Comparative studies of different quinolinone derivatives as donors in fluorescence-resonance-energy transfer (FRET) - Systems in combination with a (bathophenanthroline)ruthenium(II) complex as acceptor

Article abstract of DOI:10.1002/hlca.200900235

We describe the preparation as well as a detailed photophysical study of Fmoc-amino acid building blocks carrying different carbostyril (=quinolin-2(1H)-one) heterocycles as donors in a FRET (fluorescence-resonance- energy transfer) system in combination

Full text of DOI:10.1002/hlca.200900235

Helvetica Chimica Acta – Vol. 92 (2009)
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Comparative Studies of Different Quinolinone Derivatives as Donors
in Fluorescence-Resonance-Energy Transfer (FRET) – Systems in
Combination with a (Bathophenanthroline)ruthenium(II) Complex as
Acceptor
by Rolf A. Kramera), Roman Flehrb), Myriam Laya), Michael U. Kumkeb), and Willi Bannwarth*a)
a) Institut fꢀr Organische Chemie und Biochemie, Albert-Ludwigs-Universitꢁt Freiburg, Albertstr. 21,
D-79104 Freiburg (phone: þ 497612036073; fax: þ 497612038705;
e-mail: willi.bannwarth@organik.chemie.uni-freiburg.de)
b) Institut fꢀr Chemie, Universitꢁt Potsdam, Karl-Liebknecht-Str. 24 – 25, D-14476 Potsdam-Golm
(phone: þ 493319775209; e-mail: Kumke@uni-potsdam.de)
We describe the preparation as well as a detailed photophysical study of Fmoc-amino acid building
blocks carrying different carbostyril (¼quinolin-2(1H)-one) heterocycles as donors in a FRET
(fluorescence-resonance-energy transfer) system in combination with a [RuII(bathophenanthroline)]
complex (bathophenanthroline ¼ 4,7-diphenyl-1,10-phenanthroline). The efforts resulted in a clear
preference for building block 16 due to its ease of synthesis (Scheme 2), its chemical robustness, and the
FRET efficiency when incorporated into peptides.
Introduction. – Luminescent chromophores as entities of fluorescence-resonance-
energy transfer (FRET) systems are important tools to study supramolecular
interactions with a special emphasis in the realm of biomolecules like DNA, RNA,
and proteins [1]. FRET Systems allow to monitor distance-dependent interactions on
the molecular level, and in a real-time mode. Therefore, they are especially suited for
the characterization of biochemical events both in vitro and in vivo. Meanwhile, a
myriad of applications has been reported. It involves binding of ligands to their
pertinent protein receptors [2], DNA – protein complexation [3], and RNA-folding
and catalysis [4]. Other applications are enzyme assays based, e.g., on the Fçrster
resonance-energy-transfer principle [5] and monitoring of polymerase chain reactions
(PCR) [6]. The FRET technology is based on the nonemissive transfer of energy
between a donor (D) and an acceptor (A) fluorophore. It decreases with rꢀ6, r being the
distance between the donor and the acceptor [7]. A prerequisite for an efficient
transfer is an intensive overlap between the emission of the donor with the absorption
of the acceptor as well as the correct orientation of their dipole transition moments
relative to each other. A plethora of different donor– acceptor pairs have been
reported up to date, but despite the multitude of available systems, sensitivity –
especially in the presence of background luminescence from matrix constituents – still
remains an issue. A further concern is robustness of the applied dyes as well as the
possibility to employ them in a modular way as broadly as possible via stable covalent
bonds and without interference of the spectral properties of the labelled molecules.
ꢂ 2009 Verlag Helvetica Chimica Acta AG, Zꢀrich

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Helvetica Chimica Acta – Vol. 92 (2009)
One way to solve the sensitivity issue are FRET systems of which one partner
possesses a long excited-state decay time and hence, allows for measurements in a time-
resolved mode reducing background luminescence. The most prominent candidates for
this purpose are lanthanide ions with lifetimes up to milliseconds. Usually, they are
employed as caged chelates [8], but leakage of Eu can still be a problem. Furthermore,
the very long decay time might be a disadvantage in monitoring events which occur in
the ms range.
Our focus has therefore been on RuII charge-transfer (CT) complexes – and
especially [RuII(bathophenanthroline)] complex 1 [9] (Fig. 1) – as alternative acceptor
units due to their thermodynamic stability, chemical inertness, straightforward
synthetic accessibility and relatively long-lived excited states (tem ¼ 1 – 5 ms) allowing
for time-resolved measurements [10][11] (bathophenanthroline ¼ 4,7-diphenyl-1,10-
phenanthroline).
Fig. 1. [RuII(bathophenanthroline)] complex 1 as amino acid building block
As suitable donor chromophore, we had identified carbostyril (¼quinolin-2(1H)-
one) derivative 2 [12] which we turned into the amino acid building block 3 (Scheme 1)
directly applicable to solid-phase peptide synthesis. The FRET system composed of the
quinolinone donor and the [RuII(bathophenanthroline)] complex as acceptor was then
verified in peptides. It turned out to be very robust and revealed an intensive FRET
measurable with high sensitivity in a time-resolved mode [13] (for application of the
system in DNA, see [14]).
Although efficient, the system still entailed a number of disadvantages: The
synthetic route from 2 to 3 was not straightforward and yielded furthermore 3 as a
racemate. An enantiomerically pure building block would be more compatible with
defined peptide structures. In addition, we were aiming at an even more intense FRET.
Results and Discussion. – Recently, Uray, Stadlbauer, and co-workers published
their efforts on improving the photophysical properties of carbostyrils by a systematic
investigation of substituent effects which converged in a push-pull model with two

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Scheme 1. Quinolin-2(1H)-one 2 and the Pertinent Amino Acid Derivative 3
electron-donating groups at position 6 and 7 and an electron-withdrawing group at the
4-position. Their first attempts culminated in compound 4 carrying a CF3 group at C(4)
[15]. Alkylation at N(1) had no influence on the photophysical properties, and
derivatives of 4 showed an absorption maximum lmax at 359 – 361 nm in H2O and
extinction coefficients around 10000. Quantum yields up to 0.50 and a Stokes shift of
70 nm were observed. Last year, the group reported on carbostyril 5 and derivatives of
it [16]. The compound revealed a broad double maximum between 385 and 410 nm
independent of the pH value (pH 1 – 11). Furthermore, derivatives of 5 fluoresce in
H2O and polar and apolar solvents in a narrow 430 – 440 nm window and with a
quantum yield around 0.5. These data prompted us to perform a comparative study
concerning the suitability of the above mentioned chromophores 2, 4, and 5 (Scheme 2)
as donors in FRET systems in combination with [RuII(bathophenanthroline)]
complexes as acceptors.
After having synthesized the parent carbostyrils 2, 4, and 5, they were turned into
their pertinent Fmoc – Lys – OH building blocks according to Scheme 2 and following
essentially the procedures published by Uray, Stadlbauer, and co-workers [15][16]. In
the alkylation reaction with BrCH2COOEt, we used lithium diisopropylamide (LDA)
for the deprotonation instead of NaOH/CH2Cl2 or K2CO3/MeCN. This led to a
quantitative deprotonation under mild conditions and avoided at the same time the
formation of the O-alkylated product. The intermediates 6 – 8 were thus obtained in
yields of 97, 86, and 88%, respectively. After saponification with NaOH (! 9 – 11), the
carboxyl functions were activated by reaction with N-hydroxysuccinimide in the
presence of N,N’-diisopropylcarbodiimide (!12 – 14). The activated esters were then
treated without further purification with Fmoc – Lys – OH using an aqueous DMF
solution and Titrisolꢃ buffer (pH 7) to yield the desired amino acid building blocks 15 –
17 in pure form. Compared to our previous synthesis route for the amino acid building
block 3 in racemic form and comprising 7 steps, the preparation according to Scheme 2
is rather straightforward involving just two chromatographic steps and yielding the
enantiomerically pure compounds.
During preliminary experiments, we learned that building blocks 16 and 17 were
completely stable towards 95% CF3COOH solution, whereas in 15, the amide bond
was partially cleaved under these conditions. Hence, the latter is not compatible with
peptide synthesis and was for this reason in this study replaced by building block 3.

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Scheme 2. Synthesis of the Amino Acid Building Blocks 15 – 17
i) LDA, THF, 08, 1 h; BrCH2COOEt, 08, 30 min; r.t., 16 h. ii) NaOH, EtOH/H2O 9 :1, reflux, 16 h. iii) N-
Hydroxysuccinimide, N,N’-diisopropylcarbodiimide, THF, 08, 15 h. iv) Fmoc – Lys – OH, Titrisolꢃ buffer
(pH 7.0), DMF/H2O 9 :1, r.t., 20 h.
For the evaluation of the different donor chromophores in the FRET system with
the [RuII(bathophenanthroline)] complex, we synthesized peptides 18 – 20 (Fig. 2)
with standard Fmoc-building blocks and introducing the Ru-complex-modified l-lysine
Fig. 2. FRET Peptides 18 – 20

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1 in the third position. The carbostyril entities were inserted by using an excess of 1.2
equiv. and TBTU as coupling reagent [17], whereupon complete coupling was
achieved. After deprotection of the side chains and removal from the support, the
peptides were obtained in pure form after semiprep. HPLC.
Photophysical Investigations. Fig. 3 shows the absorption spectra of building blocks
3 and 15 – 17. Only the absorption of the CN-substituted building block 17 is
significantly shifted to longer wavelengths. The data obtained for building blocks 16
and 17 agree well with the ones reported for the pure dyes by Uray, Stadlbauer, and co-
workers [15][16]. Since these dyes are intended to be used as fluorescence donors, their
fluorescence properties are important. The location of the fluorescence maximum is
very similar and is found in the spectral range of 430 nm < lem < 445 nm (Table). The
fluorescence quantum efficiencies Fem are around 0.18 for compounds 3 and 15 but
significantly higher for compound 16 and 17 for which an efficiency Fem of roughly 0.5
was determined (Table). The fluorescence decay curves found for 3 and 15 – 17 follow
first-order kinetics. The obtained fluorescence decay times t correspond to the
observed trends found for Fem: For 3 and 15, short decay times t of ca. 600 ps were
determined, while for 16 and 17, t of ca. 3.5 ns were found (Table).
Fig. 3. Absorption spectra of building blocks 3, 15, 16, and 17
Based on the absorption and fluorescence spectra of the building-block dyes 3 and
15 – 17, first the critical Fçrster distance R0 was calculated with Eqns. 1 and 2. Here, R0 is
the distance, at which the energy transfer occurs with a transfer efficiency E of 50%. As
a rule of thumb, a specific D/A pair can be used for distance determinations within
R0/2 < R < 2R0.

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Table. Photophysical Properties of the Building Blocks 3, 15, 16, and 17
labs [nm]
eA (l) [l molꢀ1 cmꢀ1
]
lexc [nm]
lem [nm]
t [ns]
Fem
R0a) [ꢄ]
3
15
16
17
367
366
368
398
14500
19000
9500
367
367
368
380
433
435
440
442
0.6
0.7
3.5
3.7
0.17
0.19
0.50
0.47
37
38
45
45
13000
a) Based on Eqn. 1 calculated critical Fçrster distance R0 with 1 as acceptor.
ð9000 ln 10Þk2F
R60 ¼
D JðlÞ
(1)
(2)
128p5n4NA
1
R
JðlÞ ¼ FDðlÞeAðlÞl4dl
0
In Eqn. 1, k2 is the orientation factor describing the relative orientation of the
transition dipol moments of D and A, FD is the fluorescence quantum efficiency of the
donor, n represents the refractive index of the solvent, and NA is the Avogadro
constant. J(l) is the spectral-overlap integral calculated from the area-normalized
fluorescence spectrum FD(l) of the donor and the absorption spectrum eA(l) of the
acceptor (see Eqn. 2). The values of R0 for the investigated compounds are
summarized in the Table. At this point, 16 and 17 seemed to be the preferred donors
for the envisaged FRET system because of the largest R0 and a quantum yield of ca. 0.5.
By incorporation of the FRET chromophores into (bio)macromolecules, a further
effect might come into play which may influence the effective transfer efficiency of the
FRET pair. Such an effect could be the limitation in the rotational freedom of the dye
molecule because of specific interactions with the (bio)macromolecule or of the dyes
themselves. To compare these building blocks as potential FRET donors (D) in
combination with the [RuII(bathophenanthroline)] complex as FRET acceptor (A),
FRET peptides 18 – 20 were further characterized with respect to their spectroscopic
properties.
The absorption spectra of the FRET peptides 18 – 20 show the typical double peak
of the RuII-complex absorption at l 445 and 467 nm as well as a less resolved peak
around l 367 nm of the donor entity. These absorption spectra are equal to the sum of
the absorption found for the D and A building blocks, respectively. Accordingly, it can
be concluded that no direct interaction of the dyes in their electronic ground states is
present.
Fig. 4 shows the fluoresence emission spectra of 18 – 20 excited at lex 360 nm. The
excitation wavelength of lex 360 nm was chosen to avoid direct excitation of the RuII
complex, which has an absorption minimum around 360 nm. For a better overview, the
spectra were normalized to the donor emission maximum. In addition to the emission
around lem 440 nm of the donor entities, a second emission feature at lem 620 nm was
observed, which belongs to the luminescence of the RuII-complex acceptor.
To determine the FRET efficiencies E of the dye-labeled peptides 18 – 20, Eqn. 3
was used. This ratiometric method introduced by Clegg and co-workers allows the

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Fig. 4. Fluorescence emission spectra of FRET peptides 18 – 20 at 620 nm after excitation at 360 nm
(normalized to donor emission maximum, not corrected for the quantum yield)
direct determination of the FRET efficiency of DA pairs labeled to (bio)macromo-
lecules by only two separate measurements [18][19]. From the measurement of the
fluorescence of the samples exciting i) the donor and ii) the acceptor, E can be
determined without further need for reference samples labeled only with D or A,
respectively.
ꢀ
ꢁ
eDðl1Þ eAðl1Þ FA
þ
FAFRETðl1Þ
FAdirectðl2Þ
¼ E
(3)
FD
eAðl2Þ eAðl2Þ
In Eqn. 3, FFRET is the fluorescence intensity of the acceptor excited via FRET (lex
360 nm corresponding to optimal excitation of the donor) and Fdirect for direct exitation
of the acceptor at lex 450 nm, E is the energy-transfer efficiency, e the molar extinction
coefficient at the according wavelength l, and F is the fluorescence quantum yield. The
resulting FRET efficiencies are 0.28 for 20, 0.78 for 18, and 0.30 for 19. Based on the
photophysical data presented, the CF3-substituted derivative 16 shows the best donor
properties for the FRET pair with the [RuII(bathophenanthroline)] complex.
Conclusions. – For the evaluation of different carbostyril chromophores, i.e., 2, 4,
and 5, as donors in a FRET system with a [RuII(bathophenanthroline)] complex as
acceptor, the carbostyrils were turned into Fmoc-amino acid building blocks 15 – 17
directly applicable to solid-phase peptide synthesis and avoiding postsynthetic label-
ling. The photophysical properties of the buildings blocks 3 and 15 – 17 did not differ
significantly from those of the parent heterocycles but indicated the superiority of 16

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and 17 as far as Fçrster radii and quantum yields were concerned. Comparative studies
of FRET efficiencies after incorporation of the donor building blocks into a reference
peptide revealed the highest FRET efficiency for building block 16. Together with its
straightforward synthesis and its chemical robustness, it turned out to be the building
block of choice for the FRET system in combination with [RuII(bathophenanthroline)]
complex 1. This building block will, therefore, be used in the aspired robust and highly
sensitive three-color FRET system we are currently pursuing.
Experimenal Part
General. All reagents were purchased from commercial sources, with the exception of compounds 1,
2, and 6 which were synthesized according to established procedures [13b][16]. The molarity of BuLi was
determined by titration with salicylaldehyde phenylhydrazone [20]. Amine-free DMF (Roth) was
employed throughout peptide syntheses. HPLC: Agilent-1100 system; Nucleosil-100-5-C18-PPN column
(Machery-Nagel). Column chromatography (CC): silica gel 60 (SiO2; Merck). NMR Spectra: Brucker
AM400; at 400 (1H) and 100.6 MHz (13C); chemical shifts d in ppm rel. to the respective solvent signals, J
in Hz. MS: Finnigan MAT-8200 (EI), Thermo LCQ Advantage (ESI); Thermo TCQ 7000 (APCI ¼
atmospheric-pressure chemical ionization); in m/z (rel. %).
All photophysical measurements were carried out in DMSO (spectroscopic grade) without further
purification. UV/VIS Spectra: Lambda-750 UV/VIS spectrometer (Perkin-Elmer). Steady-state
fluorescence measurements: Fluoromax3 spectrometer (Jobin Yvon). A FL920 fluorescence spectrom-
eter (Edinburgh Instruments) in time-correlated single-photon-counting (TCSPC) mode was used for
the measuring of the fluorescence decay times. For the excitation, the frequency-doubled output (second
harmonic generation, SHG) of a titanium – sapphire laser (l 720 nm, lSHG 360 nm) was focused into the
sample. The resulting luminescence was detected in a right-angle configuration to the incoming beam.
For the detection, a multichannel plate (Europhoton) was used. In the data analysis, the resulting decay
curves were deconvoluted with a least-square fitting procedure based on the Levenberg – Marquardt
algorithm. The fluorescence quantum yields were estimated with a photo-luminescence (PL) quantum-
yield measurement system C9920-02 (Hamamatsu, Japan).
i
Ethyl 6,7-Dimethoxy-3-(4-methoxyphenyl)-2-oxoquinoline-1(2H)-acetate (6). To a soln. of Pr2NH
(1.35 ml, 9.64 mmol, 3.0 equiv.) in anh. THF (10 ml) was added at ꢀ 788 dropwise 1.18m BuLi in hexane
(6.80 ml, 8.02 mmol, 2.5 equiv.). The mixture was stirred for 1 h at ꢀ 788 and then allowed to warm to 08.
The soln. was added dropwise to a soln. of quinolin-2(1H)-one 2 (1.00 g, 3.21 mmol, 1.0 equiv.) in anh.
THF (15 ml). The resulting mixture was stirred at 08 for 1 h, before BrCH2COOEt (1.77 ml, 16.04 mmol,
5.0 equiv.) was added. The mixture was stirred for 30 min at 08, and at r.t. for additional 15 h. The reaction
was quenched by the addition of an aq. NH4Cl soln. (30 ml). The aq. phase was extracted with CH2Cl2
(4 ꢁ 100 ml), the combined org. phase dried (MgSO4) and concentrated, and the residue purified by CC
(SiO2, CH2Cl2 ! CH2Cl2/MeOH 98 :2 ! 95 :5): 6 (1.24 g, 97%; [21]: 97%). Pale yellow solid. M.p. 1818
1
([21]; 182 – 1838). H-NMR (CDCl3): 1.26 (t, J ¼ 7.0, MeCH2); 3.84 (s, MeO); 3.94 (s, MeO); 3.95 (s,
MeO); 4.25 (q, J ¼ 7.1, MeCH2); 5.11 (s, CH2N); 6.56 (s, HꢀC(8)); 6.95 (dt, J ¼ 9.1, 2.4, HꢀC(3’,5’)); 7.02
(s, HꢀC(5)); 7.69 (d, J ¼ 8.9, HꢀC(2’,6’)); 7.73 (s, HꢀC(4)). EI-MS: 397 (100, Mþ). Spectroscopic data in
accordance with [21].
6,7-Dimethoxy-3-(4-methoxyphenyl)-2-oxoquinoline-1(2H)-acetic Acid (9). A suspension of
6
(1.40 g, 3.52 mmol, 1.0 equiv.), NaOH (0.86 g, 22 mmol, 6.1 equiv.), H2O (5.5 ml), and EtOH (36 ml)
was heated under reflux for 7 h (!clear soln.). The solvent was evaporated, H2O (80 ml) added, and the
pH adjusted to 1 – 2 with conc. HCl soln. The resulting precipitate was filtered, washed with ice water
(80 ml), and dried by co-evaporation with MeCN: 9 (921 mg, 71%; [21]: 93%). Pale yellow solid. M.p.
1
2038 ([21]: 205 – 2068). H-NMR ((D6)DMSO): 3.79 (s, MeO); 3.82 (s, MeO); 3.89 (s, MeO); 5.10 (s,
CH2N); 6.92 (s, HꢀC(8)); 6.98 (d, J ¼ 8.0, HꢀC(3’,5’)); 7.34 (s, HꢀC(5)); 7.69 (d, J ¼ 8.2, HꢀC(2’,6’));
8.00 (s, HꢀC(4)). APCI-MS: 370 (100, [M þ H]þ). Spectroscopic data in accordance with [21].
(2S)-6-{2-[6,7-Dimethoxy-3-(4-methoxyphenyl)-2-oxoquinolin-1(2H)-yl]acetamido}-2-{[(9H-fluo-
ren-9-ylmethoxy)carbonyl]amino}hexanoic Acid (¼ N6-{2-[6,7-Dimethoxy-3-(4-methoxyphenyl)-2-oxo-

Helvetica Chimica Acta – Vol. 92 (2009)
1941
quinolin-1(2H)-yl]acetyl}-N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-l-lysine; 15). To a suspension of 9
(369 mg, 1.00 mmol, 1.0 equiv.) in anh. THF (20 ml) was added slowly at 08 N-hydroxysuccinimide
(115 mg, 1.00 mmol, 1.0 equiv.) and N,N’-diisopropylcarbodiimide (88.1 ml, 1.00 mmol, 1.0 equiv.). The
mixture was stirred for 15 h at 08. The solvent was evaporated and the residue redissolved in DMF/H2O
9 :1 (30 ml) and Titrisolꢃ buffer (pH 7.0; 7.5 ml). To this soln., Fmoc – Lys – OH (479 mg, 1.30 mmol, 1.3
equiv.) was added, and the mixture was stirred at r.t. for 20 h. The solvent was evaporated, the residue
suspended in H2O (100 ml), and the pH adjusted to 1 – 2 with conc. HCl soln. The resulting precipitate
was filtered and washed with ice water (100 ml). The residue was purified by CC (SiO2, CH2Cl2 !
CH2Cl2/MeOH/AcOH 95 :5 :0.1): 15 (410 mg, 57% over two steps). White solid. 1H-NMR
((D6)DMSO): 1.29 – 1.48 (m, NHCHCH2CH2); 1.55 – 1.76 (m, CONHCH2CH2); 3.07 – 3.14 (m,
CONHCH2); 3.79 (s, MeO); 3.81 (s, MeO); 3.84 (s, MeO); 3.89 – 3.93 (mc, HꢀC(9) of Fmoc); 4.21 (t,
J ¼ 6.96, CHCO2H); 4.27 – 4.31 (m, CH2 of Fmoc); 5.00 (s, NCH2CON); 6.78 (s, HꢀC(8) of quin.); 6.98
(dt, J ¼ 9.8, 2.6, HꢀC(3,5) of 4-MeOC6H4); 7.31 – 7.34 (m, HꢀC(4,5) of Fmoc); 7.40 (dd, J ¼ 7.5, 7.4,
HꢀC(2,7) of Fmoc); 7.60 (d, J ¼ 8.1, NHCHCO2H); 7.68 – 7.74 (m, HꢀC(3,6) of Fmoc, HꢀC(2,6) of 4-
MeOC6H4); 7.88 (d, J ¼ 7.5, HꢀC(1,8) of Fmoc); 7.98 (s, HꢀC(4) of quin.); 8.24 (t, J ¼ 5.6, CONHCH2);
12.61 (br. s, CO2H). 13C-NMR ((D6)DMSO): 13.9; 21.0; 22.1; 23.0; 28.3; 28.7; 30.4; 31.2; 45.4; 46.6; 53.7;
55.1; 55.8; 65.6; 97.8; 110.0; 113.3; 113.5; 120.1; 125.2; 127.0; 127.2; 127.6; 129.3; 129.8; 134.6; 135.8; 140.7;
143.8; 144.7; 151.5; 156.1; 158.7; 160.4; 166.9; 172.0; 173.9. APCI-MS: 718 (100, [M ꢀ H]ꢀ). Anal. calc. for
C41H41N3O9 · 0.5 H2O (728.79): C 67.63, H 6.33, N 5.57; found: C 67.57, H 5.98, N 5.77.
Ethyl 6,7-Dimethoxy-2-oxo-4-(trifluoromethyl)quinoline-1(2H)-acetate (7). As described for 6, with
iPr2NH (1.24 ml, 8.79 mmol, 3.0 equiv.), THF (10 ml) 1.18m BuLi in hexane (6.21 ml, 7.33 mmol, 2.5
equiv.), 6,7-dimethoxy-4-(trifluoromethyl)quinolin-2(1H)-one (4; 0.80 g, 2.93 mmol, 1.0 equiv.), THF
(15 ml), and BrCH2COOEt (1.62 mmol, 16.7 mmol, 5.0 equiv.). CC (SiO2, CH2Cl2 ! CH2Cl2/MeOH
98 :2) afforded 7 (0.91 g, 86%). Pale yellow solid. M.p. 1508 ([15a]: 150 – 1518). 1H-NMR (CDCl3): 1.26
(t, J ¼ 7.2, MeCH2); 3.94 (s, MeO); 3.96 (s, MeO); 4.25 (q, J ¼ 7.1, MeCH2); 5.11 (s, CH2N); 6.58 (s,
HꢀC(3)); 7.01 (s, HꢀC(8)); 7.23 (s, HꢀC(5)). EI-MS: 359 (100, Mþ). Spectroscopic data in accordance
with [15a].
6,7-Dimethoxy-2-oxo-4-(trifluoromethyl)quinoline-1(2H)-acetic Acid (10). As described for 9, with 7
(0.90 g, 2.50 mmol, 1.0 equiv.), NaOH (0.60 g, 15 mmol, 6.0 equiv.), H2O (3.9 ml), EtOH (35 ml), H2O
(50 ml), and conc. HCl. The resulting precipitate was filtered, washed with ice water (100 ml) and dried
by azeotropic co-evaporation with MeCN. The combined aq. phase was extracted with CH2Cl2 (4 ꢁ
100 ml), the combined org. phase dried (MgSO4) and concentrated, and the residue combined with
the precipitate to yield 10 (688 mg, 83%: [15a]: 81%). Pale yellow solid. M.p. 2828 ([15a]: 284 – 2858).
1H-NMR ((D6)DMSO): 3.84 (s, MeO); 3.93 (s, MeO); 5.13 (s, CH2N); 6.97 (s, HꢀC(3)); 7.04 (s,
HꢀC(8)); 7.10 (s, HꢀC(5)); 13.16 (br. s, CO2H). APCI-MS: 330 (100, [M ꢀ H]ꢀ). Spectroscopic data in
accordance with [15a].
(2S)-6-{2-[6,7-Dimethoxy-2-oxo-4-(trifluoromethyl)quinolin-1(2H)-yl]acetamido}-2-{[(9H-fluoren-
9-ylmethoxy)carbonyl]amino}hexanoic Acid (¼ N6-{2-[6,7-Dimethoxy-2-oxo-4-(trifluoromethyl)quino-
lin-1(2H)-yl]acetyl}-N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-l-lysine; 16). As described for 15, with 16
1
(500 mg, 73% over two steps). White solid. H-NMR ((D6)DMSO): 1.22 – 1.46 (m, NHCHCH2CH2);
1.55 – 1.75 (m, CONHCH2CH2); 3.06 – 3.12 (m, CONHCH2); 3.81 (s, MeO); 3.88 (s, MeO); 3.89 – 3.92
(m, HꢀC(9) of Fmoc); 4.21 (t, J ¼ 6.95, CHCO2H); 4.26 – 4.31 (m, CH2 of Fmoc); 4.99 (s, CH2 of quin.);
6.87 (s, HꢀC(5) of quin.); 6.94 (s, HꢀC(8) of quin.); 7.07 (s, HꢀC(3) of quin.); 7.30 (d, J ¼ 7.1, H ꢀC(4)
of Fmoc); 7.32 (d, J ¼ 7.1, H ꢀC(5) of Fmoc); 7.39 (dd, J ¼ 7.5, 7.4, HꢀC(2,7) of Fmoc); 7.59 (d, J ¼ 8.0,
NHCHCO2H); 7.72 (dd, J ¼ 7.5, 7.1, HꢀC(3,6) of Fmoc); 7.87 (d, J ¼ 7.6, HꢀC(1,8) of Fmoc); 8.28 (t, J ¼
5.5, CONHCH2); 12.61 (br. s, CO2H). 13C-NMR ((D6)DMSO): 13.9; 22.1; 23.0; 23.3; 28.6; 30.4; 31.2;
45.3; 46.6; 53.7; 55.8; 65.6; 98.9; 105.5; 107.0; 127.0; 127.6; 136.6; 140.7; 143.8; 145.1; 152.7; 156.1; 159.5;
166.2; 173.9. ESI-MS: 680 (100, Mꢀ).
i
Ethyl 4-Cyano-6,7-dimethoxy-2-oxoquinoline-1(2H)-acetate (8). As described for 6, with Pr2NH
(0.92 ml, 6.5 mmol, 3.0 equiv.), THF (7 ml), 1.35m BuLi in hexane (4.0 ml, 5.4 mmol, 2.5 equiv.), 1,2-
dihydro-6,7-dimethoxy-2-oxoquinoline-4-carbonitrile (5; 0.50 g, 2.2 mmol, 1.0 equiv.), THF (10 ml), and
BrCH2COOEt (0.50 g, 2.2 mmol, 1.0 equiv.). CC (SiO2, CH2Cl2 ! CH2Cl2/MeOH 98 :2) afforded 8
(0.60 g, 88%). Pale green solid. M.p. 2178 ([16]: 220 – 2218). 1H-NMR ((D6)DMSO): 1.22 (t, J ¼ 7.1,

1942
Helvetica Chimica Acta – Vol. 92 (2009)
MeCH2); 3.83 (s, MeO); 3.94 (s, MeO); 4.18 (q, J ¼ 7.1, MeCH2); 5.19 (s, CH2N); 7.00 (s, HꢀC(3)); 7.37
(s, HꢀC(8)); 8.65 (s, HꢀC(5)). APCI-MS: 317 (100, [M þ H]þ). Spectroscopic data in accordance with
[16].
4-Cyano-6,7-dimethoxy-2-oxoquinoline-1(2H)-acetic Acid (11). A soln. of 8 (516 mg, 1.63 mmol, 1.0
equiv.), 1m aq. NaOH (3.4 ml, 3.4 mmol, 2.1 equiv.), and EtOH (35 ml) was heated under reflux for 7 h.
The solvent was evaporated, and the residue dissolved in H2O (10 ml). The mixture was acidified to
pH 1 – 2 with conc. HCl soln. The formed precipitate was filtered, washed with H2O (100 ml), and dried
by co-evaporation with MeCN. The combined aq. layer was extracted with CH2Cl2 (4 ꢁ 100 ml), the
combined org. phase dried (MgSO4) and concentrated, and the residue combined with the precipitate to
yield 11 (348 mg, 74%; [16]: 80%). Pale green solid. M.p. 2808 ([16]: 281 – 2828). 1H-NMR ((D6)DMSO):
3.83 (s, MeO); 3.93 (s, MeO); 5.08 (s, CH2N); 6.99 (s, HꢀC(3)); 7.36 (s, HꢀC(8)); 8.63 (s, HꢀC(5)). ESI-
MS: 287 (100, [M ꢀ H]ꢀ). Spectroscopic data in accordance with [16].
(2S)-6-{2-[4-Cyano-6,7-dimethoxy-2-oxoquinolin-1(2H)-yl]acetamido}-2-{[(9H-fluoren-9-ylmethoxy)-
carbonyl]amino}hexanoic Acid (¼ N6-{2-[4-Cyano-6,7-dimethoxy-2-oxoquinolin-1(2H)-yl]acetyl}-N2-
[(9H-fluoren-9-ylmethoxy)carbonyl]-l-lysine; 17). As described for 15, with 11 (188 mg, 0.652 mmol,
1.0 equiv.), THF (15 ml), N-hydroxysuccinimide (75.2 mg, 0.652 mmol, 1.0 equiv.), N,N’-diisopropyl-
carbodiimide (57.5 ml, 0.652 mmol, 1.0 equiv.), DMF/H2O 9 :1 (20 ml), Titrisol bufferꢃ (pH 7.0; 4.5 ml),
and Fmoc – Lys – OH (312 mg, 0.848 mmol, 1.3 equiv.). The pH of the suspension in H2O (70 ml) was
adjusted to 1 – 2 with conc. HCl soln. and the resulting precipitate filtered and washed with ice water
(100 ml). The residue was purified by CC (SiO2, CH2Cl2/MeOH/AcOH 98 :2 :0.1 ! 98 :5 :0.1): 17
(140 mg, 34% over two steps). Pale green solid. 1H-NMR ((D6)DMSO): 1.28 – 1.48 (m, NHCHCH2CH2);
1.54 – 1.76 (m, CONHCH2CH2); 3.06 – 3.12 (m, CONHCH2); 3.81 (s, MeO); 3.88 (s, MeO); 3.89 – 3.93
(m, HꢀC(9) of Fmoc); 4.21 (t, J ¼ 7.01, CHCO2H); 4.24 – 4.31 (m, CH2 of Fmoc); 4.97 (s, CH2N); 6.81 (s,
HꢀC(3)); 7.29 – 7.34 (m, HꢀC(8), HꢀC(4,5) of Fmoc); 7.40 (dd, J ¼ 7.5, 7.4, HꢀC(2,7) of Fmoc); 7.55 (d,
J ¼ 8.1, NHCHCO2H); 7.72 (dd, J ¼ 7.5, 7.1, HꢀC (3,6) of Fmoc); 7.88 (d, J ¼ 7.5, HꢀC(1,8) of Fmoc);
8.28 (t, J ¼ 5.4, CONHCH2); 12.61 (br. s, CO2H). 13C-NMR ((D6)DMSO): 23.0; 28.6; 30.4; 38.5; 45.4;
46.3; 53.8; 55.8; 56.2; 65.5; 97.9; 101.0; 110.2; 112.1; 116.5; 120.0; 125.2; 127.0; 127.6; 137.7; 140.7; 143.8;
145.4; 147.4; 155.0; 156.1; 158.5; 166.0; 174.0. ESI-MS: 662 (87, [M þ Na]þ), 639 (100, [M þ H]þ). Anal.
calc. for C35H34N3O8 · 3/2 H2O (665.69): C 64.61, H 5.47, N 8.61; found: C 64.88, H 5.98, N 8.22.
Peptide Syntheses. Standard peptide synthesis was employed on a 0.025 mmol scale, by using the
Fmoc/tBu protocol [22] and TentaGel S Ram resin (Rapp Polymere; loading 0.24 mmol/g) with O-(1H-
benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU) [17] as a coupling reagent.
Standard enantiomeric pure Boc and tBu side-chain-protected amino acids were employed. The modified
amino acid building blocks 1, 3, 16, and 17 were introduced with an 1.2-fold excess, with TBTU as
coupling reagent and the standard protocol with an elongated coupling time of 15 h. The peptide was
deprotected and cleaved from the solid support by exposure to CF3COOH/H2O/iPr3SiH 95 :2.5 :2.5 for
2 h. The cleavage cocktail was treated with Et2O to precipitate the peptides. The peptides 18 – 20 were
finally isolated by prep. HPLC.
Sodium {l-Histidyl-l-alanyl-N6-{1-oxo-5-[4-(7-phenyl-1,10-phenanthrolin-4-yl-kN1,kN10)phenyl]-
pentyl}-l-lysyl-l-tyrosyl-l-histidyl-N6-{2-[6,7-dimethoxy-2-oxo-4-(trifluoromethyl)quinolin-1(2H)-yl]-
acetyl}-l-lysylglycinamide}bis{(1,10-phenanthroline-4,7-diyl-kN1,kN10)bis[benzenesulfonato](2ꢀ)}ruthenate-
(2ꢀ) Chloride (4 :1 :2) (18 · 2 Clꢀ): ESI-MS: 1326 (100, [M ꢀ 4 Na þ 4 H ꢀ 2 Cl]2þ), 884 (10, [M ꢀ
4 Na þ 5 H ꢀ 2 Cl]3þ).
Sodium {l-Histidyl-l-alanyl-N6-{1-oxo-5-[4-(7-phenyl-1,10-phenanthrolin-4-yl-kN1,kN10)phenyl]-
pentyl}-l-lysyl-l-tyrosyl-l-histidyl-N6-{2-[4-cyano-6,7-dimethoxy-2-oxoquinolin-1(2H)-yl]acetyl}-l-lysyl-
glycinamide}bis{(1,10-phenanthroline-4,7-diyl-kN1,kN10)bis[benzenesulfonato](2 ꢀ)}ruthenate(2ꢀ)
Chloride (4 :1:2) (19 · 2 Clꢀ): ESI-MS: 1305 (100, [M ꢀ 4 Na þ 4 H ꢀ 2 Cl]2þ), 870 (20, [M ꢀ 4 Na þ
5 H ꢀ 2 Cl]3þ).
Sodium {l-Histidyl-l-alanyl-N6-{1-oxo-5-[4-(7-phenyl-1,10-phenanthrolin-4-yl-kN1,kN10)phenyl]-
pentyl}-l-lysyl-l-tyrosyl-l-histidyl-4-{3-[6,7-dimethoxy-3-(4-methoxyphenyl)-2-oxoquinolin-1(2H)-yl]-
propyl}-l-phenylalanylglycinamide}bis{(1,10-phenanthroline-4,7-diyl-kN1,kN10)bis[benzenesulfonato]-
(2ꢀ)}ruthenate(2ꢀ) Chloride (4 :1:2) (20 · 2 Clꢀ): ESI-MS: 1355 (100, [M ꢀ 4 Na þ 4 H ꢀ 2 Cl]2þ).

Helvetica Chimica Acta – Vol. 92 (2009)
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Received June 16, 2009