Phosphorescent thymidine triphosphate sensor based on a donor-acceptor ensemble system using intermolecular energy transfer

Article abstract of DOI:10.1002/chem.200801260

An ensemble sensor system that exhibited selective luminescence enhancement upon binding to thymidine 5′-triphosphate (TTP) in HEPES buffer over other nucleotides was developed. The ensemble system consisted of an energy acceptor (Flrpicbis(Zn²⁺

Full text of DOI:10.1002/chem.200801260

FULL PAPER
DOI: 10.1002/chem.200801260
Phosphorescent Thymidine Triphosphate Sensor Based on a Donor–
Acceptor Ensemble System using Intermolecular Energy Transfer
Tae-Hyuk Kwon,[a, b] Hee Jin Kim,[a] and Jong-In Hong*[a]
Abstract: An ensemble sensor system
that exhibited selective luminescence
enhancement upon binding to thymi-
dine 5’-triphosphate (TTP) in HEPES
buffer over other nucleotides was de-
veloped. The ensemble system consist-
ed of an energy acceptor (FIrpic–
nato-N,C2+]picolinate) derivative) and
an energy donor (mCP–Zn2+–cyclen,
mCP=N,N’-dicarbazolyl-3,5-benzene).
Among the nucleotides, the selective
recognition and luminescence enhance-
ment for TTPwas achieved by the
strong binding of the thymine unit to
Zn2+–cyclen (cyclen=1,4,7,10-tetraaza-
cyclododecane) and intermolecular
energy transfer between the mCPand
FIrpic moieties.
Keywords: donor–acceptor
sys-
tems · intermolecular energy trans-
fer · luminescence · phosphores-
cence · sensors
bis(Zn2+–dipicolylamine
conjugate,
FIrpic=bis[(4,6-difluorophenyl)-pyridi-
Introduction
cell division.[1b] Because of the central role of thymidylate
synthase in growing cells, it has been used as a target in
cancer therapy for many years.[1b] We believe that the devel-
opment of a thymidine 5’-triphosphate (TTP) sensor would
enable monitoring of thymidylate synthase. However, with
the exception of a TTPsensor, which shows fluorescence
quenching through a photoinduced-electron-transfer mecha-
nism,[4b] there is no report on a TTPsensor that shows lumi-
nescence enhancement. Furthermore, until recently, fluores-
cent sensors have been used as analytical tools for examin-
ing biological events. However, they might have several
problems, such as strong pH dependence, high photobleach-
ing rates, low photostability, small Stokes shifts, and short
lifetimes.[5] To overcome these problems, this paper introdu-
ces two concepts, energy transfer and phosphorescence
(Figure 1). The former will be very useful for enhancing
both the signal and signal-to-noise ratio.[6] In addition, tran-
sition-metal complexes using phosphorescence can over-
come the problems of organic fluorophores.[5] Based on
these concepts, this paper proposes a new paradigm for the
phosphorescent TTPsensor.
Artificial receptor molecules that enable the direct detection
of nucleotides have received considerable attention on ac-
count of their responsibility for genetic information storage
and transfer in cells. Therefore, recognition of a specific nu-
cleotide is challenging.[1a] As an example, the colorimetric
adenosine 5’-triphosphate (ATP) receptor[2] and fluorescent
quenching guanosine 5’-triphosphate (GTP) receptor[3] have
been reported. The ZnII complex of a marcrocyclic tetra-
amine (Zn2+–cyclen, cyclen=1,4,7,10-tetraazacyclodode-
cane) is known to be a thymidine receptor because of the
strong affinity of Zn2+ for the imide part of the thymine
base and the two complementary hydrogen bonds between
the two NH groups of Zn2+–cyclen and the two carbonyl
oxygen atoms of the thymine base.[1a,4] It is known that thy-
midylate synthase is necessary to produce thymidine triphos-
phate, an essential building block for DNA replication and
[a] Dr. T.-H. Kwon,+ H. J. Kim,+ Prof. Dr. J.-I. Hong
Department of Chemistry, College of Natural Sciences
Seoul National University, Seoul 151-747 (Korea)
Fax : (+82)2-889-1568
E-mail: jihong@snu.ac.kr
[b] Dr. T.-H. Kwon+
Results and Discussion
Current address: Bio21 Institute, The School of Chemistry
University of Melbourne, Parkville
Victoria, 3010 (Australia)
Ensemble system: These concepts for a TTPphosphorescent
sensor are based on an emission enhancing ensemble system
using intermolecular energy transfer, which shows a high
sensitivity and selectivity for TTPamong the nucleotides
and other various anions. This sensor system consisted of an
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200801260.
Chem. Eur. J. 2008, 14, 9613 – 9619
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
9613

Zn2+–cyclen acts as a mono-
topic receptor for deoxythymi-
dine (dT) and uridine (U) from
all the nucleosides at a physio-
logical pH in aqueous solution
through Zn2+–cyclen/imide Nꢀ
binding and two complementa-
ry hydrogen bonds between the
two NH groups of Zn2+–cyclen
and the two carbonyl oxygen
atoms of the thymine base.[4d]
The outstanding Lewis acid
properties of
a
Zn2+–cyclen
complex tend to bind with
anionic ligands at the vacant
fifth coordination site rather
than with neutral nitrogen do-
nors.[4b] Therefore, there ap-
pears to be a slight interaction
between the Zn2+–cyclen com-
plex and the donor sites of nu-
Figure 1. Proposed binding mode between the donor (1), TTP, and the acceptor (2).
energy acceptor (acceptor (A)=compound 2, Scheme 2) de-
rived from an iridium(III) complex linked covalently to the
cleobases, such as 2’-de
A
G
E
sine (dA), and 2’-deoxycytidine (dC).[4b] However, the Zn2+
–cyclen complex can also act as a good monotopic receptor
for phosphate moieties in aqueous solution.[4d] Indeed, the
Job plot shows 2:1 stoichiometry when compound 1 is titrat-
ed with TTPin aqueous solution (Figure S2 in the Support-
ing Information). Hence, in this ensemble system, there may
ZnII–DPA (DPA=N,N’-di(2-picolyl)amine) complex as a
binding site for the triphosphate unit of nucleotide guests
(ATP, CTP (cytosine 5’-triphosphate), GTP, and TTP), and
an energy donor (donor (D)=compound 1, Scheme 1) de-
rived from the N,N’-dicarbazolyl-3,5-benzene (mCP) deriva-
tive appended with the Zn2+–cyclen unit (Figure 1). Iridium-
be a competitive binding mode between the Zn2+–cyclen/tri-
2+
A
bis[(4,6-difluorophenyl)-pyridinato-N,C2+]picolinate
phosphate and bis
(Zn2+–DPA)/triphosphate. However, Zn
N
(FIrpic), which is a well-known sky-blue dopant material in
organic light-emitting diodes, was selected as an energy-ac-
ceptor moiety on account of its high quantum efficiency
(FPL =0.42). mCPwas chosen as an energy-donor unit that
demonstrates higher singlet and triplet energies than the ac-
ceptor moiety.[7]
–cyclen mainly binds with a nucleobase unit in this ensemble
system because the binding constant for bis
AHCTREUNG
phosphate (logKꢁ6–7)[8] is much larger (ca. 3 orders) than
that for Zn2+–cyclen/triphosphate (logKꢁ3–4).[4d] To fur-
ther remove any possibility of Zn2+–cyclen/triphosphate
binding, two equivalents of compound 2 were used in the
experiment.[9] As a result, it was confirmed that compound 2
binds mainly with the triphosphate unit of TTP, whereas the
Binding mode: It is expected that both bis
(Zn2+–DPA) and
A
Zn2+–cyclen units can be used as multiple bindings for the
triphosphate and nucleobase moieties. For example, the bis-
nucleobase unit of TTPinteracts with compound
1
(Figure 1).[9]
(Zn2+–DPA) moieties generate an anion-binding site
through the formation of a well-known phenoxo-bridged di-
nuclear metal complex,[8] and two sets of oxygen anions on
each phosphorus of a triphosphate moiety bind to the dinu-
clear zinc complex by binding the two metal ions to give
rise to two hexacoordinated Zn2+ ions.[8] Zn2+–DPA can
also weakly bind with a nucleobase unit of nucleotides, such
as imide nitrogen and carbonyl oxygen atoms of the thymine
base. Indeed, the Job plot shows 2:1 binding when com-
pound 2 is titrated with TTPin a buffer solution (Figure S1
in the Supporting Information). However, it is believed that
Synthesis: Schemes 1 and 2 outline the synthesis of the
phosphorescent donor-acceptor ensemble system. Donor 1
was obtained by the three consecutive reactions of alkyl-
A
(90% yield), and zinc insertion (48% yield; Scheme 1). Ac-
ceptor 2 was composed of the iridium complex (6) and bis-
(Zn2+–DPA) derivative (3). Compound 3 was synthesized
from 4-(2-aminoethyl)phenol, which was protected with
Boc2O and then treated with a solution of DPA and formal-
dehyde in ethanol. Compound 3 was finally obtained after
removing the Boc group with 10% TFA, in an overall yield
of 16% (Scheme 2). Compound 6 was coupled with ethyl
bromoacetate to give compound 7 in 58% yield after hy-
drolysis followed by an acid treatment. Amide coupling be-
tween compounds 7 and 3 in the presence of N,N’-diisopro-
pylethylamine (DIPEA), benzotriazol-1-yl-oxytripyrrolidi-
the bis
A
phosphate unit of TTP. This is because the binding affinity
[8]
between bis
(Zn2+–DPA) and the triphosphate is much
G
stronger than that between bis
moiety.
A
9614
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 9613 – 9619

Phosphorescent Thymidine Triphosphate Sensor
FULL PAPER
Scheme 1. Synthesis of Compound 1.
Scheme 2. Synthesis of Compound 2.
Chem. Eur. J. 2008, 14, 9613 – 9619
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9615

J.-I. Hong et al.
Table 1. Photophysical properties of 1 and 2.
nophosphonium hexafluorophosphate (PyBOP), and 1-hy-
droxybenzotriazole (HOBt), followed by zinc insertion af-
forded the acceptor 2 of the ensemble system in 19% yield.
1
2
absorption [nm]
(e”104 cmꢀ1 mꢀ1
)
H2O[a]
221 (29), 289
(0.45)
322 (0.11), 338
(0.11)
222 (28), 253 (21)
300 (0.86), 372
(0.12)
Photophysical studies: It was previously reported that the
phosphorescent FIrpic acceptor coupled with the dendrimer
mCPdonor by a nonconjugated bridge showed a dramatic
increase in emission intensity compared with that of the ac-
ceptor without the donor or just the donor–acceptor blend-
ing system when excited at the donor absorption (lex =
310 nm).[6] This is because the singlet–singlet (S–S) and trip-
let–triplet (T–T) energy transfer (ET) can occur efficiently.[6]
As shown in Figure 2a, there is a good overlap between the
emission spectrum of 5mCPand the absorption spectrum of
emission [nm]
H2O[a]
351, 362 (sh)
–
–
475, 507
475, 500 (sh)
4756, 527
DMSO[b]
[b]
CH2Cl2
[a] 0.01 mm of 1 or 2 in HEPES buffer solution [b] 0.01 mm solution.
photoluminescence (PL) data of compound 1 and UV and
PL data of compound 2 in solution in buffer, CH2Cl2, and
DMSO. These data are similar to Figure 2a. The lumines-
3
FIrpic over 350 nm (1MLCT and LC region), which ensures
S–S ET from mCPto FIrpic. The T–T ET from 5mCPto
FIrpic can also occur because the triplet energy of 5mCPis
higher than that of FIrpic. Table 1 and Figure 2b shows the
1
cence of compound 2 results from both MLCT (metal-to-
ligand charge transfer, dp(Ir)!p*
A
centered) in a FIrpic moiety. Photophysical properties of
compound 2 are very sensitive to the polarity of the envi-
ronment due to the MLCT character.[10] Therefore, its emis-
sion maximum is found at 475 nm, which is almost identical
to that of FIrpic, but emission spectral shape is dependent
on the solvent environment (Figure 2b).[11] The absorption
spectrum of compound 2 in buffer shows relatively weak
MLCT absorption compared with that of FIrpic in 2-methyl-
tetrahydrofuran (2-MeTHF). However, the inset of Fig-
ure 2b clearly shows the MLCT region of compound 2, and
the spectral overlap between the absorption spectrum of 2
over 350 nm and the emission spectrum of 1. We can also
assume that 1 and 2 have similar triplet energy values to
[12]
those of 5mCPand FIrpic, respectively.
Therefore, the
donor–acceptor ensemble system composed of 1 and 2 is ex-
pected to exhibit a good ET from the donor to the acceptor
through both the S–S and T–T ET mechanisms. Without
TTP, a large amount of donor emission still remained and
there is a little acceptor emission intensity upon excitation
at the donor (1) absorption peak (lex =310 nm) (Figure 4
and Figure S3 in the Supporting Information). This is be-
cause the distance between the donor and the acceptor is
too far away for efficient intermolecular energy transfer in
the absence of TTP. However, the donor emission was re-
duced dramatically in the 1:1:2 mixture of 1+TTP+2 com-
pared with that of 1+TTP( lex =310 nm), as shown in
Figure 3. In addition, the acceptor emission intensity in 1+
TTP+2 upon excitation at 310 nm increases more than
three times compared with that upon excitation at the ac-
ceptor absorption (lex =380 nm). This increase is because ef-
ficient intermolecular S–S and T–T ET can occur in this en-
semble system. Furthermore, it shows little change in lumi-
nescence upon the addition of various anions, such as Iꢀ,
ꢀ
ꢀ
Clꢀ, NO3 , CO32ꢀ, NO32ꢀ, and ClO4 , when excited at
310 nm (Figure 4). This observation means that the 1:1:1 en-
semble system does not form in the presence of other
anions. Only nucleotides with two strong binding sites can
form an ensemble system that enables intermolecular
energy transfer.
Figure 2. a) UV spectra of FIrpic and PL spectra of 5mCP and FIrpic in
0.02 mm 2-MeTHF at 298 and 77 K. The structures of 5mCPand FIrpic
are also shown.[6] b) UV spectra of 2 ( ) in 0.01 mm buffer solution, and
&
*
PL spectra of 1 in 0.01 mm buffer ( ), and PL spectra of 2 in 0.01 mm
~
!
^
buffer ( ), DMSO ( ), and CH2Cl2
( ), respectively. Inset shows the
magnification of the MLCT region of 2.
9616
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 9613 – 9619

Phosphorescent Thymidine Triphosphate Sensor
FULL PAPER
79% for TTP, ATP, GTP, and CTP, respectively (Table 2;
also see the Supporting Information). ET efficiency appears
to increase according to the binding strength between the
Zn2+–cyclen unit and nucleobase. Therefore, TTPshows the
highest ET efficiency of all nucleotides due to Zn2+–cyclen/
imide Nꢀ binding and two complementary hydrogen bonds
between the two NH groups of Zn2+–cyclen and the two
carbonyl O atoms of the thymine base.
Due to the highest ET efficiency of TTPamong the nucle-
otides examined, the residual donor emission was greatly
decreased and the emission intensity of the acceptor in 1+
TTP+2 was enhanced slightly compared with those of the
other nucleotide ensemble systems when excited at the
donor absorption (lex =310 nm). In contrast, there was no
selectivity when it was excited at the acceptor absorption
(lex =380 nm; see the Supporting Information for more de-
tails). The acceptor (2) emission intensity (IA) in the TTP
ensemble system increases only 1.2 times compared with the
other ensemble systems, as shown in the inset of Figure 5a.
However, if the ET efficiency is considered, the current en-
semble system shows good selectivity for TTPfrom all nu-
cleotides. Figure 5b shows that TTPcan be recognized selec-
tively when the acceptor (2) emission intensity (IA) is divid-
ed by the residual donor (1) emission intensity (ID). The
Figure 3. PL spectra of 1 (10 mm) and TTP(10 mm) upon excitation at
&
310 nm ( ) and 1 (10 mm), TTP(10 mm), and 2 (20 mm) upon excitation at
~
*
310 nm ( ) and 380 nm ( ) upon excitation at 310 nm. respectively, in
HEPES buffer at 298 K and pH 7.4.
Figure 4. Photoluminescence spectra of 1 (10 mm) and various anions
ꢀ
ꢀ
ꢀ
2ꢀ
ꢀ
~
!
3
&
*
^
(TTP( ), I ( ), Cl
(
), ClO4
(
), CO3
( ), NO3 ( )) (each 10 mm)
and 2 (10 mm) in HEPES buffer at 298 K and pH 7.4, upon excitation at
310 nm.
Energy transfer efficiency and TTP selectivity: Table 2
shows the ET efficiency obtained using the steady-state PL
method. The ET efficiency was determined from the extent
of luminescence quenching of the donor in the presence of
the acceptor.[6] This was measured from the relative ratio be-
tween the integrated area of 1+NTP( ID) and that of the re-
sidual donor peak in 1+NTP+2 (ID–A) at 298 K. A decrease
in donor emission intensity of the 1+TTP+2 system results
from intermolecular ET. The ET efficiency from the donor
to the acceptor at 298 K was calculated to be 91, 74, 76, and
Table 2. Energy transfer efficiency.
FET[a] [%]
TTP91
ATP74
GTP76
CTP79
Figure 5. PL spectra of the ensemble system excited at 310 nm and TTP
selectivity. a) Donor emission intensity (ID) of compound 1 (10 mm), TTP
(10 mm), and 2 (20 mm) ensemble system, excited at 310 nm. The inset
shows the relative acceptor emission intensity at 476 nm (IA). b) The rela-
tive ratios when the acceptor emission intensity (IA) at 476 nm is divided
by the residual donor emission (ID) at 348 nm.
[a] The ET efficiency was measured from the relative ratio between the
integrated area of 1+NTP( ID) and that of the residual donor peak in
1+NTP+2 (ID–A). Energy transfer efficiency (%)=(1ꢀID–A/ID)”100.
Chem. Eur. J. 2008, 14, 9613 – 9619
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9617

J.-I. Hong et al.
Compound 9: Trifluoroacetic acid (0.26 mL) was slowly added to a solu-
tion of compound 8 (118 mg, 0.132 mmol) in CH2Cl2 (5 mL). After being
stirred at room temperature for 24 h, the solvent was removed in vacuo
and dissolved in CH2Cl2. The organic phase was washed with water and
dried over Na2SO4. The reaction mixture was concentrated under re-
duced pressure. The resulting crude powder was crystallized from
CH2Cl2/hexane to provide the desired product as a white powder (70 mg,
ratio of IA/ID in the TTPensemble system shows more than
four times the enhancement compared with that of other nu-
cleotide ensemble systems. This ensemble system also dis-
played a relatively small energy transfer effect for ADP,
2ꢀ
AMP, and H PO4 (Figure S6 in the Supporting Informa-
2
tion). Therefore, TTPselectivity was achieved.
As a result, this phosphorescent ensemble system can be
used for the selective recognition of TTPdue to its intermo-
lecular ET efficiency.
89.4%). 1H NMR (300 MHz, [D3]acetonitrile): d=8.22 (d,
7.7 Hz, 4H), 7.78 (s, 3H), 7.64 (d, J(H,H)=8.2 Hz, 4H), 7.50 (t, J
8.2 Hz, 4H), 7.34 (t, J(H,H)=7.4 Hz, 4H), 4.10 (s, 2H), 3.20 (br, 4H)
J
ACHTREUNG
A
ACHTREUNG
AHCTREUNG
3.02 (m, 8H), 2.88 ppm (br, 4H); 13C NMR (125 MHz, [D6]acetone): d=
140.9, 140.8, 139.4, 127.7, 126.3, 124.9, 123.4, 120.2, 120.2, 110.1, 56.5,
48.6, 45.0, 42.7, 42.6 ppm; HRMS (FAB): m/z calcd for C39H41N6
[M+H]+: 593.3393; found: 593.3395.
Conclusion
Compound 1: Compound 9 (40 mg, 0.0612 mmol) in MeOH (1 mL) and
Zn(ClO4)2·6H2O in MeOH (1 mL) were combined and heated at reflux
for 1 h under nitrogen. Most of the solvent was removed in vacuo, and
the precipitated product was collected by filtration and dried in vacuo to
N
A phosphorescent TTPsensor was developed through a
donor–acceptor ensemble system using intermolecular ET.
Out of all nucleotides, TTPshows the best ET efficiency
yield the product as
1H NMR (300 MHz, CD3CN): d=8.24 (d, J
(H,H)=15.9 Hz, 1H), 7.75 (d, J(H,H)=11.5 Hz, 2H), 7.62 (t, J
8.2 Hz, 4H), 7.56–7.50 (m, 4H), 7.36 (t, J(H,H)=7.4 Hz, 4H), 4.21 (s,
a
white powder (22 mg, 48.4%). M.p. 2008C;
(H,H)=7.7 Hz, 4H), 8.01 (d,
(H,H)=
(>90%) due to the strong binding between Zn2+–cyclen
AHCTREUNG
J
A
A
ACHTREUNG
and the nucleobase. This system can be used for the selec-
tive sensing of TTPfrom the nucleotides on account of the
intermolecular ET.
AHCTREUNG
1H), 4.10 (s, 1H), 3.22–2.74 ppm (m, 16H); 13C NMR (125 MHz,
CD3CN): d=140.7, 139.3, 138.5, 128.8, 128.0, 126.4, 125.4, 123.4, 120.5,
109.9, 55.9, 49.2, 47.6, 45.0, 44.5, 43.7, 42.5, 42.3, 41.7 ppm; HRMS
(FAB): m/z calcd for C39H40N6ZnClO4 [M]+: 755.2091; found: 755.2072.
Compound 6: This compound was prepared according to the literature
procedure.[6]
Experimental Section
Compound 10: A mixture of 6 (100 mg, 0.141 mmol), bromoacetic acid
ethyl ester (94 mg, 0.563 mmol), and K2CO3 (29 mg, 0.210 mmol) was
heated at reflux in THF for 24 h. After cooling to room temperature, the
solvent was removed in vacuo and the residue was dissolved in CH2Cl2.
The organic phase was washed with water, washed with brine, and dried
over Na2SO4. The solvent was evaporated to give the crude product,
which was purified by column chromatography on silica gel with CH2Cl2/
methanol (100:1, v/v) as the eluent to provide the desired product as a
yellow powder (75 mg, 67%). 1H NMR (300 MHz, [D6]DMSO): d=8.59
Reagents: Buffer A solution contained 10 mm HEPES adjusted to pH 7.4
with NaOH. To make compounds 1 and 2 soluble in aqueous solvent, a
cosolvent system (H2O/DMSO=24:1) was used. The tetrasodium salt of
TTPand the di sodium salt of GTPwere purchased from Fluka. The diso-
A
dium salt of ATPwas purchased from Sigma. The disodium salt of CTP
was purchased from Aldrich. DIPEA, HOBt, and PyBOP were pur-
chased from Aldrich. Analytical thin-layer chromatography was per-
formed on Kieselgel 60F-254 plates obtained from Merck. Column chro-
matography was carried out on Merck silica gel 60 (70–230 mesh). All
solvents and reagents were commercially available and used without fur-
ther purification unless otherwise noted.
Instruments: 1H and 13C NMR spectra were recorded using an Advance
300 MHz Bruker spectrometer in CDCl3 and [D6]DMSO. UV/Vis spectra
were recorded on a Beckman DU 650 spectrophotometer. Mass spectra
were obtained using a MALDI-TOF mass spectrometer from Bruker or
(d, J
(H,H)=8.6 Hz, 1H), 7.67 (d, J
7.37–7.33 (m, 2H), 6.87–6.76 (m, 2H), 5.68 (dd, J
1H), 5.45 (dd, J(H,H)=8.6, 2.2 Hz, 1H), 4.99 (s, 2H), 4.15 (q, J
7.1 Hz, 2H), 1.18 ppm (t, J
(H,H)=7.1 Hz, 3H); 13C NMR (125 MHz,
ACHTREUNG
J
A
ACHTREUNG
AHCTREUNG
A
ACHTREUNG
AHCTREUNG
[D6]DMSO): d=170.2, 168.6, 163.5, 158.3, 153.9, 149.3, 148.5, 142.1,
139.8, 138.9, 130.4, 128.3, 126.4, 124.4, 124.0, 123.1, 114.1, 98.0, 66.2, 61.3,
14.4 ppm; MALDI-TOF: m/z calcd for C32H23F4IrN3O5 [M+H]+:
798.120; found: 798.448.
a
gas chromatography–mass spectrometer from JEOL. Fluorescence
spectra were recorded on a Jasco FP-7500 spectrophotometer.
Compound 7: A 1n aqueous solution of NaOH (1 mL) was added to a
solution of 10 (75 mg, 0.0941 mmol) in THF/H2O (1 mL/1 mL). After stir-
ring for 2 h at room temperature, the reaction mixture was acidified with
1n HCl. The solvent was removed in vacuo and dissolved in CH2Cl2. The
organic phase was washed with water and dried over Na2SO4. The solvent
was evaporated to give the desired product as a yellow powder (63 mg,
87%). 1H NMR (300 MHz, [D6]DMSO): d=13.3 (br, 1H), 8.59 (d, J-
Compound (4): This compound was prepared according to the literature
procedure.[6]
Compound (5): This compound was prepared according to the literature
procedure.[13]
Compound 8: (10-(3,5-Bis-carbazole-9-yl-benzyl)-1,4,7-tris(tert-butyloxy-
carbonyl)-1,4,7,10-tetraazacyclododecane): A mixture of 5mCP-Br (4)
(100 mg, 0.188 mmol), 3Boc–cyclen (5, 170 mg, 0.360 mmol), K2CO3
(62.4 mg, 0.451 mmol) was heated at reflux in acetonitrile for 24 h. After
cooling to room temperature, the solvent was removed in vacuo and dis-
solved in ethyl acetate. The organic phase was washed with water,
washed with brine, and dried over Na2SO4. The solvent was evaporated
to give the crude product, which was purified by column chromatography
on silica gel using ethyl acetate/hexane (1:3, v/v) as the eluent to provide
AHCTREUNG
A
ACHTREUNG
AHCTREUNG
AHCTREUNG
(125 MHz, [D6]DMSO): d=170.7, 170.0, 164.0, 158.5, 153.5, 149.4, 148.5,
142.2, 139.9, 138.7, 130.6, 128.3, 126.8, 124.5, 124.1, 123.4, 114.1, 98.0,
66.7 ppm; MALDI-TOF: calcd for C30H19F4IrN3O5 [M+H]+: 770.089;
found: 770.530.
the desired product as
(300 MHz, [D6]acetone): d=8.23 (d, J
7.61 (d, J(H,H)=8.2 Hz, 4H), 7.50 (t, J
a
white powder (132 mg, 78.5%). 1H NMR
(H,H)=7.7 Hz, 4H), 7.81 (s, 3H),
(H,H)=7.5 Hz, 4H), 7.31 (t, J-
Compound 14: A mixture of compound 7 (32 mg, 0.042 mmol), DIPEA
(11 mg, 0.085 mmol), PyBOP (21 mg, 0.041 mmol), and HOBt (3 mg,
0.040 mmol) was stirred in distilled CH2Cl2 (4 mL) for 1 h , followed by
slowly adding compound 3 in CH2Cl2 (1 mL). After stirring at room tem-
perature for 7 h, the mixture was diluted with CH2Cl2, and washed with
water and brine. The organic phase was dried over Na2SO4 and evaporat-
ed to dryness. The residue was purified by column chromatography on
AHCTREUNG
A
ACHTREUNG
A
4H), 1.47 (s, 9H), 1.28 ppm (s, 18H); 13C NMR (125 MHz, CDCl3): d=
156.3, 155.9, 141.3, 140.7, 139.2, 128.0, 126.3, 124.7, 123.6, 120.4, 120.3,
109.6, 79.7, 79.5, 56.8, 56.0, 55.2, 50.3, 47.8, 28.7, 28.4 ppm; MALDI-TOF:
m/z calcd for C54H66N6O6 [M+2H]+: 894.504; found: 894.417.
9618
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 9613 – 9619

Phosphorescent Thymidine Triphosphate Sensor
FULL PAPER
silica gel with CH2Cl2/methanol (20:1, v/v) as the eluent to provide the
desired product as a yellow powder (10 mg, 18.6%). H NMR (300 MHz,
Acknowledgements
1
[D6]DMSO): d=9.04 (br, 1H), 8.54–8.50 (m, 5H), 8.25 (d, J
8.3 Hz, 2H), 8.06–8.00 (m, 3H), 7.81 (d, J(H,H)=8.6 Hz, 1H), 7.74–7.67
(m, 6H), 7.61–7.57 (m, 1H), 7.44 (t, J(H,H)=6.7 Hz, 1H), 7.38–7.27 (m,
8H), 7.11 (s, 2H), 6.90–6.76 (m, 2H), 5.66 (dd, J(H,H)=8.7, 2.2 Hz, 1H),
5.46 (dd, J(H,H)=8.6, 2.1 Hz, 1H), 4.65 (s, 2H), 4.01 (br, 12H), 3.02 (br,
2H), 2.66 ppm (t,
(H,H)=7.1 Hz, 2H); 13C NMR (125 MHz,
[D6]acetone): d=172.1, 167.1, 164.4, 163.9, 157.9, 155.6, 155.0, 153.9,
149.3, 149.0, 148.3, 142.5, 139.2, 138.5, 137.2, 131.8, 130.6, 129.8, 128.4,
125.7, 123.7, 123.5, 123.2, 122.9, 121.4, 114.3, 97.4, 68.3, 58.1, 55.1, 46.1,
34.2; HRMS (FAB): m/z calcd for C64H54F4IrN10O5 [M+H]+: 1311.3844;
found: 1311.3820.
A
The authors wish to thank the Seoul R&BD Program for the financial
support. We also acknowledge the BK 21 fellowship grants to T.H.K. and
H.J.K.
AHCTREUNG
AHCTREUNG
AHCTREUNG
ACHTREUNG
J
ACHTREUNG
[1] a) S. Aoki, E. Kimura, Chem. Rev. 2004, 104, 769; b) S. T. Pullarkat,
J. Stoehlmacher, V. Ghaderi, Y.-P. Xiong, S. A. Ingles, A. Sherrod,
R. Warren, D. Tsao-Wei, S. Groshen, H.-J. Lenz, Pharmacogenomics
J. 2001, 1, 65.
[2] F. Sancenón, A. B. Descalzo, R. Martínez-Mµòez, M. A. Miranda, J.
Soto, Angew. Chem. 2001, 113, 2710; Angew. Chem. Int. Ed. 2001,
40, 2640.
Compound 2: An aqueous solution of Zn(NO3)2·6H2O (7 mg,
A
0.024 mmol) was added dropwise to a solution of 14 (15 mg, 0.011 mmol)
in methanol (1.5 mL), and the mixture was stirred for 30 min at room
temperature. The solvent was evaporated to give the desired product as a
yellow powder (19 mg, 100%). M.p. 2338C; MALDI-TOF: m/z calcd for
C64H52F4IrN10O5·2Zn·2NO3ꢀ [M]+: 1561.2027; found: 1561.2048.
[3] J. Y. Kwon, N. J. Singh, H. N. Kim, S. K. Kim, K. S. Kim, J. Yoon, J.
Am. Chem. Soc. 2004, 126, 8892.
[4] a) M. Shionoya, E. Kimura, M. Shiro, J. Am. Chem. Soc. 1993, 115,
6730; b) M. Shionoya, T. Ikeda, E. Kimura, M. Shiro, J. Am. Chem.
Soc. 1994, 116, 3848; c) E. Kimura, H. Kitamura, K. Ohtani, T.
Koike, J. Am. Chem. Soc. 2000, 122, 4668; d) S. Aoki, E. Kimura, J.
Am. Chem. Soc. 2000, 122, 4542.
Compound 11: This compound was prepared according to the literature
procedure.[11]
[5] K. K.-W. Lo, W.-K. Hui, C.-K. Chung, K. H.-K. Tsang, T. K.-M. Lee,
C.-K. Li, J. S.-Y. Lau, D. C.-M. Ng, Coord. Chem. Rev. 2006, 250,
1724.
Compound 12: DPA (4.17 g, 20.9 mmol) was added slowly to a solution
of 37% aqueous formaldehyde (1.86 g, 22.9 mmol) and ethanol (5 mL).
The reaction mixture was heated at reflux for 12 h, and compound 11
(2.36 g, 9.95 mmol) in ethanol (20 mL) was added. After being heated at
reflux for 5 d, the reaction mixture was cooled down to room tempera-
ture. The solvent was removed in vacuo and the residue was dissolved in
ethyl acetate. The organic phase was washed with water and dried over
Na2SO4. The solvent was evaporated to give the crude product, which
was purified by column chromatography on silica gel with CH2Cl2/metha-
nol (200:1, v/v) as the eluent to provide the desired product as a brown
sticky liquid (1.72 g, 26.2%). 1H NMR (300 MHz, [D6]acetone): d=10.95
[6] a) T.-H. Kwon, M. K. Kim, J. Kwon, S.-J. Park, D. Y. Shin, C.-L.
Lee, J.-J. Kim, J.-I. Hong, Chem. Mater. 2007, 19, 3673; b) T.-H.
Kwon, J. Kwon, J.-I. Hong, J. Am. Chem. Soc. 2008, 130, 3726.
[7] R. J. Holmes, S. R. Forrest, Y.-J. Tung, R. C. Kwong, J. J. Brown, S.
Garon, M. E. Thompson, Appl. Phys. Lett. 2003, 82, 2422.
[8] a) D. H. Lee, J. H. Im, S. U. Son, Y. K. Chung, J.-I. Hong, J. Am.
Chem. Soc. 2003, 125, 7752; b) D. H. Lee, S. Y. Kim, J.-I. Hong,
Angew. Chem. 2004, 116, 4881; Angew. Chem. Int. Ed. 2004, 43,
4777.
[9] In this system, two equivalents of compound 2 were used because
an excess of compound 2 can prevent any possible binding between
the Zn2+–cyclen moiety of compound 1 and the phosphate moiety
of TTP. Although two equivalents of compound 2 and one equiva-
lent of TTPcan form 2:1 binding, the addition of one equivalent of
compound 1 to this solution would cause weak binding between the
Zn2+–DPA unit of compound 2 and the imide unit of TTPto be
easily broken and then replaced with stronger binding between the
Zn2+–cyclen unit of compound 1 and the imide part of TTP. In addi-
tion, the residual compound 2 has little influence on the emission in-
tensity when excited at the donor absorption because of the long
distance between the donor and the acceptor, as shown in the Sup-
porting Information. Therefore, we can expect that the ensemble
system shows 1:1:1 binding mode, as shown in Figure 1.
(s, 1H), 8.52 (d, J
(d, J(H,H)=7.8 Hz, 4H), 7.22 (t, J
(s, 1H), 3.86 (s, 8H), 3.74 (s, 4H), 3.27 (q, J
(H,H)=7.3 Hz, 2H), 1.37 ppm (s, 9H); 13C NMR (125 MHz, CDCl3):
A
N
A
ACHTREUNG
AHCTREUNG
J
ACHTREUNG
d=159.0, 155.9, 154.3, 148.6, 136.3, 129.5, 128.6, 123.8, 122.8, 121.8, 78.3,
59.6, 54.7, 42.0, 35.2, 28.3; MALDI-TOF: m/z calcd for C39H46N7O3
[M+H]+: 660.366; found: 660.588.
Compound 3: Trifluoroacetic acid (2.2 mL) was added slowly to a solu-
tion of 12 (467 mg, 0.71 mmol) in CH2Cl2 (20 mL). After being stirred at
room temperature for 12 h, the solvent was removed in vacuo and the
residue was dissolved in CH2Cl2. The organic phase was washed with
water and neutralized with an aqueous solution of NaHCO3, and dried
over Na2SO4. The solvent was evaporated to give the desired product as
1
a brown sticky liquid (326 mg, 82.3%). H NMR (300 MHz, [D6]acetone):
d=8.52 (d, J
(H,H)=7.8 Hz, 4H), 7.22 (t, J
8H), 3.79 (s, 4H), 3.41 (t, J
U
G
[10] a) W. R. Glomm, S. Volden, J. Sjçblom, M. Lindgren, Chem. Mater.
2005, 17, 5512; b) J. Li, P. I. Djurovich, B. D. Alleyne, M. Yousufud-
din, N. N. Ho, J. C. Thomas, J. C. Peters, R. Bau, M. E. Thompson,
Inorg. Chem. 2005, 44, 1713.
[11] I. R. Laskar, S.-F. Hsu, T.-M. Chen, Polyhedron 2005, 24, 189.
[12] We assume that the mCPmoiety of 1 and the FIrpic moiety of 2 are
located within the effective Dexter energy-transfer distance
J
A
ACHTREUNG
G
A
upon 1:1:1 complexation, as shown in Figure 1.[6]
[13] E. Kimura, S. Aoki, T. Koike, M. Shiro, J. Am. Chem. Soc. 1997,
119, 3068.
Received: June 25, 2008
Published online: September 18, 2008
Chem. Eur. J. 2008, 14, 9613 – 9619
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9619