www.ccdc.
cam.ac.uk/conts/retrieving.html (or from the Cambridge Crys-
tallographic Data Centre, 12, Union Road, Cambridge, CB2
1EZ, U.K.; Fax: +44 1223 336033; e-mail: deposit@ccdc.
cam.ac.uk).
We have previously reported structural and physicochemical
properties of a nickel(II) complex supported by a pyridine-
containing macrocyclic tetradentate ligand LO (=7-oxa-
3,11,17-triazabicyclo[11.3.1]heptadeca-1(17),13,15-triene),
[Ni(LO)(X)2]2+ 34
In that paper, we mentioned that the affinity
.
of the coordinating solvent molecules (X) located at the apical
positions is significantly stronger, and the stable octahedral
high-spin nickel(II) is given both in the solid state and in
solution. We expected that the core structure of this nickel(II)
complex (NiLO, Figure 1) would be applicable as a fluores-
cence quencher of DCF as well as a novel interacting partner of
the polyhistidine sequence. When NiLO is conjugated to the
DCF dye through a flexible linker, the internal NiII-DCF
complex would be formed and the fluorescence would be
quenched. In the presence of His-tagged proteins, on the other
hand, the folded structure is expanded by coordinating the
histidine imidazoles of the His-tag, and the enhanced emission
of DCF can be observed.
Results and Discussion
Selection of Nickel(II) Complex for the Probe.
We
attributed the lower binding affinity of Ni-NTA for anionic
compounds to electrostatic repulsion, namely the anionic

390
Bull. Chem. Soc. Jpn. Vol. 84, No. 4 (2011)
Fluorescent Probe for Polyhistidine Tag
(a)
(b)
N(1)
N(1)
N(3)
N(4)
Ni(1)
O(1)
N(2)
N(4)
N(5)
N(2)
N(3)
Ni(1)
N(5)
O(1)
Figure 2. (a) ORTEP drawing of [Ni(LO)(bpy)](ClO4)2 showing 50% thermal ellipsoids and selected atom labels. The counter
anions and the hydrogen atoms except on nitrogen atoms are omitted for clarity. Selected bond lengths (¡) and angles (deg) for
[Ni(LO)(bipy)](ClO4)2: Ni(1)-N(1) = 1.9849(18), Ni(1)-N(2) = 2.1032(18), Ni(1)-N(3) = 2.1134(16), Ni(1)-N(4) = 2.0543(18),
Ni(1)-N(5) = 2.0636(18), Ni(1)-O(1) = 2.2852(15), N(1)-Ni(1)-N(2) = 81.47(7), N(1)-Ni(1)-N(3) = 81.38(7), N(1)-Ni(1)-
N(4) = 97.17(7), N(1)-Ni(1)-N(5) = 176.71(7), N(2)-Ni(1)-N(3) = 162.79(7), N(4)-Ni(1)-N(5) = 79.54(7), N(1)-Ni(1)-O(1) =
89.52(6). (b) Reported crystal structure of [Ni(LO)(CH3CN)2](ClO4)(BPh4) (Ref. 32).
Table 1. X-ray Crystallographic Data for [Ni(LO)(bpy)](ClO4)2
the macrocyclic ligand LO and two pyridine nitrogen atoms of
the external bpy ligand. No solvent molecule is present in the
crystal. Interestingly, the ether oxygen atom of LO is located
perpendicular to the pseudo-plane defined by the metal ion, the
pyridine ring, and two secondary amine nitrogens (N1-Ni-O1:
89.52(6)°). The other two coordination sites in a relative cis-
orientation are occupied by one external bpy ligand. The bent
configuration of the macrocyclic framework of LO observed
in the bpy-bound form is different from the reported crystal
structure of [Ni(LO)(CH3CN)2]2+, where the LO framework is
a flat configuration (Figure 2b).34 These findings indicate that
the structure of the nickel(II) complex can vary depending on
the nature of the external ligand. Thus, we worked on the
assumption that the His-tag-bound form of the NiLO complex
could adopt a similar bent configuration as observed for
[Ni(LO)(bpy)]2+ and that the two histidine imidazoles could
similarly coordinate in a bidentate fashion.41
Empirical formula
Formula weight
Crystal size/mm3
Crystal system
Space group
a/¡
b/¡
c/¡
¢/deg
V/¡3
C23H29O9N5NiCl2
649.12
0.30 © 0.10 © 0.10
Monoclinic
P21/c (#14)
11.7741(11)
16.1901(12)
14.2419(13)
93.541(3)
2709.7(3)
4
Z
T/K
133
1.591
9.739
1344.00
¹3
Dcalcd/g cm
¹1
® (Mo K¡)/cm
F(000)
Total reflections
26687
Design and Synthesis of NiLODCF.
We decided to
Unique reflections (Rint)
Refln./param. ratio
6181 (0.050)
44.15
0.0596
0.1641
1.003
substitute the 4-position of the pyridine ring to link with the
DCF dye, because the pyridine substitution would not interfere
with the coordination of the His-tag sequence to the NiII ion.
The synthetic scheme of NiLODCF is outlined in Scheme 1.
The reaction of chelidamic acid diethyl ester with Z-protected
propylamine and the following reduction with NaBH4 afforded
diol 2. Pyridine-containing 14-membered macrocyclic ligands
are usually synthesized by a one-pot template condensation
reaction using 2,6-pyridinedicarbaldehyde, 4-X-1,7-heptanedi-
amine (X = aza, thia, and oxa), and a metal ion such as
copper(II) and nickel(II).34,42 However, these attempts at using
4-substituted 2,6-pyridinedicarbaldehyde were unsuccessful.
Instead, synthesis of the macrocyclic unit 6 was achieved by
slowly adding dibromide 4 to a solution of nosyl (Ns)-protected
diamine 5 in DMF at room temperature. After Z was removed
to give 7, a reaction with an NHS-activated ester of pivaloyl
(Piv)-protected fluorescein derivative 8 was performed to
afford the fully protected compound 9. Finally, deprotection
a)
R1 (I > 2.00·(I))
b)
wR2 (I > 2.00·(I))
GOF on F2
2
2 2
a) R = -««Fo« ¹ «Fc««/-«Fo«. b) wR2 = [-{w(Fo ¹ Fc ) }/
2 2 1/2
-w(Fo ) ]
.
Structural Studies with a Model Ligand. To investigate
the structural features of the ternary complex between the NiLO
moiety and the His-tag, we employed 2,2¤-bipyridyl (bpy) as
the model compound of polyhistidine, which can coordinate to
the NiII center in a bidentate fashion, as found in the Ni-NTA/
His-tag pair.21 Suitable crystals of the nickel(II) complex for
X-ray analysis were obtained by vapor diffusion of ether into
an acetonitrile solution of [Ni(LO)(bpy)]2+. The crystal data and
the experimental parameters are given in Table 1. As shown in
Figure 2a, the nickel(II) complex [Ni(LO)(bpy)](ClO4)2 is in a
distorted octahedral geometry defined by N3O1 donor atoms of

M. Taki et al.
Bull. Chem. Soc. Jpn. Vol. 84, No. 4 (2011)
391
Z
Z
O
N
N
H
Z
O
N
H
OH
N
O
N
H
a
b
c
X = OTs: 3
X = Br: 4
N
d
EtOOC
COOEt
EtOOC
N
COOEt
X
X
OH
OH
1
2
O
PivO
O
O
N
O
O
Z
N
O
O
O
O
8:
NH2
Cl
O
N
Cl
Cl
H
OPiv
Cl
PivO
O
OPiv
O
NHNs
5:
2
O
N
e
f
g
O
H
N
O
Ns
N
N Ns
Ns
N
N
Ns
N
O
Ns
N
N Ns
O
O
6
7
9
HO
O
O
Cl
OH
O
N
N
O
h
i
H
NiLODCF
Cl
O
NH
HN
•2TFA
LODCF
O
Scheme 1. (a) Benzyl 3-bromopropylcarbamate, K2CO3, CH3CN, reflux, overnight, 62%; (b) NaBH4, EtOH, THF, rt, 1 h, 93%;
(c) TsCl, NaOH, water, THF, rt, 4 h, 77%; (d) LiBr, acetone, reflux, 8 h, 100%; (e) 5, Na2CO3, DMF, rt, overnight, 52%;
(f) 25%HBr/AcOH, rt, 1 h, 100%; (g) 8, DMF, rt, overnight, 50%; (h) KOH, PhSH, CH3CN/H2O, 50 °C, overnight, then 1 M HCl,
81%; (i) Et3N, Ni(ClO4)2¢6H2O, MeOH, 81%.
of Ns and Piv groups gave the desired fluorescein-macrocyclic
ligand LODCF, which was purified by preparative reverse-
phase HPLC eluted by H2O/CH3CN containing 0.1% trifluoro-
acetic acid (TFA). Because LODCF was isolated as a TFA salt,
it was neutralized with NaOH prior to preparation of the
nickel(II) complex. Isolation with reverse-phase LC eluted by
H2O/MeOH and following lyophilization afforded desired
complex NiLODCF as a red-orange powder. It is advantageous
for fluorescence labeling experiments that this probe can be
used without Ni-complexation, unlike in the case for fluores-
cent NTA compounds, which need an additional complexation
step with the metal ion to label the proteins.23,24
Spectroscopic Characterization. All spectroscopic mea-
surements were performed in aqueous buffer solution (50 mM
HEPES, pH 7.20). Figure 3 shows the absorption spectra of
LODCF and NiLODCF, where a characteristic band of LODCF
Wavelength/nm
shifted from 509 (¾ = 48300 M¹1 cm¹1) to 488 nm (¾ = 44300
¹1 cm¹1) upon complexation with a nickel ion. Such a
Figure 3. Absorption spectra of LODCF (2.5 ¯M, dashed
line) and NiLODCF (2.5 ¯M, solid line) in an aqueous
solution (50 mM HEPES, pH 7.20).
M
hypochromic shift (¦ = 21 nm) in the absorption wavelength
indicated that the electronic structure (³-system) of the
xanthene core was perturbed in the nickel complex;43 namely,
the phenolic oxygen atom of DCF, rather than the carboxy
group at the 2-position of the benzene ring, was directly
coordinated to the nickel center in the aqueous buffered
solution. The optimized structure of NiLODCF by a DFT
calculation at the B3LYP/LANL2DZ level also provided the
possibility of an interaction between the phenolate oxygen of
DCF and the NiLO moiety (Figure S1 in Supporting Informa-
tion). It should be noted that the hypochromic shift as shown
for NiLODCF was not observed for the corresponding cobalt(II)
complex of LODCF (Figure S2). The fact that there was little
change from the original ligand in the absorption spectrum
suggested that the fluorescein moiety, in this case, was located
from the metal center. The fluorescence of LODCF was
significantly quenched by complexation with NiII, as expected

392
Bull. Chem. Soc. Jpn. Vol. 84, No. 4 (2011)
Fluorescent Probe for Polyhistidine Tag
Scheme 2. Displacement of coordinated DCF with histidine imidazoles results in fluorescence enhancement.
600
400
200
0
0
50
100
150
Wavelength/nm
[H6Y1]/µM
Figure 4. Fluorescence spectral change of NiLODCF (1.0
¯M) upon addition of the hexahistidine peptide (H6Y1).
[H6Y1] = 0, 1.4, 4.1, 6.9, 9.6, 13.7, 20.5, 27.2, 40.4, 53.3,
and 126.1 ¯M. The spectra were monitored with an
excitation wavelength at 509 nm in 50 mM HEPES buffer
(pH 7.20). The dashed spectrum is the macrocyclic ligand
LODCF (1.0 ¯M) observed under the same conditions.
Figure 5. Fluorescence response at 528 nm (-ex = 509 nm)
observed by the titration of NiLODCF (1.0 ¯M) with H6Y1
peptide in 50 mM HEPES buffer (pH 7.20). The data were
analyzed by nonlinear least-square fitting to determine a
dissociation constant of 24 « 1 ¯M.
relevant ions, such as Na+, K+, Mg2+, Ca2+, phosphate (Pi),
and pyrophosphate (PPi) (Figure S3), demonstrating that this
sensing system will be useful in a wide range of biological and
microscopic applications. The intensity of the fluorescence
of DCF increased as the concentration of H6Y1 increased,
reaching a maximum in the presence of an excess amount of the
peptide. The final fluorescent intensity was as high as that
observed for the metal-unbound form of LODCF, indicating no
interaction between the DCF fluorophore and the NiII ion in the
H6Y1-NiLODCF ternary complex. The dissociation of the
phenolic oxygen of DCF from the NiII center in this ternary
complex was also confirmed by the absorption spectrum, which
showed the disappearance of the characteristic peak at 488 nm
attributed to NiII-bound DCF. The binding stoichiometry of
NiLODCF and H6Y1 was determined to be 1:1 by a Job’s plot
(Figure S4). Based on this finding, the apparent dissociation
constant (Kd) of NiLODCF for H6Y1 was calculated by the
curve fitting analysis of the change of the fluorescence intensity
at 528 nm to be 24 ¯M as shown in Figure 5. This value is
from the absorption data. The quantum yields were 0.60 and
0.084 for LODCF and NiLODCF, respectively. These results
(86% quenching efficiency) were in contrast to those reported
by Lippard et al. showing that the fluorescence of the NTA-
functionalized DCF decreased only 5% upon complexation
with NiII.23 Such a difference in the photochemical properties
of DCF was due to the nature of the supporting ligands, LO and
NTA. The positively charged NiLO complex coordinated with
the DCF dye, as anticipated in the design strategy of the probe.
Fluorescence Response toward His6 Peptide. To model a
His-tag-fused protein, we prepared a hexahistidine peptide
(H6Y1: YHHHHHH), in which tyrosine was incorporated to
determine the accurate concentration of the peptide24 and
evaluated the binding behavior of NiLODCF by means of
fluorescent titration (Scheme 2). The addition of H6Y1 to a
1 ¯M solution of NiLODCF resulted in a 7-fold increase in the
emission intensity (Figure 4). The fluorescent properties of
NiLODCF were little affected by the presence of biologically

M. Taki et al.
Bull. Chem. Soc. Jpn. Vol. 84, No. 4 (2011)
393
4, 491.
comparable to those reported for Ni-NTA and the hexahistidine
peptide (Kd = 13 « 5 ¯M),21-23 suggesting that the two histi-
dine imidazole side chains of the His-tag bind NiLO core with
a manner similar to the case of Ni-NTA. In addition, we
performed a labeling experiment of His6-coated resin beads
with NiLODCF to evaluate the binding properties of the probe.
When the NiLODCF-labeled His6-beads were washed several
times with a neutral aqueous buffered solution, a red color
remained on the resin, indicating that the probe is still
immobilized on the resin surface. In contrast, the probe was
easily dissociated from the beads by washing with an EDTA-
containing buffer, which is a direct evidence for the Ni-
mediated interaction between NiLODCF and the His6 peptide
on the resin. Thus, these results suggest that NiLODCF
undergoes a slow dissociation from the His6-NiLODCF
complex and demonstrate the usefulness of NiLODCF for
optical imaging of specific protein expressed on the cell
surface.
6
O. Agbulut, A. Huet, N. Niederländer, M. Puceat, P.
Menasché, C. Coirault, J. Biol. Chem. 2007, 282, 10465.
M. Fernández-Suárez, A. Y. Ting, Nat. Rev. Mol. Cell Biol.
2008, 9, 929.
7
8
E. M. Sletten, C. R. Bertozzi, Angew. Chem., Int. Ed. 2009,
48, 6974.
9
N. Soh, Sensors 2008, 8, 1004.
10 B. A. Griffin, S. R. Adams, R. Y. Tsien, Science 1998, 281,
269.
11 A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H.
Vogel, K. Johnsson, Nat. Biotechnol. 2003, 21, 86.
12 I. Chen, M. Howarth, W. Y. Lin, A. Y. Ting, Nat. Methods
2005, 2, 99.
13 C.-W. Lin, A. Y. Ting, J. Am. Chem. Soc. 2006, 128, 4542.
14 A. Ojida, K. Honda, D. Shinmi, S. Kiyonaka, Y. Mori,
I. Hamachi, J. Am. Chem. Soc. 2006, 128, 10452.
15 C. T. Hauser, R. Y. Tsien, Proc. Natl. Acad. Sci. U.S.A.
2007, 104, 3693.
16 G. V. Los, L. P. Encell, M. G. McDougall, D. D. Hartzell,
N. Karassina, C. Zimprich, M. G. Wood, R. Learish, R. F. Ohane,
M. Urh, D. Simpson, J. Mendez, K. Zimmerman, P. Otto, G.
Vidugiris, J. Zhu, A. Darzins, D. H. Klaubert, R. F. Bulleit, K. V.
Wood, ACS Chem. Biol. 2008, 3, 373.
17 A. Ojida, S. Fujishima, K. Honda, H. Nonaka, S.
Uchinomiya, I. Hamachi, Chem. Asian J. 2010, 5, 877.
18 K. Honda, E. Nakata, A. Ojida, I. Hamachi, Chem.
Commun. 2006, 4024.
19 D. L. Johnson, L. L. Martin, J. Am. Chem. Soc. 2005, 127,
2018.
20 A. N. Kapanidis, Y. W. Ebright, R. H. Ebright, J. Am.
Chem. Soc. 2001, 123, 12123.
21 S. Lata, A. Reichel, R. Brock, R. Tampé, J. Piehler, J. Am.
Chem. Soc. 2005, 127, 10205.
22 E. G. Guignet, R. Hovius, H. Vogel, Nat. Biotechnol. 2004,
22, 440.
23 C. R. Goldsmith, J. Jaworski, M. Sheng, S. J. Lippard,
J. Am. Chem. Soc. 2006, 128, 418.
Conclusion
We have designed a novel “turn-on” fluorescent probe
NiLODCF for the His-tag sequence of proteins on the basis of
the fluorophore displacement mechanism. We demonstrated
that a macrocyclic nickel(II) complex moiety NiLO incorpo-
rated into the probe can act as the fluorescence quencher
through a labile coordinative interaction and can serve as the
His-tag binding site. NiLODCF exhibits an increase in fluores-
cence in the presence of the His-tag. The 1:1 stoichiometry and
dissociation constant (Kd) for the interaction between NiLODCF
the hexahistidine peptide are comparable to those for the Ni-
NTA/His6 pair. These features for NiLODCF, which include
the switchable fluorescence response, the reasonable Kd value,
and the visible light excitation, would allow fluorescent
labeling of the His-tag-fused proteins, as well as optical cell
imaging. We consider further applications of NiLODCF to
fluorescence-based enzyme assay tools in which enzymatic
reactions result in an alteration of the binding affinity of the
fluorophore, triggering an increase in fluorescence.
24 M. Kamoto, N. Umezawa, N. Kato, T. Higuchi, Chem.®
Eur. J. 2008, 14, 8004.
25 M. Kamoto, N. Umezawa, N. Kato, T. Higuchi, Bioorg.
Med. Chem. Lett. 2009, 19, 2285.
26 N. Soh, D. Seto, K. Nakano, T. Imato, Mol. BioSyst. 2006,
2, 128.
This work was financially supported by a Grant-in-Aid for
Young Scientists (B) (No. 21750168 to M.T.) and Grant-in-Aid
for JSPS Fellows (T.H.).
27 L. Fabbrizzi, M. Licchelli, P. Pallavicini, L. Parodi, Angew.
Chem., Int. Ed. 1998, 37, 800.
Supporting Information
DFT optimized structure for NiLODCF, UV-vis spectrum of
cobalt(II) complex, fluorescence response toward biologically
relevant ions, Job’s plot, and mass spectra for LODCF and
NiLODCF. This material is available free of charge on the web
at http://www.csj.jp/journals/bcsj/.
28 K. J. Franz, N. Singh, S. J. Lippard, Angew. Chem., Int. Ed.
2000, 39, 2120.
29 S. C. Burdette, G. K. Walkup, B. Spingler, R. Y. Tsien, S. J.
Lippard, J. Am. Chem. Soc. 2001, 123, 7831.
30 T. Hirano, K. Kikuchi, Y. Urano, T. Nagano, J. Am. Chem.
Soc. 2002, 124, 6555.
31 L. Zeng, E. W. Miller, A. Pralle, E. Y. Isacoff, C. J. Chang,
J. Am. Chem. Soc. 2006, 128, 10.
References
1
J. Zhang, R. E. Campbell, A. Y. Ting, R. Y. Tsien, Nat. Rev.
Mol. Cell Biol. 2002, 3, 906.
B. N. G. Giepmans, S. R. Adams, M. H. Ellisman, R. Y.
Tsien, Science 2006, 312, 217.
32 Y. Urano, D. Asanuma, Y. Hama, Y. Koyama, T. Barrett, M.
Kamiya, T. Nagano, T. Watanabe, A. Hasegawa, P. L. Choyke, H.
Kobayashi, Nat. Med. 2009, 15, 104.
33 G. Sharma, J. P. Tandon, J. Inorg. Nucl. Chem. 1970, 32,
1273.
34 M. Taki, Y. Kawashima, N. Sakai, T. Hirayama, Y.
Yamamoto, Bull. Chem. Soc. Jpn. 2008, 81, 590.
35 A.-S. Chauvin, S. Comby, B. Song, C. D. B. Vandevyver,
2
3
4
R. Y. Tsien, Annu. Rev. Biochem. 1998, 67, 509.
A. Chiesa, E. Rapizzi, V. Tosello, P. Pinton, M. de Virgilio,
K. E. Fogarty, R. Rizzuto, Biochem. J. 2001, 355, 1.
C. S. Lisenbee, S. K. Karnik, R. N. Trelease, Traffic 2003,
5

394
Bull. Chem. Soc. Jpn. Vol. 84, No. 4 (2011)
Fluorescent Probe for Polyhistidine Tag
J.-C. G. Bünzli, Chem.®Eur. J. 2008, 14, 1726.
36 C. C. Woodroofe, R. Masalha, K. R. Barnes, C. J.
Frederickson, S. J. Lippard, Chem. Biol. 2004, 11, 1659.
37 T. Hirayama, M. Taki, A. Kodan, H. Kato, Y. Yamamoto,
Chem. Commun. 2009, 3196.
38 J. R. Lakowicz, Principles of Fluorescence Spectroscopy,
3rd ed., Springer, New York, 2006.
39 CrystalStructure 3.8.2: Crystal Structure Analysis Package,
Rigaku and Rigaku/MSC, The Woodlands, TX 77381, USA,
2007.
adopts a flat conformation in the His-tag bound form, where two
histidine imidazoles occupy both axial positions of the NiN3O1
plane, and these bent- and flat-configurations are hardly distin-
guishable from each other in the spectroscopic properties. How-
ever, we considered that for the flat conformation the steric
repulsion between the ligand framework and the histidine residues
of His-tag would make it less favorable for the tag sequence to
adopt a hairpin-like structure.
42 V. Félix, M. J. Calhorda, J. Costa, R. Delgado, C. Brito,
M. T. Duarte, T. Arcos, M. G. B. Drew, J. Chem. Soc., Dalton
Trans. 1996, 4543.
43 V. Balzani, F. Scandola, Supramolecular Photochemistry,
Ellis Horwood, New York, 1991.
40 SIR92: A. Altomare, G. Cascarano, C. Giacovazzo, A.
Guagliardi, M. C. Burla, G. Polidori, M. Camalli, J. Appl.
Crystallogr. 1994, 27, 435.
41 It is also possible that the macrocyclic ligand of NiLO