A fluorescence sensor for detection of geranyl pyrophosphate by the chemo-ensemble method

Article abstract of DOI:10.1021/jo802173b

Hexaamide receptor 4 and coumarin phosphate 5 form an ensemble for effective sensing of geranyl pyrophosphate (GPP). Fluorescence resonance energy transfer in the 4 + 5 ensemble diminishes when 5 is replaced by GPP. Receptor 4 binds selectively with GPP o

Full text of DOI:10.1021/jo802173b

the design of effective receptors. For selective binding with
phosphate and pyrophosphate ions, many synthetic receptors
contain polyaza, quanidinium, imidazolium, urea, thiourea, and
amide groups in appropriate dispositions to exert the desired
electrostatic interactions and hydrogen bondings.1,2 Alterna-
tively, the metal-ligand interaction is widely applied to
recognize the phosphate groups.1,3
A Fluorescence Sensor for Detection of Geranyl
Pyrophosphate by the Chemo-Ensemble Method
Kuan-Hung Chen, Jen-Hai Liao, Hsin-Yu Chan, and
Jim-Min Fang*
Department of Chemistry, National Taiwan UniVersity,
Taipei, 106, Taiwan
The methods for effective sensing of biologically important
organic phosphate molecules are in embryonic development,
and most studies focus on the design of optical chemosensors
for detection of nucleotides,5-7 such as adenosine triphosphate
(ATP). A nucleotide chemosensor often incorporates a moiety
to bind with the nitrogenous base in the specific nucleotide, in
addition to the moiety for binding with mono-, di-, or triphos-
phates. Crick-Watson pairs6 and (hetero)aromatic hydrocar-
bons7 are often designed to provide multiple hydrogen bonding,
hydrophobic, and π-π interactions with nitrogenous bases.
Furthermore, dinuclear zinc-dipicolylamine complexes have
been employed as the effective sensors of adenosine mono-
phosphate (AMP) and uridine diphosphate (UDP) in enzyme
activity assays.8 Several research groups have also explored the
optical chemosensors for inositol-1,4,5-trisphosphate,9 lyso-
phosphatidic acid (LPA),10 2,3-bisphosphoglycerate,11 and phos-
phatidylserine on membranes.12 However, the optical method
for sensing isoprenyl pyrophosphates has not yet been reported.
jmfang@ntu.edu.tw
ReceiVed October 09, 2008
Isoprenyl pyrophosphates are known to involve in the
regulation of cell growth and division.13 Consecutive condensa-
Hexaamide receptor 4 and coumarin phosphate 5 form an
ensemble for effective sensing of geranyl pyrophosphate
(GPP). Fluorescence resonance energy transfer in the 4 + 5
ensemble diminishes when 5 is replaced by GPP. Receptor
4 binds selectively with GPP over other anions, including
fatty acids. The 4-GPP complex in 1:1 stoichiometry is
inferred by NMR and fluorescence titrations. Receptor 4
contains a pseudotetrahedral cleft to accommodate GPP via
multiple hydrogen bonds, and the two aliphatic chains exert
additional hydrophobic interactions.
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10.1021/jo802173b CCC: $40.75
Published on Web 12/02/2008
2009 American Chemical Society
J. Org. Chem. 2009, 74, 895–898 895

tion of isoprenyl pyrophosphates produces geranyl pyrophos-
phate (GPP), farnesyl pyrophosphate, and many other important
derivatives. The common methods for determination of isoprenyl
pyrophosphates include HPLC, GC-MS, and capillary elec-
trophoresis, which often require prior treatments with enzyme,
acid, or radioisotope.14-18 It is demanding to explore an effective
method for the sensing of isoprenyl pyrophosphates. To
demonstrate this idea, we utilize a hexaamide receptor 4 (R1 )
C11H23, R2 ) pyrenyl) and an indicator of coumarin phosphate
5 to build up an ensemble system for the sensing of isoprenyl
pyrophosphate GPP (6). The binding behavior of hexaamide
molecules 1 (R1 ) R2 ) H), 2 (R1 ) R2 ) pyrenyl), and 3 (R1
) R2 ) C11H23) was also examined for comparison.
We have previously reported that molecule 2 having the
scaffold of pyridine 2,6-bis-carboxamide with four pyrene-
annexed amido groups provides a preorganized cleft to hold
the incoming phosphate and pyrophosphate ions via multiple
hydrogen bondings.2a,b The detailed structural analysis of
molecule 2 indicates that the pyrene rings stack in two pairs
(R1/R1 and R2/R2) by π-π interactions to give a significant
excimer emission (λmax ) 477 nm). When receptor 2 binds with
phosphate or pyrophosphate ions, in 1:1 stoichiometry, ratio-
metric changes of the monomer and excimer emissions occur
in response to the segregation of pyrene rings. We conceived
that hexaamide 4 having a similar structure to 2 would also
bind effectively with the pyrophosphate group via multiple
hydrogen bondings. Furthermore, the pair of aliphatic chains
(R1 ) C11H23) in 4 would provide additional hydrophobic
interactions with isoprenyl pyrophosphates. The binding event
would be readily detected by the fluorescence change of the
pyrene rings in 4.
FIGURE 1. 1H NMR titration of receptor 4 (2.4 × 10-3 M) with GPP
(0.3 M as the tetrabutylammonium salt) in DMSO-d6 solution.
TABLE 1. Association Constants Ka (M-1) in DMSO-d6 Solutions
a
1
As Determined by the H NMR Titrations
-
receptor
H2PO4
DHP
GPP
NDb
1280 ( 500
2500 ( 240
3300 ( 270
1
2
3
4
950 ( 140
1370 ( 200
960 ( 140
880 ( 100
150 ( 20
780 ( 120
840
1830 ( 150
a Individual receptor in DMSO-d6 solution (2.4 × 10-3 M) was
titrated with incremental additions of respective substrate in DMSO-d6
solution (0.3 M). b Not determined.
pyridinecarboxamides > pyrenyl amides > aliphatic amides.
This result was consistent with incorporation of GPP inside the
cleft of receptor 4 as a 1:1 complex. According to the nonlinear
least-squares method,19 the association constants (Ka) were
determined by using the chemical-shift changes of N2,N5-H in
an individual receptor as a function of the concentration of
phosphate substrates based on a 1:1 stoichiometry of complex-
ation (Table 1).
In comparison, receptor 2 exhibited the strongest binding with
phosphate ion, presumably due to the preorganization effect
rendered by stacking its pyrene rings in pairs.2b,d On the other
hand, receptor 4 showed the highest affinity in binding with
aliphatic phosphates DHP and GPP. This result might be
attributable to the synergistic effect of preorganization by a pair
of pyrene rings and the hydrophobic interactions exerted by the
long hydrocarbon chains. Even though receptor 3 has four
aliphatic chains to render strong hydrophobic interactions, it
lacks pyrene rings for structural preorganization, and thus shows
lower affinity with GPP and DHP than receptor 4. Receptor 1
with methylamido groups at the termini only had weak binding
with DHP due to lacking effective hydrophobic interactions.
-
The binding affinity of receptors 1-4 with H2PO4 , di(hexa-
1
decyl) phosphate (DHP), and GPP was first evaluated by H
NMR titrations in DMSO-d6 solutions. Upon complexation with
phosphate substrates, the amido protons in the receptor mol-
ecules showed substantial changes of the chemical shifts. For
example, addition of GPP (1.375 equiv) to receptor 4 (Figure
1) caused significant downfield shifts of N2,N5-H (∆δ ) 1.1
ppm), N3,N6-H (∆δ ) 0.6 ppm), and N4,N7-H (∆δ ) 0.4 ppm).
The chemical-shift changes might result from the interactions
of the corresponding protons with GPP in different degrees, i.e.,
(14) (a) Bruenger, E.; Rilling, H. C. Anal. Biochem. 1988, 173, 321. (b)
McCaskill, D.; Croteau, R. Anal. Biochem. 1993, 215, 142–149.
(15) Fisher, A. J.; Rosenstiel, T. N.; Shirk, M. C.; Fall, R. Anal. Biochem.
2001, 292, 272–279.
(16) Anderson, P. E.; Pfeffer, W. D.; Blomberg, L. G. J. Chromatogr. A
1995, 699, 323–330.
(17) (a) Zhang, D.; Poulter, C. D. Anal. Biochem. 1993, 213, 356–361. (b)
Saisho, Y.; Morimoto, A.; Umeda, T. Anal. Biochem. 1997, 252, 89–95.
(18) Song, L. Anal. Biochem. 2003, 317, 180–185.
(19) Connors, K. A. Binding Constants-The Measurement of Molecular
Complex Stability; John Wiley: New York, 1987.
896 J. Org. Chem. Vol. 74, No. 2, 2009

The photophysical properties of 4 were investigated. Upon
excitation at 328 nm, receptor 4 showed the monomer emission
at λmax ) 377 and 397 nm, as well as the excimer emission at
λmax ) 477 nm. As the intensity ratio of monomer-to-excimer
emissions remained constant in various concentrations (1.25 ×
10-6 to 10-5 M), the excimer emission should occur via
intramolecular interactions of pyrenes, but not intermolecularly
in this concentration range. Though receptor 4 had good binding
affinity with GPP as shown by the NMR titration experiments,
the binding event did not induce appreciable fluorescence change
as we expected. A possible explanation is that receptor 4 has
only one pair of pyrenes, which may still remain in proximity
in the 4-GPP complex presumably by assistance of the
interactions of aliphatic chains.
We thus designed a chemosensing ensemble11,20 using
fluorescence resonance energy transfer (FRET)21 for an effective
detection of the binding event. There are several criteria for
choosing the appropriate indicator in an ensemble system.22
After we surveyed several possible fluorogenic indicators, we
chose coumarin phosphate 5 as a FRET acceptor of the
monomer emission of pyrenes to establish the ensemble for
displacement assay. By 1H NMR titration, the association
constant between receptor 4 and phosphate 5 was deduced to
be 1100 ( 207 M-1 in DMSO-d6 solution. Thus, the ensemble
of 4 + 5 was reasonably stable, but coumarin 5 would be readily
replaced by GPP to form a stronger 4-GPP complex, Ka )
3300 ( 535 M-1 in DMSO-d6 solution.
In a typical fluorescence titration experiment, a DMSO
solution of phosphate 5 was added in incremental portions to
receptor 4 (5 × 10-6 M in DMSO). The fluorescence spectra
showed consistent decreases in the monomer emission of
pyrenes (λmax ) 395 nm) along with concurrent increases of
the coumarin emission at λmax ) 470 nm (Figure 2), supporting
a FRET process upon their complexation.23 When GPP was
added to the 4 + 5 ensemble (1:1 complex in DMSO solution),
the monomer emission of pyrenes was recovered at the expense
of the 470-nm emission, indicating that the indicator 5 was
displaced by GPP in the binding cavity of receptor 4. On the
basis of the emission change at 395 nm, the association constant
of the 4-GPP complex was deduced to be 2.3 ( 0.8 × 104
M-1 in DMSO by a competitive spectrophotometric method.19
The 4 + 5 ensemble was similarly determined to have a Ka
value of 4.1 ( 0.4 × 103 M-1 in DMSO. In comparison, the 2
+ 5 ensemble (Ka ) 3.3 ( 0.7 × 104 M-1) was less sensitive
to GPP due to its lower binding affinity to receptor 2.
FIGURE 2. (a) Fluorescence titration of 4 (5 × 10-6 M in DMSO)
with incremental addition of 5 (up to 5 equiv, 1 × 10-3 M solution in
DMSO). (b) Fluorescence titration of the ensemble 4 + 5 (1:1 complex,
5 × 10-6 M in DMSO) with incremental addition of GPP (up to 3
equiv, 1 × 10-3 M solution in DMSO). The excitation wavelength was
328 nm
FIGURE 3. Fluorescence calibration curves at 395 nm for the 4 + 5
ensemble (1:1 complex, 5 × 10-6 M) upon addition of various anions
-
In comparison with GPP, the response of phosphate and
pyrophosphate ions to the 4 + 5 ensemble was relatively low
(Figure 3). Other examined anions, including F-, Cl-, Br-,
SCN-, AcO-, NO3-, ClO4-, and the carboxylate ions of fatty
acids (C10H21CO2H, and C11H23CO2H), were insensitive to the
4 + 5 ensemble. Thus, the high binding affinity of 4-GPP was
attributable not only to the hydrophobic interactions of the
aliphatic chains but also to the multiple hydrogen bondings of
the tetrahedral pyrophosphate in the hexaamide cleft.
in DMSO solution (up to 3 equiv). Other anions: CH3CO2
,
C10H21CO2-, C11H23CO2-, F-, Cl-, Br-, SCN-, NO3-, and ClO4
.
-
In summary, we have designed a chemo-ensemble system
using FRET as the sensing mechanism to detect isoprenoid
pyrophosphates, e.g., GPP in this study. The hexaamide
molecule 4 has a well-defined structure with pseudotetrahedral
cleft to accommodate the pyrophosphate. Receptor 4 also
incorporates two long hydrocarbon chains to enhance the
interactions with the aliphatic moiety of GPP. The coumarin
phosphate 5 is selected as an appropriate fluorescence indicator
because it binds modestly with the receptor 4 to render a FRET
from the pyrene rings. Thus, the present chemo-ensemble system
provides an efficient, selective, and sensitive method for
detection of GPP. This method is potentially useful for selective
sensing of other biologically important aliphatic phosphates and
pyrophosphates.
(20) Wiskur, S. L.; Ait-Haddou, H.; Lavigne, J. J.; Anslyn, E. V. Acc. Chem.
Res. 2001, 34, 963–972.
(21) (a) Wu, P.; Brand, L. Anal. Biochem. 1994, 218, 1–13. (b) Li, I. T.;
Pham, E.; Truong, K. Biotechnol. Lett. 2006, 28, 1971–1982.
(22) Fabbrizzi, L.; Marcotte, N.; Stomeo, F.; Taglietti, A. Angew. Chem.,
Int. Ed. 2002, 41, 3811–3814.
(23) In comparison with the 4 + 5 ensemble, coumarin phosphate 5 showed
much less increase of fluorescence intensity (λmax ) 470 nm) by excitation at
328 or 340 nm (see Figure S13 in the SI). The excess increment of fluorescence
intensity in the ensemble system was attributable to the FRET effect.
J. Org. Chem. Vol. 74, No. 2, 2009 897

were monitored as a function of the substrate concentrations.
Nonlinear regression analyses were used to determine the binding
constants.19
UV-Vis Spectral Measurement. The stock solutions of receptor
4 in anhydrous THF at various concentrations were prepared. Their
corresponding UV-vis spectra were recorded at 298 K in a quartz
cell (1 cm width).
Fluorescent Titration for the Binding Studies. A solution of
receptor was prepared, e.g., 5× 10-6 M of receptor 4 in DMSO
(99%), and an aliquot (2 mL) was transferred to a 1-cm fluorescence
tube. To the receptor solution was added a stock solution of
substrate, e.g., coumarin phosphate 5 in DMSO (1 × 10-3 M), by
small portions in an incremental fashion. The fluorescence spectra
were recorded by excitation at the maximum absorption (328 nm).
The relative fluorescence changes were recorded as a function of
substrate concentrations. On the basis of 1:1 stoichiometry of the
complex, the binding constant was calculated according to the
following equation, and determined by the nonlinear least-squares
curve-fitting method.21
Experiment Section
N,N′-Bis{[3-(pyren-1-yl)methylcarbamoyl-5-dodecylcarbam-
oyl]benzyl}pyridine-2,6-dicarboxamide (4). By a procedure simi-
lar to that described previously,2a,b compound 4 was synthesized
in a straightforward manner. In brief, 5-(azidomethyl)isophthalic
acid was activated to its dipentafluorophenyl ester and subjected
to coupling reaction with a mixture of (1-pyrenyl)methylamine and
n-dodecylamine to give the corresponding diamide. The azido group
was reduced to amine, and the subsequent coupling reaction with
2,6-pyridinedicaboxyl dichloride afforded the desired hexaamide
receptor 4.
White solid, mp 204-206 °C; TLC (CH2Cl2/CH3OH, 95:5) Rf
1
0.8; H NMR (DMSO-d6, 400 MHz) δ 9.92 (2 H, t, J ) 5.9 Hz),
9.32 (2 H, t, J ) 5.2 Hz), 8.52 (2 H, t, J ) 5.7 Hz), 8.47 (2 H, d,
J ) 9.2 Hz), 8.27-8.02 (23 H, m), 7.91 (2 H, s), 5.21 (4 H, d, J
) 5.2 Hz), 4.67 (4 H, d, J ) 5.6 Hz), 3.19-3.17 (4 H, m), 1.44 (4
H, m), 1.18-1.15 (36 H, m), 0.80 (6 H, t, J ) 6.8 Hz); 13C NMR
(DMSO-d6, 100 MHz) δ 166.3, 166.2, 164.0, 149.0, 140.3, 135.7,
135.1, 133.2, 131.2, 130.7, 130.6, 129.1, 129.0, 128.6, 128.0, 127.8,
127.5, 127.2, 126.7, 125.7, 125.6, 125.2, 125.1, 124.5, 124.4, 123.7,
42.7, 41.6, 31.7, 29.5, 29.4, 29.2, 29.1, 26.9, 22.5, 14.4; HRMS
calcd for C83H92N7O6 1282.7031, found m/z 1282.7043 [M + H]+.
y ) f + [(d - f)/(2c)]{K-1+c + x - [(K-1+c + x)2-4cx)]0.5
}
where c is the receptor concentration; d is the maximum change of
fluorescence intensity at saturation; f is the initial fluorescence
intensity; K is the association constant; x is the substrate concentra-
tion; and y is the fluorescence intensity.
4-[(7-Diethylaminocoumarin-3-carbonyl)amino]butyl Phos-
phate (5). The coupling reaction of 7-diethylaminocoumarin-3-
carboxylic acid with 4-aminobutanol was performed in the presence
of HBTU and diisopropylethylamine. Treatment with di-tert-butyl
N,N-diisopropyl phosphoramidite in the presence of 1H-tetrazole,
followed by oxidation with t-BuOOH, gave the phosphorylation
product (57% yield). After removal of the tert-butyl groups by TFA,
the desired phosphate 5 was obtained in 96% yield.
Job’s Plot for Determination of the Stoichiometry of
Complexes.19 The stock solutions of receptor compound (e.g.,
receptor 4, 1 × 10-5 M) and the examined substrate [e.g., GPP, 1
× 10-5 M] in anhydrous DMSO were prepared in separate
volumetric flasks. Nine sample solutions containing both receptor
and substrate in different ratios (1/1 to 9/1) were prepared to
maintain a total volume of 2.0 mL. The mixture was shaken well,
and the corresponding fluorescence emission curves were recorded.
A plot according to F - F0(1 - X) versus X was made, where F
is the maximum change of fluorescence intensity at saturation, F0
is the initial fluorescence intensity, and X is the mole fraction of
receptor.
Yellow solid, mp 190-192 °C; 1H NMR (DMSO-d6, 400 MHz)
δ 8.66-8.63 (2 H, m), 7.66 (1 H, d, J ) 8.9 Hz), 6.78 (1 H, d, J
) 8.9 Hz), 6.59 (1 H, s), 3.83-3.80 (2 H, m), 3.49-3.44 (4 H,
m), 3.31-3.30 (2 H, m), 1.56 (4 H, br s), 1.12 (6 H, t, J ) 6.9
Hz); 13C NMR (DMSO-d6, 100 MHz) δ 162.1, 161.8, 157.2, 152.4,
147.6, 131.5, 110.1, 109.5, 107.7, 95.8, 64.8 (d, J ) 5.1 Hz), 44.3
(2×), 38.4, 27.5 (d, J ) 6.8 Hz), 25.6, 12.3 (2×); 31P NMR (DMSO-
d6, 162 MHz) δ 17.9; HRMS calcd for C18H24N2O7P 411.1399,
found m/z 411.1387 [M - H]-.
Acknowledgment. We thank the National Science Council
for financial support.
1H NMR Titration for the Binding Studies. A solution of
receptor was prepared, e.g., 2.4 × 10-3 M of receptor 4 in DMSO-
d6 (99%), and an aliquot (0.5 mL) was transferred to a 5-mm NMR
tube. To the receptor solution was added a stock solution of
substrate, e.g., GPP in DMSO-d6 (0.3 M), by small portions in an
incremental fashion. The chemical shifts of N2,N5-H in the receptor
Supporting Information Available: Synthetic procedures,
1
compound characterization, H NMR titrations, fluorescence
titrations, absorption spectra, and NMR spectra. This material
is available free of charge via the Internet at http://pubs.acs.org.
JO802173B
898 J. Org. Chem. Vol. 74, No. 2, 2009