Synthesis and spectroscopic properties of a FRET pair based on PPO and DBD dyes

Article abstract of DOI:10.1016/j.dyepig.2015.07.022

Abstract A new pair of F?rster resonance energy transfer (FRET) performing fluorophores is introduced: An amine functionalized 2,5-diphenyloxazole (PPO) donor tethered via a flexible linker to a carboxylic acid functionalised ester-[1,3]-dioxolo[4.5-f][1,

Full text of DOI:10.1016/j.dyepig.2015.07.022

Dyes and Pigments 123 (2015) 39e43
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Synthesis and spectroscopic properties of a FRET pair based on PPO
and DBD dyes
*
Robert Wawrzinek, Pablo Wessig
Institut für Chemie, Universit a€ t Potsdam, Karl-Liebknecht-Str. 24e25, D-14476 Potsdam, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 May 2015
Received in revised form
A new pair of F o€ rster resonance energy transfer (FRET) performing fluorophores is introduced: An amine
functionalized 2,5-diphenyloxazole (PPO) donor tethered via a flexible linker to a carboxylic acid func-
tionalised ester-[1,3]-dioxolo[4.5-f][1,3]benzodioxole (ester DBD) acceptor. Synthesis and spectroscopic
properties of this proof-of-concept molecule in acetonitrile are presented and compared to those of its
independent chromophores. The system exhibits a F o€ rster distance of 2.9 nm, high FRET efficiency of 0.88
11 July 2015
Accepted 16 July 2015
Available online 28 July 2015
and a pseudo Stokes shift of 190 nm.
©
2015 Elsevier Ltd. All rights reserved.
Keywords:
FRET
DBD
Pseudo Stokes shift
Microenvironmental probing
1
. Introduction
opportunity to measure the distance between FRET pair mole-
cules. This interaction is possible if donor and acceptor address
certain parameters: the resonance frequency of the donor in
the excited state needs to match the corresponding frequency of
the acceptor in the ground state which is usually the case
when the donor's emission spectrum overlaps with the acceptor's
absorption spectrum. Also crucial for an effective FRET is the
donor's fluorescence quantum yield and lifetime in absence of
the acceptor, the relative alignment of the transition dipole
moments of both chromophores, and of course the distance be-
tween them [8].
Herein we report on a new pair of FRET active chromophores
showing a highly efficient energy transfer combined with a pseudo
Stokes shift of 190 nm in acetonitrile. Fig. 1 shows the chemical
structure of the independent donor and acceptor molecules as well
as the FRET active proof-of-concept molecule 1.
DBD dyes are a relatively new class of small molecules exhib-
iting large Stokes shifts, fluorescence in the VIS region and other
interesting properties, such as high fluorescence lifetime (FLT)
and in some cases extraordinary sensitivity of the latter to their
microenvironment [1,2]. This behaviour has been successfully
exploited in biological applications to show conformational
changes in proteins and to develop an FLT based assay for ace-
tylpolyamine amidohydrolases [3,4].
Such highly sensitive probes provide important information
about their molecular surrounding and are powerful tools for
understanding biological mechanisms. However, the most popu-
lar approach to investigate microenvironmental changes is to
measure exchange of energy between two fluorophores via
F o€ rster resonance energy transfer (FRET) [5e7]. Here an ener-
getically excited donor transfers its energy radiationless via
dipoleedipole interaction to an acceptor molecule. Through this
mechanism it is possible to observe acceptor emission (or donor
emission quenching) despite irradiation in the wavelength region
of donor absorption. Due to its strong distance dependency FRET
systems are often called ‘spectroscopic rulers’ as they provide the
The motivation to design 1 was to find an appropriate donor for
the ester-DBD fluorophore. The latter was chosen because it
shows high fluorescence quantum yields not only in organic sol-
vents but also in water [3]. As we will show 2,5-diphenyloxazole
(PPO) turns out to be an ideal partner for the ester-DBD
acceptor as it can be easily functionalised and matches with the
spectroscopic requirements to function as a donor. Although its
absorption maximum is around 300 nm, it has been shown that
PPO can be excited with two-photon spectroscopy (hence pushing
its theoretical absorption to 600 nm) making it interesting in
*
Corresponding author.
E-mail address: wessig@uni-potsdam.de (P. Wessig).
http://dx.doi.org/10.1016/j.dyepig.2015.07.022
143-7208/© 2015 Elsevier Ltd. All rights reserved.
0

40
R. Wawrzinek, P. Wessig / Dyes and Pigments 123 (2015) 39e43
O
O
O
O
O
O
O
O
O
O
O
O
O
O
N
O
N
O
HN
O
O
O
PPO (donor)
Ester DBD (acceptor)
1
Fig. 1. Chemical structure of used chromophores and the target molecule 1.
biological applications [9,10]. To the best of our knowledge this is
the first time that PPO is used as a donor in an FRET system.
Although this specific system is not soluble in water, the proof-of-
concept, carried out in acetonitrile, is transferable to aqueous
conditions because both chromophores show low solvatochrom-
ism and should behave in a similar manner.
2. Results and discussion
2.1. Synthesis
The synthetic route to target molecule 1 is depicted in Scheme 1.
Commercially available BOC-protected p-aminomethyl benzoic
O
O
O
O
DCM,
Oxalylchlorid,
DMF
O
O
O
O
O
O
O
O
OH
quant.
Cl
O
O
O
O
O
O
2
3
O
O
O
2
H N
O
EDC, HOBt,
DIPEA, DMF
N
H
OH
H
N
H
O
O
N
73%
O
O
4
5
C2Cl6, PPh3,
NEt3, DCM
77%
N
N
O
O
TFA
DCM
H
N
O
H2N
quant.
7
O
6
3
DIPEA,
DCM
76%
O
O
O
O
O
O
O
O
NH
N
O
O
1
Scheme 1. Synthetic route to target molecule 1.

R. Wawrzinek, P. Wessig / Dyes and Pigments 123 (2015) 39e43
41
Fig. 2. Absorption and emission spectra of PPO 7 (dashed lines) and ester-DBD 2 (solid lines) in acetonitrile. The inset shows the overlap integral J used in Eq. (1).
acid 4 was coupled to 2-aminoacetophenone using 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC) and N-hydroxybenzo-
triazole (HOBt) under classic peptide coupling conditions [11].
The following Appel cyclisation to oxazole 6 under mild condi-
tions and subsequently deprotection gave amino PPO 7 in higher
yields compared to previously reported approaches [12,13]. 7 was
then coupled to the acid chloride 3 of ester-DBD 2 [3]. The resulting
target molecule 1 contained the donor and acceptor fluorophores
separated by a flexible non-conjugated amide linker.
This becomes evident in Fig. 3 where absorption and emission
spectra of target molecule 1 are presented. Due to the non-
conjugated linker the absorption spectrum is almost the sum of
the absorptions of the individual chromophores. Moreover,
regardless which excitation wavelength was chosen, emission was
almost exclusively detected at 500 nm, which indicated that
nearly all energy absorbed by the PPO donor was transferred to
the DBD acceptor. The small amount of detected donor emission
(around 370 nm) arises when the orientation of its transition
dipole moment is not matching with the acceptor's. This could be
caused by a restricted conformational flexibility of the linker be-
tween the fluorophores and therefore weakening the FRET. To
confirm our assumption that an FRET takes place we measured the
excitation spectrum depicted in Fig. 4, which clearly shows the
acceptor's strong emission when excited at the donor's absorption
maximum around 300 nm.
2.2. Spectroscopic properties
As mentioned before the reason we selected PPO as a donor and
the ester-DBD as an acceptor is their matching spectral overlap and
the fact that both systems exhibit high fluorescence quantum yields
(
in acetonitrile 0.99 (7) and 0.56 (2), respectively). Fig. 2 shows the
absorption and emission spectra of the independent chromo-
phores. It is notable that the two broad absorption maxima of 2
*
*
(
p
/
p
around 240 nm and n /
p
around 400 nm) are baseline
*
separated at the
p / p absorption maximum of 7 (301 nm), which
sits almost exactly in between. This ensures that donor excitation
will not accidentally excite the acceptor.
Fig. 3. Absorption (blue) and emission (red) spectra of 1 (excitation wavelength:
307 nm) in acetonitrile (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.).
Fig. 4. Excitation spectrum of 1 in acetonitrile.

42
R. Wawrzinek, P. Wessig / Dyes and Pigments 123 (2015) 39e43
Table 1
Spectroscopic properties of 1, 2, 7 and calculated FRET parameters of 1.
[M 1 cm
ꢀ
ꢀ1
]
R
0
[nm]
E
Compound
labs [nm]
lem [nm]
F
F
t
F
[ns]
3
1
2
7
307, 403
403
307
495
495
378
0.74a (0.62)
0.56
0.99
1.54b (15.40)
15.40
1.66
e
2.9
0.88
[3]
4680 (403 nm)
35700 (301 nm)
a
Value includes donor and acceptor emissions. In brackets: only acceptor emission.
Excitation at 254 nm, detection at 353 nm. In brackets: detection at 495 nm.
b
ꢀR
ꢁ
To further characterise the system we determined the fluores-
6
k
T
ðrÞ ¼ 1
(3)
0
cence quantum yield at different excitation wavelengths and its
fluorescence lifetime. The F o€ rster distance R
(e.g. the distance
t
r
D
0
between donor and acceptor in which the FRET efficiency is 0.5)
was derived from the spectral overlap integral J using the nor-
malised spectra of the donor's emission and the acceptor's molar
extinction coefficient plotted against the fourth power of the
wavelength (inset Fig. 2) and used in Eq. (1) [8].
3
. Conclusion
In this work we presented a new pair of FRET active fluo-
rophores containing two small molecules; PPO as an UV absorbing
donor and a green emitting ester-DBD dye acting as an acceptor.
Using mild reaction conditions and peptide coupling techniques
gave an easily accessible proof-of-concept molecule which than
was spectroscopically investigated. Irradiated with light at the
donor's excitation wavelength of 307 nm this molecule emitted
acceptor fluorescence at 495 nm with a pseudo Stokes shift close to
∞
Z
2
9
$F $k
D
5
6
4
R ¼
0
FDðlÞ
3
ðlÞl dl
(1)
A
4
1
28$p $N $n
A
D
0
2
Here
k
is the orientation factor (2/3), n
D
the refractive index of
the used solvent (1.344 for acetonitrile) and
D
F the fluorescence
quantum yield of the donor in absence of an acceptor (0.99). The
integral term represents the mentioned spectral overlap J in which
190 nm and a fluorescence lifetime of 15 ns in acetonitrile. An FRET
efficiency of 0.88 and a F o€ rster distance of 2.90 nm were found. This
new promising FRET pair could be a very useful addition to the DBD
based tool box for microenvironmental probing. We are currently
working on first applications and other FRET systems containing
DBD dyes. Their large Stokes shifts, high fluorescence quantum
yields and long fluorescence lifetimes provide huge advantages
especially in FLT based microscopic and spectroscopic
investigations.
Especially systems where a DBD moiety would function as FRET
donor are of interest because the long FLT of DBD dyes would be
transferred to the acceptor allowing for time gated temporal
discrimination of background emission at a more red-shifted range
of the visual spectrum.
F
D
(
l
) is the donor's normalised fluorescence intensity and
acceptor's molar extinction coefficient. R was determined to be
.90 nm.
The fluorescence quantum yield (
A
3 (l) the
0
2
F
F ) of 1 revealed a remarkable
high value of 0.74 which, however, needed to be corrected as there
was little donor emission detected. Measurements using excitation
at 403 nm gave the fluorescence quantum yield
A
F exclusively
caused by the acceptor (0.62). This value was then subtracted from
the initial measurement to obtain
in presence of the acceptor (0.12).
FDA, the donor's quantum yield
Because this system shows no ‘crosstalk’ (the simultaneous
excitation of donor and acceptor) and F (the donor’s quantum
D
yield in absence of an acceptor) is 0.99, we used Eq. 2 to calculate a
FRET efficiency E of 0.88.
4. Experimental
F
DA
E ¼ 1 ꢀ
(2)
4.1. Spectroscopy
F
D
We have chosen the fluorescence quantum yield to determine E
rather than the often used fluorescence lifetime of the donor. The
reason for that is the marginal influence of the acceptor on the
already short lifetime of the donor (see Table 1), which causes
larger errors due to a poor signal-noise ratio. Furthermore, it would
be only legitimate to use lifetime measurements for a fixed distance
between donor and acceptor and a monoexponential decay which
is not complied by compound 1 due to its flexible linker.
UVevis measurements were performed with a JASCO V-630
spectrophotometer and analysed with Spectra Manager 2 (v
2.08.01). For all measurements square quartz cells (1 ꢁ 1 cm) were
used.
Steady-state fluorescence spectra were measured with a Horiba
Jobin Yvon Fluoromax 4 and analysed with FluorEssence (v 2.5.2.0).
Fluorescence lifetime spectroscopy was performed on a Horiba
Jobin Yvon Single Photon Counting Controller (TCSPC) Fluorohub
with various LASER-diodes (NanoLED-254/372/447) used in com-
bination with the mentioned spectrometer and the Datastation (v
2.5) software. To analyse the spectra DAS6 (v 6.4) was used. Fluo-
rescence quantum yields were measured with a Hamamatsu Pho-
tonic Multi-Channel Analyzer C10027.
At this point it is important to note that an efficiency of
greater than 0.99 was expected because the distance between
the fluorophores' centres could be maximal 1.3 nm (found by
computational modelling). This means the in Eq. (1) used
2
assumption of
[
k
¼ 2/3 for that particular molecule 1 is not correct
14]. However, it is legitimate to determine R
0
to generally
describe a donoreacceptor system under the assumption of fully
randomised orientation [15]. Furthermore, compound 1 falls into
the category of FRET systems in which donor and acceptor
are considered to be “too close” to each other (r > 2 nm) causing
deviation from the ideal dipole approximation (IDA) [16]. As
a consequence it is not valid for that particular system 1 to
calculate parameters based on Eq. (1) such as the transition rate
4.2. Syntheses
4.2.1. Diethyl 2-(2-oxo-2-((4-(5-phenyloxazole-2-yl)benzyl)amino)
ethyl)benzo-[1,2-d:4,5-d ]bis([1,3]dioxol)4,8-dicarboxylate (1)
Acid chloride 3 (53 mg, 0.136 mmol) was dissolved in 10 mL dry
dichloromethane and cooled down to 0 C. Amine 7 (68 mg,
0
ꢂ
0.271 mmol) and N,N-diisopropylethylamine (23
mL, 0.136 mmol)
T
k (r) (Eq. (3)).
were added and the mixture was stirred over night at room

R. Wawrzinek, P. Wessig / Dyes and Pigments 123 (2015) 39e43
43
temperature. After adding 50 mL dichloromethane the organic
layer was separated and washed with saturated NaHCO solution
and brine. The separated organic layers were dried over MgSO , and
the solvent evaporated in vacuo. The residue was purified using
flash column chromatography (dichloromethane/methanol 25:1)
16.21 mmol), NEt
3
(2.25 mL, 16.21 mmol) and amide 5 (905 mg,
3
2.46 mmol, dissolved in 30 ml dry dichloromethane) were added to
the solution. After stirring for 30 min 10 mL dry dichloromethane
was added and again stirred for 2 h at room temperature. After that
4
period of time the mixture was washed with saturated NH
subsequently saturated NaHCO solution.
The separated organic layers were dried over MgSO
evaporated in vacuo and the residue purified using flash column
chromatography (hexane/ethyl acetate 3:1 (þ10% CHCl )) to obtain
660 mg of PPO derivate 6 (1.88 mmol, 77%) as a bright yellow solid.
4
Cl and
to obtain 30 mg of compound 1 (50 mol, 37%), and starting ma-
terial 2 (25 mg, 67 mol, 50%).
6 5
/ppm, DMSO-d /pyridine-d , 300 MHz): 1.21 (t,
m
3
m
4
, the solvent
1
H NMR (
d
3
3
3
J ¼ 7.11, 6H, eCH
3
); 3.19 (d, J ¼ 5.30, 2H, eCH
2
); 4.55 (d, J ¼ 5.90,
3
3
2
7
H, eCH
2
); 4.60 (d, J ¼ 5.96, 4H, eCH
2
); 6.16 (s, 2H, eCH );
2
ꢂ
1
.30e8.20 (10H, eCH). Mp: 239 C. TLC: dichloromethane/meth-
H NMR (
2H, eCH ); 7.30e7.48 (6H, eCH); 7.65e8.10 (4H, eCH). C NMR (
ppm, CDCl , 75 MHz): 28.4 (3C); 44.4 (1C); 79.7 (1C); 123.2 (1C);
3 3
d/ppm, CDCl , 300 MHz): 1.47 (s, 9H, eCH ); 4.36 (s,
þ
13
anol (100:4) R
6
1
f
¼ 0.31. MS: (ESI) m/z ¼ 601.1814 [M þ H] , calc.:
2
d/
01.1822. IR: 3274, 2923, 1723, 1638, 1551, 1496, 1453, 1416, 1294,
3
ꢀ
1
174, 1081, 1023, 955, 759 cm
.
124.18e128.9 (10C); 141.5 (1C); 151.2 (1C); 155.9 (1C); 160.9 (1C).
ꢂ
Mp: 121 C. TLC: hexane/ethyl acetate 3:1 R
f
¼ 0.2. MS: (EI) m/
þ
4.2.2. Compound 2
z ¼ 350.1642 [M ], calc.: 350.1630. IR: 3341, 2977, 2927, 1696, 1497,
ꢀ
1
Compound 2 was synthesised according to the literature [3].
1366, 1271, 1251, 1169, 1135, 953, 866, 829, 762, 689, 487 cm .
0
4
.2.3. Diethyl 2-(2-chloro-2-oxoethyl)benzo[1,2-d:4,5-d ]bis([1,3]
4
.2.6. (4-(5-Phenyloxazole-2-yl)phenyl)methanamine (7)
dioxole)-4,8-dicarboxylate (3)
632 mg of compound 6 (1.8 mmol) was dissolved in 12 mL dry
Compound 2 (50 mg, 0.137 mmol) was dissolved in 5 mL dry
dichloromethane and oxalylchloride (82 mL, 0.95 mmol) as well as
one drop dry DMF were added. After stirring for 4 h the solvents
were carefully evaporated in vacuo (max. 50 mbar) to obtain acid
chloride 3, which was immediately used without any further
purification.
dichloromethane, 2 mL trifluoroacetic acid (TFA) was added and the
mixture stirred for 2.5 h at room temperature. The solvents and
remaining TFA were evaporated in vacuo to obtain 450 mg of amine
7
(1.8 mmol, quant.) which was immediately used without further
purification [13].
4.2.4. tert-Butyl (4-((2-oxo-2-phenylethyl)carbamoyl)benzyl)
Acknowledgements
carbamate (5)
Carboxylic acid
4
(1.2 g, 4.78 mmol) was dissolved in
We thank Kathlen Brennenstuhl for the fluorescence quantum
yield measurements and Prof. Michael U. Kumke for inspiring
discussions.
4
0 mL dry DMF and 1-ethyl-3-(3-dimethylaminopropyl)carbo-
diimide$HCl (916 mg, 4.78 mmol), N,N-diisopropylethylamine
(
(
1
mL, 5.73 mmol) as well as N-hydroxybenzotriazole
645 mg, 4.78 mmol) were added. After 30 min additional 676
m
L
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N,N-diisopropylethylamine (3.82 mmol) and subsequently 2-
aminoacetophenone$HCl (682 mg, 4.78 mmol) were added and
the mixture was stirred over night at room temperature. After
adding 60 mL ethyl acetate and washing with 50 mL saturated
[
[
[
[
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); 7.30e8.08 (9H, eCH); 7.59
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[
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6
648, 1534, 1503, 1450, 1392, 1365, 1251, 1225, 1168, 1002, 866, 751,
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.2.5. tert-Butyl (4-(5-phenyloxoazole-2-yl)benzyl)carbamate (6)
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.14 g PPh
3
(8.11 mmol) was dissolved in 25 mL dry dichloro-
[
ꢂ
methane and cooled to ꢀ10 C. Hexachloroethane (3.84 g,