A new robust and highly sensitive FRET donor-acceptor pair: Synthesis, characterization, and application in a thrombin assay

Article abstract of DOI:10.1002/hlca.200690275

The synthesis of a new, robust fluorescence-resonance-energy-transfer (FRET) system is described. Its donor chromophore is derived from an N-allyl-substituted quinolinone attached to 4-bromophenyl-alanine via Heck cross-coupling. The resulting Fmoc-protected derivative 11 was used as building block in solid-phase peptide synthesis (SPPS). As FRET acceptor, a sulfonylated ruthenium(II)-bathophenanthroline complex with a peripheral COOH function was prepared for covalent attachment to target molecules. The UV/VIS absorption and emission spectra of peptides bearing only the donor (D) or acceptor (A) dye showed a good overlap of the emission band of the donor with the absorption band of the acceptor. The fluorescence spectra of a peptide bearing both dyes revealed an additional emission after excitation of the donor, which is due to indirect excitation of the acceptor via FRET. The long fluorescence lifetime of the Ru^II complex (0.53 μs) makes it well-suited for time-resolved measurements. As a first application of this new FRET system, the peptide 18, with the recognition sequence for the protease thrombin, flanked by the two dyes, was synthesized and successfully cleaved by the enzyme. The change in the ratio of the fluorescence intensities could be determined.

Full text of DOI:10.1002/hlca.200690275

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A New Robust and Highly Sensitive FRET Donor–Acceptor Pair: Synthesis,
Characterization,and Application in a Thrombin Assay
by Eva K. Kainmüller and Willi Bannwarth*
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität Freiburg, Albertstr. 21,
D-79104 Freiburg
(phone: +49-761-2036073; fax: +49-761-2038705; e-mail: willi.bannwarth@organik.chemie.uni-
freiburg.de)
The synthesis of a new, robust fluorescence-resonance-energy-transfer (FRET) system is described.
Its donor chromophore is derived from an N-allyl-substituted quinolinone attached to 4-bromophenyl-
alanine via Heck cross-coupling. The resulting Fmoc-protected derivative 11 was used as building
block in solid-phase peptide synthesis (SPPS). As FRET acceptor, a sulfonylated ruthenium(II)–batho-
phenanthroline complex with a peripheral COOH function was prepared for covalent attachment to tar-
get molecules. The UV/VIS absorption and emission spectra of peptides bearing only the donor (D) or
acceptor (A) dye showed a good overlap of the emission band of the donor with the absorption band of
the acceptor. The fluorescence spectra of a peptide bearing both dyes revealed an additional emission
after excitation of the donor, which is due to indirect excitation of the acceptor via FRET. The long fluo-
rescence lifetime of the RuII complex (0.53 ms) makes it well-suited for time-resolved measurements. As a
first application of this new FRET system, the peptide 18, with the recognition sequence for the protease
thrombin, flanked by the two dyes, was synthesized and successfully cleaved by the enzyme. The change
in the ratio of the fluorescence intensities could be determined.
1. Introduction. – A very important task in molecular biology is the elucidation of
reaction pathways and mechanisms involved in living cells and whole organisms.
Research on this topic not only gives fascinating insight into the fundamental question
of how life works, but also has a great impact in medicine and pharmacology. This is due
to the fact that a multitude of biologically relevant molecules involved in these proc-
esses have been identified as potential drug targets against various diseases.
As analyses of these kinds of mechanisms and the identification of the molecules
involved therein are difficult, the development of new methods is an ongoing task. In
this context, fluorescence-resonance-energy-transfer (FRET) systems have proven to
be important tools for the elucidation of distance-dependent interactions on the molec-
ular level. With these systems, changes in distances on a nanometer scale and in real-
time mode can be monitored, and are, therefore, especially well-suited for the charac-
terization of biochemical events both in vitro and in vivo. Among these have been the
binding of ligands to proteins as well as their subsequent conformational changes
[1–5], DNA–protein complexation [6–10], and RNA folding and catalysis [11–13].
Another subject of current interest is the study of interactions between three different
biomolecules [14], a task that is impossible to accomplish by any other biochemical
method at present. Apart from this, FRET has also been applied in diagnostics and
ꢀ 2006 Verlag Helvetica Chimica Acta AG, Zürich

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drug research, where tools like molecular beacons [15], TaqMan probes [16], or fluo-
rescent enzyme substrates [17–22] use the same principle.
The FRET technique is based on the transfer of fluorescence energy between a
donor (D) fluorophore and an acceptor (A) fluorophore. The dyes must be chosen in
a way such that the emission wavelength of the donor overlaps with the absorption
wavelength of the acceptor, thereby enabling excitation energy to be transferred
from the donor to the acceptor. The intensity of FRET depends on the quality of the
spectral overlap of the dyes, their extinction coefficients, and their orientation to and
distance from each other. According to Fçrsterꢁs equation [23], FRET decreases with
rꢀ6, when r is the distance between the two chromophores. For the detection of a mean-
ingful signal, r should be in the range of 10–100 Š.
An extension of this principle is represented in ternary systems comprising a donor,
an acceptor I, and an acceptor II. Energy transfer is possible to occur between the first
and the second pair of chromophores, provided that their optical properties match the
requirements described above. In this way, the measurement of two different distances
can be achieved.
Another feature of a FRET system is the possibility to detect signal changes in a
ratio-dependent way, i.e., to monitor both the decrease in the emission of the donor
and the increase in the emission of the acceptor, or vice versa. This enhances the sen-
sitivity of the method in comparison to probes bearing only one reporter dye, and it is
especially interesting when dealing with highly diluted samples. A number of different
donor–acceptor pairs have been reported and successfully employed [24–26], whereas
only very few ternary systems have been developed up to now [27][28]. In any case, the
need for even more sensitive probes remains. This problem could be overcome by using
chromophores that can be studied in a time-resolved mode. Among these, lanthanide-,
especially Eu and Tb complexes, have been reported to be suitable candidates due to
their strong fluorescence and excited-state lifetimes of up to milliseconds [29][30].
Usually, they are employed as caged chelates [31][32], but leakage of Eu can still
occur. Furthermore, their very long fluorescence lifetimes make it impossible to resolve
events faster than those in the range of milliseconds.
We, therefore, focused on RuII charge-transfer (CT) complexes as alternative
FRET acceptors. These substances not only exhibit high thermodynamic stabilities,
they are chemically inert, can be readily prepared, and exhibit excited states with a
decay time in the microsecond range that allows for time-resolved measurements
[33]. In a preliminary communication [34], we have described the use of the rutheniu-
m(II)–bathophenanthroline complex 1 as a FRETacceptor in combination with the car-
bostyril derivative 2 as donor [34].
Herein, we report the details of the syntheses of the two dyes, their physical proper-
ties, their insertion into peptide substrates, and substrate optimization for a thrombin
assay. Our final aim is to extend this binary FRET system into the first part of a ternary
system.
2. Results and Discussion. – 2.1. Synthesis of the Acceptor. The Ru complex 1 was
synthesized according to Scheme 1 by adaptation of a simple known two-step procedure
starting from RuCl3 ·H2O [35][36]. In the first step, two ligands based on bathophenan-
throlinedisulfonic acid disodium salt (bpds) were introduced to enhance solubility in

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aqueous media, affording [Ru(bpds)2Cl2] (3). Next, complexation with 4 was carried
out, which gave 1 in 80% overall yield over both steps. Complex 1 is equipped with a
COOH function allowing for covalent attachment (via an amide bond) to target mol-
ecules bearing an amino group. Activation of the COOH group of 1 was performed
with O-(N-succinimidyl)-N,N,N’,N’-tetramethyluroniumtetrafluoroborate (TSTU),
which yielded the succinyl-activated ester 5 in 73% yield [37].
The RuII complex 1 exhibits a characteristic metal-to-ligand CT absorption band at
440–464 nm, and a strong red emission at 618 nm. It is completely stable, even under
strongly acidic and basic conditions, as corroborated by exposure to 95% CF3COOH
(TFA) or 2N aq. NaOH, which resulted in no decomposition at all. Of the two different
ligands in 1, sulfonylated bathophenanthroline (bpds) is commercially available, and
compound 4 was synthesized according to Scheme 2 [38].
The key steps of the synthesis of 4 were two Skraup reactions: in the first one, 2-
nitroaniline and b-chloropropiophenone (=3-chloro-1-phenylpropan-1-one) were
reacted to 6 in 83% yield, followed by hydrogenation to afford the intermediate 7 in
77% yield. Next, 7 was combined with the Friedel–Crafts product 8, obtained from 9,
which afforded a mixture of 4 (46%) and 10 (27%). The pure ligand 4 was then
obtained in 72% yield by saponification.
2.2. Synthesis of the Donor. The carbostyril derivative 2 was envisaged as donor
entity due to its chemical and spectroscopic properties. Being a lactam, it is chemically
more stable than the corresponding lactones (coumarins). With an absorption maxi-
mum at 368 nm and an emission ꢀm1 aximum at 435 nm, in combination with a high
extinction coefficient (e=20800M cmꢀ1), compound 2 is well-suited as a FRET
donor [39]. For its use in solid-phase peptide synthesis (SPPS), the Fmoc-protected1)
unnatural amino acid 11, containing the donor as a pendant arm, was synthesized
1
)
Abbreviations: Fmoc, Boc, and Su refer to [(9H-fluoren-9-yl)methoxy]carbonyl, (tert-butoxy)car-
bonyl, and succinimide residues, resp.

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Scheme 1. Synthesis of the Activated Ruthenium(II)–Bathophenanthroline Complex 5 for Solid-Phase
Peptide Synthesis
i) LiCl, DMF; quant. ii) MeOH/H2O; 80%. iii) TSTU (=O-(N-succinimidyl)-N,N,N’,N’-tetramethyl-
uroniumtetrafluoroborate), i-Pr2NEt, DMF; 73%.
according to Scheme 3. It is stable under the conditions employed during peptide syn-
thesis and during cleavage and workup (95% TFA, 20% piperidine in DMF), thus being
applicable directly during peptide assembly on solid support.
Briefly, the quinolinone derivative 2 [39][40] was N-alkylated under microwave
assistance to yield the allyl intermediate 12 in 83% yield. Subsequent cross-coupling
of 12 with Boc-protected1) racemic 4-bromophenylalanine methyl ester [41] via Heck
reaction turned out to be the crucial step of the synthesis. Initial experiments afforded
the desired product 13 in only ca. 40% yield. However, optimization of the reaction
conditions (temperature, base, ratio of reagents) finally led to 13 in a yield of ca.
90%, both when performed in a microwave oven or with conventional heating. Hydro-
genation of 13 over Pd/C then afforded 14 in 92% yield. This reduction step was nec-
essary to impart both chemical stability and flexibility to the molecule. Treatment of
14 with 50% TFA led to cleavage of the Boc group, and insertion of the Fmoc group
with Fmoc-OSu1) as reagent yielded the ꢂdonor-labeledꢁ target amino acid 11 in 97%
yield over two steps. Compared to the starting material 2, no change of the absorption
and emission characteristics could be observed for neither 11 nor 12, as can be seen
from Fig. 1. The additional bands at ca. 300 nm in 11 are caused by the aromatic moi-
eties of the Fmoc group.
2.3. Spectroscopic Properties of FRET Dyes. To evaluate the applicability of the
chromophores as FRET donor and acceptor, respectively, the model peptides 15–17

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Scheme 2. Synthesis of the Ligand 4
i) H3PO4, As2O5 ·(H2O)n; 83%. ii) H2, Pd/C, MeOH, toluene; 77%. iii) MeOH, H2SO4; 93%. iv) 3-
Chloropropanoyl chloride, AlCl3, CH2Cl2; 83%. v) H3PO4, As2O5 ·(H2O)n; 73% (10+4). vi) NaOH,
EtOH, H2O; 72%.
were prepared by standard Fmoc/Boc chemistry [42]. They bear the donor and/or the
acceptor dye. Incorporated into 15 was the recognition sequence and cleavage site of
the protease thrombin. Peptides 16 and 17 represent the labeled fragments to be
expected as cleavage products (Scheme 4). For the coupling of the donor, the Fmoc-
protected unnatural amino acid 11 was employed as building block during SPPS,
whereas 5 was used to attach the Ru complex 1 to the N-terminus of the peptides.
Excitation and emission spectra of 16 and 17 revealed a very good overlap of the
emission band of the donor and the absorption band of the acceptor (Fig. 2), which
is a crucial requirement for strong FRET. Furthermore, the excitation spectrum of
the Ru complex showed a minimum at 350 nm, the wavelength at which the donor
has its excitation maximum. Hence, there will be hardly any direct excitation of the
acceptor. Finally, the Stokes shift of the N-terminally attached Ru acceptor complex
is rather large: the excitation at 450 nm and the emission at ca. 620 nm are well-sepa-
rated, allowing for undisturbed fluorescence detection, without interference of the
donor emission.

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Scheme 3. Synthesis of the FRET-Donor Amino Acid 11
i) KHMDS (=potassium hexamethyldisilazane), allyl bromide, THF, microwave heating; 83%. ii)
Boc-protected racemic 4-bromophenylalanine methyl ester, [Pd(OAc)2], Ph3P, Cs2CO3, DMF, H2O,
microwave heating; 90%. iii) H2, Pd/C, MeOH; 92%. iv) 1. CF3COOH/CH2Cl2/i-Pr3SiH 10 :10 :0.5; 2.
Fmoc-OSu1), aq. Na2CO3/acetone; 97%.
Fig. 1. UV/VIS Absorption spectra of 2, 11, and 12 in DMF
As desired, comparison of the fluorescence spectra of 15 with a mixture of 16 and 17
indicated an efficient FRET in peptide 15 (Fig. 3). The spectrum recorded after excita-
tion of the donor at 350 nm displayed only the emission of the donor at 420 nm for the

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Scheme 4. Sequences of Peptides 15–17, and Representative Synthesis of 15. The FRET donor and
acceptor chromophores are represented by D and A, resp.
i) 20% Piperidine in DMF, 15 min. ii) 5, i-Pr2NEt, DMF, 2 d. iii) CF3COOH/CH2Cl2/i-Pr3SiH 95 :3 :2,
3 h.
Fig. 2. UV/VIS Excitation/emission spectra of peptides 16 (green/blue) and 17 (black/red)
mixture of 16/17, but an additional band at 618 nm for 15. This emission results from the
indirect excitation of the acceptor by the donor due to FRET, and not from direct exci-
tation of the Ru complex, which has a negligible absorbance at 350 nm, as mentioned
above. This indicated that the two chromophores not only possess complementary spec-
troscopic properties, but also that their distance and orientation in the peptide is favor-
able for efficient energy transfer.
The lifetime t of the donor in the absence of FRET (peptide 17) was found to be
0.61 ns; in the presence of FRET (peptide 15), t was found to be 0.54 ns (Fig. 4). For

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Fig. 3. Fluorescence-emission spectra of 15 (…) and of a mixture of 16/17 (—)
Fig. 4. Fluorescence-relaxation curves of the donor chromophore in 17 (filled circles) and in 15 (open
circles) at excitation and emission wavelengths of 381 and 420 nm, resp.
the lifetime of the acceptor in peptide 15, a value of 0.53 ms was obtained (Fig. 5). This
factor of 103 between the lifetimes of the donor and the acceptor is well-suited for time-
gated spectroscopy.
Unfortunately, peptide 15 was not soluble in the reaction buffer used for the pro-
tease reaction, nor was it accessible to cleavage by thrombin, probably due to insolubil-
ity and steric hindrance by the terminal Ru complex. Therefore, further optimization of
the protease substrate was necessary.

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Fig. 5. Fluorescence-relaxation curve of the acceptor chromophore in 15 at excitation and emission
wavelengths of 358 and 620 nm, resp.
2.4. Optimization of the Peptide Substrate and Thrombin Assay. To determine the
optimal distance between the donor and acceptor dyes in the protease substrate regard-
ing the intensity of the FRET and the cleavability by the enzyme, a range of peptides
were synthesized. All sequences comprised the recognition sequence for thrombin,
as well as the FRET donor and the FRET acceptor, but they varied in the number
and nature of the amino acids between the two chromophores.
Our studies showed that the sequence of peptide 18 was the best compromise
between cleavability by the enzyme and FRET intensity (Scheme 5, Fig. 6).
Scheme 5. Enzymatic Cleavage of Peptide 18
i) Thrombin, 25 mM Tris·HCl (pH 8.0), 100 mM NaCl, 308, 24 h.
The substrate 18 was hydrolyzed to ca. 95% by thrombin, the ratio of its FRET
intensities before and after the reaction being about 11:1. Such a signal change is read-
ily detectable and can be used to monitor the reaction of the substrate with the appro-

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Fig. 6. Fluorescence-emission spectra of peptide 18 before (…) and after (—) cleavage with thrombin
priate protease. Furthermore, the knowledge gained during this work should be helpful
to establish similar assays for other hydrolytic enzymes.
3. Conclusions. – A new pair of acceptor (1) and donor (2) dyes was developed for
FRET measurements in both normal and time-resolved modes. Both dyes were readily
synthesized in reasonable-to good yields over all steps, and exhibit very good chemical
stability towards strongly acidic and basic conditions. They were designed in a way
allowing incorporation into peptides, as verified by the synthesis of a peptide substrate
for the protease thrombin. Enzymatic hydrolysis was then followed by fluorescence
measurements before and after the reaction with thrombin, corroborating the excellent
spectroscopic properties of the dyes. Due to the possibility of time-resolved FRET
measurements, the present dye combination might serve as a very good alternative
to conventional systems, especially when high sensitivity is required.
The main future goal will be to employ these chromophores as donor and acceptor-I
dyes in a ternary FRET system. A suitable acceptor II has already been identified, and
work in our group is currently underway to establish a system that can be applied to the
measurement of two different distances and their changes relative to each other.
Finally, these dyes will be used to label three biomolecules and monitor their supra-
molecular interactions.
The authors wish to thank Prof. L. Moroder for a short introduction into peptide chemistry, Prof. P.
Gräber, Dr. S. Steigmiller, and R. Bienert for the measurement of excitation, emission, and fluorescence
spectra. We also thank Prof. C. Seidel, Dr. S. Kalinin, and Dr. B. Zimmermann for recording fluorescence-
lifetime-decay curves.

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Experimental Part
General. All reagents were purchased from commercial sources and used without further purifica-
tion. Amine-free DMF (Roth) was employed throughout peptide syntheses, water for the protease assays
was purified via a Direct-Q system (Millipore), and THF was dried over Na/benzophenone before use.
Microwave-assisted reactions were performed with a Discover apparatus (CEM GmbH). Column chro-
matography (CC): silica gel 60 (Merck). Peptide synthesis: semi-automatic SP-4000 synthesizer
(Labortec AG). Semi-prep. HPLC: Agilent-1100 system, with Nucleosil 100-5 C18 PPN columns
(Macherey-Nagel) for peptides or a Source 5RPC ST 4.6/150 column (Amersham Pharmacia Biotech)
for Ru complexes. Fluorescence-emission spectra: Perkin-Elmer LS45 spectrometer. Fluorescence-life-
time decay: instrument from IBH Consultants, Ltd. (Glasgow, Scotland), with an N2 flash lamp as exci-
tation source (microsecond range) or a FluoTime-200 (Picoquant) pulsed LED (nanosecond range).
NMR Spectra: at 300 or 400 MHz (1H), and at 75.5, 100.6, or 125.7 MHz (13C); chemical shifts d in
ppm rel. to the respective solvent signals, J in Hz. MS: Finnigan MAT-8200 (EI), TSQ-7000 (ESI), or
LTQ FT (HR-ESI) mass spectrometers; in m/z.
Synthesis of Complex 5. Starting from bathophenanthrolinedisulfonic acid disodium salt trihydrate
(bpds; 460 mg, 0.78 mmol) and RuCl3 ·H2O (100 mg, 0.38 mmol), 3 was synthesized in quant. yield. Com-
pound 3 (1.34 g, 1.07 mmol) was then reacted with 4 (607 mg, 1.4 mmol) under modified work-up condi-
tions, i.e. instead of precipitation, the complex was purified by CC (SiO2; CH2Cl2/MeOH 75 :25 contain-
ing 0.05% of CF3COOH (TFA)) to yield 1 (1.44 g, 80%). Activation of 1 as the O-succinimide ester with
an excess of TSTU was carried out to afford 5 in 73% yield, the progress of the reaction being monitored
by LC/MS.
8-Nitro-4-phenylquinoline (6). A suspension of 2-nitroaniline (2.0 g, 14.5 mmol, 1.0 equiv.), As2O5
hydrate (4.0 g, 17.4 mmol, 1.2 equiv.), and 85% H3PO4 (14.5 ml) was heated at 1008. Then, b-chloropro-
piophenone (3.4 g, 20.3 mmol, 1.4 equiv.) was added within 5 min, and the mixture was stirred at 1308 for
2 h. After cooling to r.t. and addition of ice-water (100 ml), the mixture was made to alkaline with conc.
NH3. The org. phase was extracted with toluene (5”50 ml), and the combined org. layers were washed
with H2O (100 ml), dried (Na2SO4), and concentrated under reduced pressure. Dissolution of the remain-
ing black oil in CH2Cl2 and filtration through a short plug of SiO2 gave a crude, which, after removal of
the solvent, was recrystallized from EtOH to yield pure 6 (3.0 g, 83%). Light-yellow crystals. M.p. 1268
1
(EtOH). H-NMR (300 MHz, CDCl3): 7.47–7.50 (m, 3 arom. H); 7.53–7.59 (m, 4 arom. H); 8.01 (dd,
J=7.5, 1.2, HꢀC(5)); 8.13 (dd, J=8.6, 1.2, HꢀC(7)); 9.08 (d, J=4.4, HꢀC(2)). 13C-NMR (100.6 MHz,
CDCl3): 122.92; 123.31; 125.23; 127.87; 128.94; 129.14; 129.55; 130.11; 136.88; 140.08; 148.93; 152.08.
EI-MS: 250 (100, M+), 220 (33), 203 (35), 192 (95), 176 (71), 165 (42), 151 (26), 88 (21). Anal. calc. for
C15H10N2O2: C 72.0, H 4.0, N 11.2; found: C 71.8, H 4.2, N 11.1.
4-Phenylquinolin-8-amine (7). Compound 6 (10.0 g, 40.0 mmol) was dissolved in MeOH/toluene 1:1
(120 ml), and 10% Pd on carbon (250 mg) was added. The suspension was agitated under H2 atmosphere
(1 bar) for 24 h. The crude mixture was adsorbed on SiO2, and then subjected to CC (SiO2; cyclohexane/
AcOEt 5 :1) to afford 7 (6.8 g, 77%). Yellow solid. 1H-NMR (300 MHz, CDCl3): 5.08 (br. s, NH2); 6.94
(dd, J=7.3, 1.4, HꢀC(7)); 7.20 (dd, J=8.5, 1.3, HꢀC(5)); 7.28–7.30 (m, 2 arom. H); 7.48–7.51 (m, 5
arom. H); 8.78 (d, J=4.2, HꢀC(2)). 13C-NMR (100.6 MHz, CDCl3): 109.85; 114.09; 121.64; 127.28;
128.13; 128.36; 129.48; 138.59; 138.77; 144.26; 146.85; 148.28. EI-MS: 220 (100, M+), 191 (15), 165
(16), 110 (11), 96 (13). Anal. calc. for C15H12N2: C 81.8, H 5.5, N 12.7; found: C 81.6, H 5.8, N 12.5.
Methyl 5-[4-(3-Chloropropanoyl)phenyl]pentanoate (8). To a mixture of AlCl3 (5.15 g, 0.04 mol, 3.7
equiv.) in CH2Cl2 (5.5 ml), 3-chloropropanoyl chloride (1.1 ml, 11.4 mmol, 1.1 equiv.) was added. Then,
after cooling with a water-bath, methyl 5-phenylpentanoate (9; 2.0 g, 10.4 mmol, 1.0 equiv.) was added
over 10 min. The mixture was stirred at r.t. for 1 h. The reaction was quenched with a mixture of H2O
(35 ml), ice (30 g), and CH2Cl2 (30 ml), and the aq. layer was separated and extracted with CH2Cl2
(3”30 ml). The combined org. phases were washed with H2O (30 ml), aq. 5% NaHCO3 soln. (30 ml),
and H2O (30 ml), and the solvent was removed under reduced pressure. The remaining solid was recrys-
1
tallized at 08 from Et2O to afford 8 (2.43 g, 83%) as colorless crystals. M.p. 418 (Et2O). H-NMR (300
MHz, CDCl3): 1.68 (m, CH2CH2); 2.34 (m, CH2COOMe); 2.70 (m, CH2Ph); 3.43 (t, J=6.8, CH2COPh);
3.66 (s, COOMe); 3.92 (t, J=6.9, ClCH2); 7.28 (d, J=8.5, 2 arom. H); 7.88 (d, J=8.2, 2 arom. H). 13C-

Helvetica Chimica Acta – Vol. 89 (2006)
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NMR (100.6 MHz, CDCl3): 24.51; 30.43; 33.82; 35.63; 38.84; 41.22; 51.55; 128.31; 128.79; 134.36; 148.52;
173.89; 196.33. EI-MS: 282 (42, M+), 219 (100), 195 (32), 187 (41), 159 (19), 131 (37), 91 (54). Anal. calc.
for C15H19ClO3: C 63.7, H 6.8; found: C 63.6, H 6.9.
5-[4-(7-Phenyl-1,10-phenanthrolin-4-yl)phenyl]pentanoic Acid (4) and Methyl 5-[4-(7-Phenyl-1,10-
phenanthrolin-4-yl)phenyl]pentanoate (10). Compound 7 (2.8 g, 12.7 mmol, 1.0 equiv.) and As2O5 hydrate
(3.5 g, 15.23 mmol, 1.2 equiv.) were suspended in 85% H3PO4 (14.5 ml), and then heated at 1108. To the
resulting black soln. was added 8 (5.0 g, 17.7 mmol, 1.4 equiv.) within 5 min, and the mixture was stirred
at 1308 for another 1.5 h. After cooling to r.t., ice-water (150 ml) was added, the pH was adjusted to 5.0
with 4N aq. NaOH soln., and the aq. phase was extracted with CH2Cl2 (5”100 ml). The combined org.
layers were washed with brine (200 ml), dried (Na2SO4) and concentrated under reduced pressure.
The residue was adsorbed on SiO2 and purified by CC (SiO2; CH2Cl2/MeOH 300 :1 ! 30 :1) to afford
a crude, which was recrystallized from MeOH to yield 2.51 g (46%) of pure 4, together with 10 (1.54
g, 27%), which contained a trace amount of an nonpolar impurity, but was hydrolyzed without further
purification. Thus, 10 (1.5 g, 3.36 mmol, 1.0 equiv.) in EtOH (9 ml) was treated with NaOH (0.45 g,
11.25 mmol, 3.3 equiv.) in H2O (2.8 ml) and heated at reflux for 2 h. After concentration under reduced
pressure, the residue was taken up in a mixture of CH2Cl2 (30 ml) and H2O (40 ml). The aq. layer was
adjusted to pH 3 with H3PO4, and extracted with CH2Cl2 (30 ml). The combined org. phases were washed
with brine (20 ml), dried (Na2SO4), and concentrated under reduced pressure. The crude product was
adsorbed on SiO2 and subjected to CC (SiO2; CH2Cl2/MeOH 50 :1) to afford 4 (1.04 g, 72%).
Data of 4. Pale-yellow solid. 1H-NMR (300 MHz, CDCl3): 1.77 (m, CH2CH2); 2.45 (m, CH2COOH);
2.76 (m, CH2Ph); 7.35 (d, J=8.1, 2 arom. H); 7.45 (d, J=8.1, 2 arom. H); 7.53 (m, 5 arom. H); 7.63 (d,
J=4.5, HꢀC(3)); 7.64 (d, J=4.5, HꢀC(8)); 7.88 (d, J=9.4, HꢀC(6)); 7.92 (d, J=9.5, HꢀC(5)); 9.30 (d,
J=4.7, HꢀC(2)); 9.31 (d, J=4.5, HꢀC(9)); 10.9 (br. s, COOH). 13C-NMR (100.6 MHz, CDCl3): 24.41;
30.75; 33.89; 35.35; 123.53; 123.57; 123.93; 124.19; 126.42; 126.51; 128.49; 128.62; 128.68; 129.68;
129.71; 135.44; 137.96; 142.63; 146.74; 146.76; 148.51; 148.54; 149.69; 149.73; 178.58. EI-MS: 432 (100,
M+), 388 (8), 345 (46), 331 (8). Anal. calc. for C29H24N2O2: C 80.5, H 5.6, N 6.5; found: C 80.0, H 5.7,
N 6.4.
Data of 10. 1H-NMR (300 MHz, CDCl3): 1.70–1.76 (m, CH2CH2); 2.38 (m, CH2COOMe); 2.68–2.77
(m, CH2Ph); 3.68 (s, COOMe); 7.35 (d, J=8.1, 2 arom. H); 7.45 (d, J=8.2, 2 arom. H); 7.53 (m, 5 arom.
H); 7.61 (d, J=4.5, HꢀC(3)); 7.62 (d, J=4.5, HꢀC(8)); 7.87 (d, J=9.5, HꢀC(6)); 7.91 (d, J=9.4, Hꢀ
C(5)); 9.28 (d, J=4.5, HꢀC(2)); 9.29 (d, J=4.7, HꢀC(9)).
6,7-Dimethoxy-3-(4-methoxyphenyl)-1-(prop-2-en-1-yl)-1H-quinolin-2-one (12). To a suspension of 2
(883 mg, 2.84 mmol, 1.0 equiv.) in THF (10 ml), a 0.5M soln. of potassium hexamethyldisilazane
(KHMDS; 6.8 ml, 3.4 mmol, 1.2 equiv.) in toluene was added at ꢀ788. The mixture was stirred for 30
min at this temp., and then allowed to reach r.t. Allyl bromide (0.75 ml, 8.5 mmol, 3.0 equiv.) was
added, and the white suspension was placed for 15 min in a microwave oven at 1208 (max. 15 bar, 200
W). Then, H2O (70 ml) and CHCl3 (70 ml) were added, the phases were separated, and the aq. layer
was extracted with CHCl3 (2”70 ml). The combined org. layers were dried (MgSO4), filtered, and the
solvent was removed under reduced pressure. The residue was purified by CC (SiO2; cyclohexane/
AcOEt 2 :1) to yield 12 (828 mg, 83%). Light-yellow solid. 1H-NMR (400 MHz, CDCl3): 3.84 (s,
MeO); 3.94 (s, MeO); 3.96 (s, MeO); 5.02 (dt, J=4.8, 1.5, CH2); 5.20 (dd, J=17.2, 1.1, 1 H of CH=
CH2); 5.26 (dd, J=10.4, 1.1, 1 H of CH=CH2); 6.00 (ddt, J=17.3, 10.4, 5.2, CH=CH2); 6.80 (s, Hꢀ
C(5)); 6.95 (m, HꢀC(3’,5’)); 6.99 (s, HꢀC(8)); 7.70 (m, HꢀC(2’,6’)); 7.71 (s, HꢀC(4)). 13C-NMR
(100.6 MHz, CDCl3): 45.66; 55.40; 56.15; 56.28; 97.98; 109.36; 113.62; 114.43; 117.23; 129.27; 129.54;
130.17; 132.35; 134.47; 135.60; 145.23; 151.68; 159.40; 161.18. EI-MS: 351 (97, M+), 336 (100), 320
(11), 310 (11), 292 (9). Anal. calc. for C21H21NO4: C 71.8, H 6.0, N 4.0; found: C 71.6, H 6.4, N 3.9.
4-{(1E)-3-[6,7-Dimethoxy-3-(4-methoxyphenyl)-2-oxoquinolin-1(2H)-yl]prop-1-enyl}-N-[(1,1-dime-
thylethoxy)carbonyl]phenylalanine (13). A mixture of 12 (150 mg, 0.42 mmol, 1.0 equiv.), Boc-protected,
racemic 4-bromophenylalanine methyl ester (229 mg, 0.66 mmol, 1.5 equiv.), [Pd(OAc)2] (7.2 mg, 0.04
mmol, 0.09 equiv.), Ph3P (25.2 mg, 0.1 mmol, 0.24 equiv.), and Cs2CO3 (416 mg, 1.26 mmol, 3.0 equiv.)
was suspended in DMF (3 ml) and H2O (1.5 ml). The mixture was heated at 808 for 10 h in a microwave

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Helvetica Chimica Acta – Vol. 89 (2006)
oven (30 W)2), the solvent was removed under reduced pressure, and the remaining solid was taken up in
a mixture of CH2Cl2 (10 ml) and 2N aq. HCl (10 ml). The aq. phase was extracted with CH2Cl2 (5”5 ml),
and the combined org. layers were dried (Na2SO4). After removal of the solvent under reduced pressure,
the crude product was purified by CC (SiO2; CH2Cl2/MeOH 100 :1 ! 95 :5) and subsequently precipi-
1
tated with pentane to afford 13 (232 g, 90%). Light-yellow powder. H-NMR (300 MHz, CDCl3): 1.39
(s, Me3C); 2.94–3.17 (m, PhCH2CH); 3.84 (s, MeO); 3.94 (s, MeO); 3.95 (s, MeO); 4.47–4.56 (m,
NHCHCOOH); 4.96 (br. d, J=7.0, NH); 5.17 (d, J=5.3, NCH2CH); 6.30 (dt, J=16.1, 5.8, CH2CH=
CH); 6.57 (d, J=16.0, CH=CHPh); 6.90 (s, HꢀC(5)); 6.95 (d, J=8.8, HꢀC(3’,5’)); 7.01 (s, HꢀC(8));
7.07 (d, J=8.1, 2 arom. H); 7.22 (d, J=8.1, 2 arom. H); 7.69 (d, J=8.8, HꢀC(2’,6’)); 7.74 (s, HꢀC(4)).
13C-NMR (75.5 MHz, CDCl3): 28.49; 37.77; 45.62; 54.63; 55.54; 56.38; 56.45; 80.19; 98.11; 109.60;
113.81; 114.78; 123.64; 126.64; 128.60; 129.40; 129.64; 129.91; 130.35; 132.72; 134.60; 135.03; 136.10;
145.56; 152.02; 155.80; 159.57; 161.52; 176.33. HR-MS: 615.2700 ([M+H]+, C35H39N2O8þ ; calc. 615.2706).
4-{(1E)-3-[6,7-Dimethoxy-3-(4-methoxyphenyl)-2-oxoquinolin-1(2H)-yl]propyl}-N-[(1,1-dimethyle-
thoxy)carbonyl]phenylalanine (14). Compound 13 (2.11 g, 3.4 mmol) was dissolved in MeOH (60 ml),
and 10% Pd on carbon (318 mg) was added to the soln. The mixture was agitated under H2 atmosphere
(1 bar) for 16 h. The catalyst was removed by filtration over Kieselguhr, and the solvent was distilled off at
reduced pressure. The residue was purified by CC (SiO2; CH2Cl2/MeOH 100 :1 ! 80 :20) to yield 14
1
(1.96 g, 92%). Light-yellow powder. H-NMR (300 MHz, CDCl3): 1.40 (s, Me3C); 2.10 (tt, J=7.5, CH2-
CH2CH2); 2.78 (t, J=7.3, CH2CH2Ph); 3.03–3.17 (m, PhCH2CH); 3.79 (s, MeO); 3.84 (s, MeO); 3.91
(s, MeO); 4.31 (t, J=7.9, NCH2CH2); 4.49–4.60 (m, NHCHCOOH); 5.02 (br. d, J=4.8, NH); 6.53 (s,
HꢀC(5)); 6.92–6.96 (m, HꢀC(3’,5’), HꢀC(8)); 7.12 (d, J=8.1, 2 arom. H); 7.18 (d, J=8.1, 2 arom.
H); 7.66 (m, HꢀC(2’,6’)); 7.67 (s, HꢀC(4)). 13C-NMR (100.6 MHz, CDCl3): 28.38; 28.58; 32.98; 37.58;
42.87; 54.47; 55.42; 56.19; 56.32; 80.28; 97.28; 109.63; 113.70; 114.75; 128.75; 129.30; 129.52; 129.69;
130.21; 134.04; 134.08; 135.67; 140.02; 145.37; 151.96; 155.51; 159.47; 161.42; 174.33. HR-MS:
617.28575 ([M+H]+; C35H41N2Oþ8 ; calc. 617.28574).
4-{(1E)-3-[6,7-Dimethoxy-3-(4-methoxyphenyl)-2-oxoquinolin-1(2H)-yl]propyl}-N-{[(9H-fluoren-9-
yl)methoxy]carbonyl}phenylalanine (11). a) To a soln. of 14 (0.70 g, 1.14 mmol) in CH2Cl2 (10 ml),
CF3COOH (10 ml) and i-Pr3SiH (0.5 ml) were added, and the mixture was stirred at r.t. TLC Examina-
tion (SiO2; CH2Cl2/MeOH 4 :1) showed complete conversion after 2 h. The mixture was co-evaporated
with MeCN (3”30 ml) to dryness under reduced pressure, the residual solid was suspended in H2O (30
ml), and extracted with Et2O (5”20 ml). The org. phase was extracted with H2O (20 ml), and the com-
bined aq. layers were evaporated to dryness by azeotropic distillation with MeCN under reduced pres-
sure. The resulting crude intermediate, 4-{(1E)-3-[6,7-dimethoxy-3-(4-methoxyphenyl)-2-oxoquinolin-
1(2H)-yl]propyl}phenylalanine, was used in the next step without further purification.
b) The N-deprotected amino acid (1.14 mmol, 1.0 equiv.) was suspended in aq. 9% Na2CO3 soln. (20
ml) and cooled in an ice bath. To this mixture, a suspension of Fmoc-OSu3) (0.58 g, 1.70 mmol, 1.5 equiv.)
in acetone (10 ml) was added dropwise. After 1 h of stirring, the ice-bath was removed, more acetone (20
ml) was added, and the suspension was stirred for a further 3 h. Then, conc. HCl was added to neutralize
the mixture, and the solvent was removed under reduced pressure. The residue was suspended in a NaCl
soln. containing 2N HCl (60 ml), and extracted with CH2Cl2 (5”40 ml). The combined org. layers were re-
extracted with brine (40 ml), dried (Na2SO4), warmed to 408, and filtered. The residue was purified by CC
1
(SiO2; CH2Cl2/MeOH 100 :1 ! 90 :10) to afford 11 (0.82 g, 97%). Light-yellow powder. H-NMR (300
MHz, CDCl3): 2.08 (tt, J=7.5, 7.3, CH2CH2CH2); 2.78 (t, J=7.0, CH2CH2Ph); 3.13 (m, PhCH2CH);
3.79 (s, MeO); 3.83 (s, MeO); 3.90 (s, MeO); 4.15 (dd, J=6.7, HꢀC(9) of Fmoc); 4.24–4.33 (m, 1 H of
CH2 of Fmoc, NCH2CH2); 4.44 (dd, J=10.3, 7.0, 1 H of CH2 of Fmoc); 4.66 (m, NHCHCOOH); 5.34
(d, J=8.1, NH); 6.51 (s, HꢀC(5)); 6.93–6.96 (m, HꢀC(3’,5’), HꢀC(8)); 7.10 (d, J=7.5, 2 arom. H);
7.17 (d, J=7.5, 2 arom. H); 7.24–7.29 (m, 2 arom. H of Fmoc); 7.36 (t, J=7.4, 2 arom. H of Fmoc);
7.51–7.54 (m, 2 arom. H of Fmoc); 7.63–7.66 (m, HꢀC(2’,6’), HꢀC(4)); 7.71 (d, J=7.5, 2 arom. H of
2
)
The same result was obtained when the mixture was heated in a sealed vessel for 21 h at 908 with an
oil bath.
Systematic name: 1-({[(9H-fluoren-9-yl)methoxy)]carbonyl}oxy)pyrrolidine-2,5-dione.
3
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Helvetica Chimica Acta – Vol. 89 (2006)
3069
Fmoc). 13C-NMR (125.7 MHz, CDCl3): 28.41; 32.89; 37.54; 42.89; 47.13; 54.76; 55.33; 56.02; 56.16; 67.03;
97.01; 109.32; 113.64; 114.68; 119.93; 125.03; 127.02; 127.68; 128.70; 129.41; 129.52; 129.68; 130.17;
133.86; 133.88; 135.84; 139.91; 141.25; 143.72; 145.29; 151.88; 155.80; 159.38; 161.39; 174.10. HR-MS:
739.3014 ([M+H]+, C45H43N2Oþ8 ; calc. 739.3019).
Peptide Synthesis. The syntheses were carried out on a 0.02-mmol scale using Fmoc protocol and
Wang resin (loading 0.75 mmol/g), with O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluroniumtetrafluoro-
borate (TBTU) as coupling reagent. Standard Boc and t-Bu side-chain-protected amino acids were
employed. As building block for the incorporation of the donor, compound 11 (30 mg, 0.04 mmol, 2.0
equiv.) was used. For the coupling of the Ru complex to the solid-phase-bound peptide at the N-terminus,
a soln. of 5 (40 mg, 0.022 mmol, 1.1 equiv.) in DMF (1 ml) and i-Pr2NEt (70 ml, 0.40 mmol, 20 equiv.) were
added to the resin, which was agitated for 2 d. The peptides were deprotected and cleaved from the solid
support by exposure to CF3COOH/CH2Cl2/i-Pr3SiH 95 :3 :2, and then purified by RP-HPLC and ana-
lyzed by LC/MS.
Data of 15. ESI-MS: 1402 (100, [Mꢀ4 Na+4 Hꢀ2 Cl]2+), 935 (26, [Mꢀ4 Na+5 Hꢀ2 Cl]3+).
Data of 16. ESI-MS: 1017 (100, [Mꢀ4 Na+4 Hꢀ2 Cl]2+).
Data of 17. ESI-MS: 789 (100, [M+H]+), 395 (12, [M+2H]2+).
Data of 18. ESI-MS: 1582 (100, [Mꢀ4 Na+4 Hꢀ2 Cl]2+), 1055 (30, [Mꢀ4 Na+5 Hꢀ2 Cl]3+).
Thrombin Assay. To a soln. of the peptide (0.125 mmol) in a mixture of 330 ml of H2O and 50 ml of
buffer (0.2M Tris·HCl (pH 8), 0.8M NaCl), 20 ml of human thrombin (20 U) was added. Before and
after incubation of the soln. at 308 for 24 h, aliquots were taken, deactivated at 1008 for 30 s, and diluted
with the same volume of MeCN. The samples were analyzed by RP-HPLC to determine the cleavage effi-
ciency, and by LC/MS to identify the cleavage fragments. For the measurement of fluorescence-emission
spectra, the samples were further diluted with H2O/MeCN 1:1 to give a final peptide concentration of ca.
0.4 mM. Excitation was performed at 350 nm.
Spectroscopic Measurements. Excitation/emission spectra were recorded at a peptide concentration
of 5 mM in H2O/MeCN 1:1. The excitation (lex) and emission (lem) wavelengths were 520 and 615 nm for
peptide 16, resp., and 350 and 440 nm for peptide 17, resp. Fluorescence-emission spectra were recorded
at a peptide concentration of 0.4 mM in H2O/MeCN 1:1 at lex 350 nm. Fluorescence-relaxation curves
were established at a peptide concentration of 0.4 mM in H2O/MeCN 1:1 at lex 381 and lem 420 nm for
the donor in both peptides 15 and 17, and at lex 358 and lem 620 nm, resp., for the acceptor in peptide 15.
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Received August 4, 2006