Application of a highly robust and efficient fluorescence-resonance-energy- transfer (FRET) system in DNA

Article abstract of DOI:10.1002/hlca.200790107

We report a feasibility study for the application of our newly developed highly efficient and robust fluorescence-resonance-energy-transfer (FRET) system to DNA. A 2′-oligodeoxynucleotide, 12, equipped with a quinolinone derivative as donor and a (bathoph

Full text of DOI:10.1002/hlca.200790107

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Application of a Highly Robust and Efficient Fluorescence-Resonance-
Energy-Transfer (FRET) System in DNA
by Lilia Clima, Caroline Hirtz-Haag, Andrea Kienzler, and Willi Bannwarth*
Institut für Organische Chemie und Biochemie, Albert-Ludwigs Universität Freiburg, Albertstr. 21,
D-79104 Freiburg (phone: þ 497612036073; fax: þ 497612038705;
e-mail: willi.bannwarth@organik.chemie.uni-freiburg.de)
Dedicated to Professor Wolfgang Pfleiderer
We report a feasibility study for the application of our newly developed highly efficient and robust
fluorescence-resonance-energy-transfer (FRET) system to DNA. A 2’-oligodeoxynucleotide, 12,
equipped with a quinolinone derivative as donor and a (bathophenanthroline)ruthenium(II) complex
as acceptor and having a single uridine as potential cleavage site under basic conditions revealed an
intensive FRET, which almost vanished after cleavage of the oligonucleotide under basic conditions
(Fig. 7). Furthermore, in the arrangement of a molecular beacon (MB) DNA (see 13), a significant
decrease of the FRET was observed after hybridization to a target sequence (Fig. 9). Due to the long
decay times of the fluorescence of the Ru-complex, the system allows for highly sensitive time-gated
measurements.
Introduction. – Fluorescence resonance energy transfer (FRET) is based on the
transfer of fluorescence energy between a donor chromophore and a corresponding
acceptor fluorophore. According to the Fçrster equation, the intensity of this transfer is
highly dependent on the distance of the two chromophores and decreases with rÀ6, r
being the distance between the donor chromophore and the acceptor entity [1].
Furthermore, suitable spectral properties of the two dyes are required, i.e., the emission
wavelength of the donor should sufficiently overlap with the absorption wavelength of
the acceptor. An additional requirement is the correct orientation of these two to each
other.
What makes such systems especially attractive is their sensitivity towards slight
changes in the distance of donor and acceptor allowing to follow biochemical events in
a real time mode. Even the folding process of a protein was investigated in vivo by
FRET measurements [2]. The myriad applications of FRET have been summarized in
a number of reviews [3 – 6]. FRET has been used as a tool for the development of
DNA-based assays, DNA probes, and DNA sequencing [7][8]. The concept of
molecular beacons (MB) as DNA probe has been largely used. It consists of a
fluorophore at one end of a hairpin DNAwith a quencher attached to the other end [9 –
11]. Although advantageous, it depends on one fluorophore which has its emission at a
specific wavelength. We describe the extension of our FRET system to molecular
beacons. Our system has the advantage that it offers the possibility to monitor the
fluorescence at two wavelengths [12]. Upon binding to a complementary sequence, the
ꢀ 2007 Verlag Helvetica Chimica Acta AG, Zürich

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FRET will be reduced, hence the emission of acceptor will decrease, whereas the
emission of the donor will increase.
Meanwhile, a number of different donor– acceptor combinations for FRET have
been reported. The most prominent ones are fluorescein – rhodamine, Cy3 – Cy5,
dabcyl-edans, and blue and green fluorescent protein [13]. However, despite of the
number of reported systems, sensitivity and robustness still remains an issue. A possible
solution to the sensitivity problem are FRET systems which can be measured in a time-
resolved mode. This requires a long excited-state lifetime but yields excellent signal-to-
noise ratios. Suitable candidates for this purpose are lanthanide complexes [14]. They
are commonly employed as caged chelates; however, they have relatively low
extinction coefficients, and relatively low stability, resulting in dissociation at low
concentrations, so that leakage of the lanthanide metal can occur.
Recently, we have published an alternative donor– acceptor pair which can be
measured in a time-resolved mode [15][16]. The system is based on the (batho-
phenanthroline)ruthenium(II) complex 1a as acceptor entity (bathophenanthroline ¼
4,7-diphenyl-1,10-phenanthroline) [17] (Fig. 1). After its excitation at 450 nm, it
reveals a metal-to-ligand charge transfer (MLCT) causing an emission at ca. 620 nm.
Depending on the conditions, the lifetime for the excited state is in the range of 0.5 –
5 ms allowing for time-gated measurements with high sensitivity since interferences
caused by autofluorescent compounds, color quenchers, or by scattered light from
precipitated particles can be avoided. In addition, the [RuII(bathophenanthroline)]
complexes are thermodynamically very stable and chemically inert. A further
advantage is their high stability towards light and their large Stokes shift. An additional
feature is that the Ru-complex as acceptor reveals an absorption minimum at the
Fig. 1. DonorÀacceptor pair

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wavelength of the excitation of the donor so that direct excitation of the acceptor is
minimal. RuII Complex 1a is soluble in aqueous systems due to the sulfonyl groups and
can be coupled covalently to target molecules via stable amide functions. As suitable
donor molecule, we have identified a substituted quinolinone of which we prepared the
derivative 2a (Fmoc¼ (9H-fluoren-9-ylmethoxy)carbonyl). Building block 2a repre-
sents a phenylalanine analogue and can be inserted during solid-phase synthesis into
peptides. The donor molecule 2a is very robust and has a high extinction coefficient.
When donor 2a and acceptor 1a were incorporated into a peptide at abuttal sides to the
recognition sequence for thrombin, a high FRET could be observed and the ratio of
signal to noise was ca. 12.
2. Results and Discussion. – Here we demonstrate a feasibility study for the
application of the newly developed system in DNA by using the [RuII(bathophenan-
throline)] complex 1b as acceptor and the quinolinone derivative 2b as donor. Complex
1b differs from 1a in the absence of sulfonate groups which were used with peptides for
solubility reasons. They are not necessary while working with DNA since DNA
sufficiently mediates the solubility in aqueous solution. Complex 1b was prepared like
1a but starting from unmodified bathophenanthroline as ligand [16].
The donor entity was synthesized according to Scheme 1. The quinolinone
derivative 3, which showed an absorption maximum at 368 nm and an emission with
a maximum at 435 nm and a high extinction coefficient (e 20800 mÀ1 cmÀ1), was
transformed into the allyl-substituted compound 4 as previously described [15][16]. A
Heck reaction with 4-bromobenzeneacetic acid yielded compound 5 which was
hydrogenated to give 2b. Activation of the carboxy function of 2b with TSTU yielded
the desired activated ester 6 [18] (TSTU ¼ N,N,N’,N’-tetramethyl-O-succinimidouro-
nium tetrafluoroborate). During this transformation, no change in the spectral
properties could be observed.
The concept for a feasibility study for the FRET system in a single-stranded
synthetic DNA fragment is outlined in Fig. 2. The activated donor 2b (i.e., 6) was
coupled to a 5-(3-aminoprop-1-ynyl)-2’-deoxyuridine (¼a-(2-aminoethylidyne)thymi-
dine) unit via an amide bond. The 5’-end of the oligonucleotide consisting of a modified
5’-amino-5’-deoxythymidine was coupled with the activated acceptor 1b (as the
hydroxysuccinimide ester). As potential cleavage site, we inserted a uridine unit which
could be specifically cleaved under basic conditions [19][20]. In this construct, both
donor and acceptor are in close proximity so that a FRET is possible, whereas after
cleavage under basic conditions, the donor and acceptor are separated. As a result no
FRET is observed.
To evaluate our strategy, we had to investigate first the cleavage conditions, and,
therefore, we synthesized the oligonucleotide 5’-d(CCGAT)Ud(TATCAT)-3’ (7)
consisting of 2’-deoxynucleotide building blocks, except the uridine at the potential
cleavage point. Oligonucleotide 7 was subjected to basic conditions, resulting in the
expected fragments as shown by HPLC (Fig. 3).
For the insertion of the amino functions into DNA allowing to attach the donor and
the acceptor, we needed the two modified building blocks 5-(3-aminoprop-1-ynyl)-2’-
deoxyuridine phosphoramidite 8 and 5’-amino-5’-deoxythymidine phosphoramidite 9,
respectively (Fig. 4). Phosphoramidite 8 was synthesized by performing first a

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Scheme 1. Synthesis of Donor Molecule 6
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a) KHMDS (potassium hexamethyldisilazanide), allyl bromide, À 788, 1.5 h, r.t., 30 min, microwave
1208, 15 min; 83%. b) [Pd(OAc)2], PPh3, Cs2CO3, H2O, DMF, 1208, 16 h; 99%. c) H2, Pd/C, MeOH,
i
overnight; quant. d) TSTU, Pr2EtN, DMF, r.t., 2 h, (79%).
Sonogashira coupling between 5-iodo-2’-deoxyuridine and 2,2,2-trifluoro-N-(prop-2-
ynyl)acetamide [21]. Under ligand-free conditions (Pd/C) and by using resin-bound
Et3N (Amberlite IRA-67) instead of Et3N, the desired nucleoside was obtained after
chromatographic purification (silica gel) in 79% yield [22]. This step was followed by

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Fig. 2. Design for the feasibility study of the FRET in single-stranded DNA
regioselective introduction of the 4,4’-dimethoxytrityl ((MeO)2Tr) group into the 5’-
position and conversion to the pertinent phosphoramidite at the 3’-position with 2-

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Fig. 3. HPLC for the cleavage of oligonucleotide 7 under basic conditions. Oligonucleotide 7 treated with
NaOH (pH 12) overnight at r.t. Ion-exchange HPLC 0 ! 60% B/A in 35 min; A ¼ 20 mm KH2PO4 in
MeCN/H2O 1:4 (pH 6); B ¼ 1m KCl 20 mm KH2PO4 in MeCN/H2O 1:4 (pH 6).
cyanoethyl tetraisopropylphosphorodiamidite) in the presence of diisopropylammo-
nium 1H-tetrazolide for the phosphinylation step. Phosphoramidite 9 was synthesized
by a route reported previously with an overall yield of 53% [23].
To evaluate the spectral overlap between the donor and the acceptor when
incorporated into single-stranded DNA, we synthesized the two DNA fragments 10 and
11 (Fig. 5,a). Fragment 10 was synthesized bearing a phosphate group at the 3’
terminus and corresponds to the fragment obtained after cleavage of oligonucleotide
12. In both fragments 10 and 11, the modified building blocks 8 and 9 were incorporated
at specific sites to yield a primary amino function after deprotection for the attachment
of the donor 2b and the acceptor 1b. The coupling of both donor as well as acceptor was
performed via their hydroxysuccinimide-derived ester in DMF/dioxane/H2O in the

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Fig. 4. Modified phosphoramidites 8 and 9 employed for the insertion of amino functions allowing the
coupling of the donor and acceptor
presence of iPr2EtN [17]. Both oligonucleotides 10 and 11 were purified by HPLC and
analyzed by polyacrylamide-gel electrophoresis (PAGE, Fig. 5,b). An excitation and
emission spectrum of the two sequences (Fig. 5,c) revealed a strong overlap of the
emission band of the donor with the absorption band of the acceptor which is a
prerequisite for an efficient FRET.
Having confirmed the sufficient spectral overlap (Fig. 5,c), we synthesized
oligonucleotide 12 (Scheme 2) which bears both the donor and the acceptor and has,
furthermore, a uridine as specific cleavage point located between the donor and the
acceptor. Oligonucleotide 12 was synthesized by standard phosphoramidite techniques
incorporating the modified building blocks 8 and 9 in their appropriate positions.
Uridine was implemented as 2’O-[(tert-butyl)dimethylsilyl]-protected phosphorami-
dite. After insertion of the last building block, the 4-monomethoxytrityl (MeOTr)
group was not removed. After deprotection and removal from the support with base,
the activated donor 2b (i.e., 6) was coupled to the aminopropynyl group. This was
followed by the removal of the MeOTr group and the attachment of the acceptor 1b to
the 5’-amino function. Finally, the (tert-butyl)dimethylsilyl (tbdms) group was cleaved
off with a mixture of Et3N · 3 HF, 1-methylpyrrolidin-2-one (NMP), and Et N to yield
3
crude fragment 12 which was purified by HPLC and PAGE (Fig. 6).
When evaluating the spectroscopic properties of 12 (Fig. 7), a high FRET with an
emission maximum at 620 nm could be observed after excitation at 350 nm. After
cleavage of 12 under basic conditions, the FRET vanished. The ratio of the FRET
signal before and after cleavage was ca. 17:1. This weak fluorescence signal after
cleavage was in the same range as that observed with an equimolar ratio of the
independently synthesized fragments 10 and 11.
In a further evaluation, the FRET donor and the FRET acceptor were extended to
the MB approach. Therefore, the hairpin sequence 13 was synthesized (Scheme 3),
carrying the quinolinone-derived FRET donor at the 3’-end, and the [RuII(bathophe-
nanthroline)] FRET acceptor at the 5’-end. Oligonucleotide 13 was synthesized by a

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Fig. 5. a) Oligonucleotides 10 and 11 for the evaluation of the spectral overlap (X ¼ 5’-amino-5’-
deoxythymidine building block, Y¼ 5-(3-aminoprop-1-ynyl-2’-deoxyuridine building block).
b) Polyacrylamide-gel electrophoresis (20%): Lane 1, fragment 10; Lane 2, fragment 11 (at 366 nm);
Lane 3, fragment 10; Lane 4, fragment 11; Lane 5, oligonucleotide 9-mer as reference (stained with 3,3’-
diethyl-9-methyl-4,5,4’,5’-dibenzothiacarbocyanine bromide solution). c) Excitation/emission spectra of
oligonucleotide 10 (- - - for excitation, – · – for emission) and oligonucleotide 11 (— for excitation,
· · · for emission).

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Scheme 2. Synthesis of Oligonucleotide 12
a) 2m NH4OH in EtOH, 30 min, 658. b) 7m MeNH2 in EtOH, 30 min, 658. c) 6 (from 2b), iPr2EtN, DMF/
i
dioxane/H2O 1:1:1, 16 h, 258. d) 80% AcOH. e) 1b (as hydroxysuccinimide-derived ester), Pr2EtN,
DMF/dioxane/H2O 1:1:1, 24 h, 258. f) Et3N · 3 HF, 1-methylpyrrolidin-2-one (NMP), Et N.
3
Fig. 6. Polyacrylamide-gel electrophoresis (20%). Lane 1, fragment 12; Lane 2, fragment 12 labelled with
donor (without acceptor, used as the reference; at 366 nm); Lane 3, fragment 12; Lane 4, fragment 12
labelled with donor (without acceptor, used as the reference, stained with 3,3’-diethyl-9-methyl-4,5,4’,5’-
dibenzothiacarbocyanine bromide solution).

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Fig. 7. a) Cleavage of fragment 12 under basic conditions. b) Fluorescence emission spectrum of
oligonucleotide 12 (solid line), and of fragments 10 and 11 after cleavage (dotted line).
standard phosphoramidite protocol, however, by using the amino-ON CPG1) to
introduce a primary amino group at the 3’-end and modified phosphoramidite 9 at the
5’-end. After insertion of the last building block, the MeOTr group was not removed.
After deprotection and removal from the support with base, the donor 2b (i.e., 6) was
coupled to the amino group at the 3’-end. This was followed by the removal of the
MeOTr group and the attachment of the acceptor 1b to the 5’-amino function to yield
crude fragment 13 which was purified by HPLC and analyzed by PAGE (Fig. 8).
In the backfolding situation, sequence 13 revealed a strong FRET as can be seen
from Fig. 9,b. The addition of a noncomplementary sequence had no influence on the
FRET, but hybridization to the complementary oligonucleotide sequence 14 led to a
reduced FRET due to the increase of the distance between the donor and the acceptor.
The excitation lifetime (t) of the donor in fragment 11 was estimated to be 0.52 ns,
whereas the one for the acceptor in fragment 10 was 0.40 ms (Fig. 10). This huge
difference in the lifetime is the basis for the possibility to measure the fluorescence of
1
)

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Scheme 3. Synthesis of Oligonucleotide 13
i
a) NH4OH. b) 6 (from 2b), Pr2EtN, DMF/dioxane/H2O 1:1:1, 16 h, 258. c) 80% AcOH. d) 1b (as
i
hydroxysuccinimide-derived ester), Pr2EtN, DMF/dioxane/H2O 1:1:1, 24 h, 258; hybridization: 5 mm
phosphate buffer, 0.1 mm EDTA, 100 mm NaCl.
the system in a time-gated mode with high sensitivity due to the possibility to eliminate
the background fluorescence.
Conclusions. – We described the application of a new, robust, and highly sensitive
FRET system in DNA. The system is based on the [RuII(bathophenanthroline)]
complex 1b as FRETacceptor and the quinolinone derivative 2b as FRET donor. Both
dyes were synthesized by straightforward routes in high yields.
Absorption and emission spectra of oligonucleotides bearing only the donor or the
acceptor dye showed a good spectral overlap of the emission band of the donor with the
absorption band of the acceptor which is a crucial requirement for a strong FRET. In
addition, at the excitation wavelength of the donor (350 nm), the absorption spectrum
of the Ru-complex reveals a minimum, thereby preventing direct excitation of the
acceptor at 350 nm almost completely. The FRET was demonstrated in a single-
stranded oligonucleotide bearing donor, acceptor, and a uridine moiety as the specific
cleavage site. A remarkable intense FRET was observed in the intact DNA but
vanished almost completely after cleavage under basic conditions.
A molecular-beacon DNA revealed also an intense FRET due to the close
proximity between donor and acceptor, but it decreased significantly after hybrid-
ization to a complementary sequence. Compared to donor-quencher systems, the
change of two fluorescence intensities can be monitored. The reported system allows
for measurements in normal as well as in a time-resolved mode because of the long

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Fig. 8. Polyacrylamide-gel electrophoresis (20%). Lane 1, fragment 13; Lane 2, fragment 13b (at
366 nm); Lane 3, oligonucleotide 13; Lane 4, fragment 13b (stained with 3,3’-diethyl-9-methyl-4,5,4’,5’-
dibenzothiacarbocyanine bromide solution).
fluorescence lifetime of the Ru-complex, thereby increasing the sensitivity due to the
elimination of background fluorescence. We intend to apply the FRET system to the
investigation of supramolecular interactions and focus furthermore on extensions to
multi-FRET systems.
The authors would like to thank Prof. P. Gräber and R. Bienert for the fluorescence-spectra
measurements and for recording the fluorescence-lifetime-decay curve of the donor chromophore and
Prof. C. Seidel and Dr. S. Kalinin for recording the fluorescence-lifetime-decay curves of the acceptor
chromophore.
Experimental Part
General: Oligonucleotide synthesis was carried out on an Expedit-6800 DNA synthesizer on a 1 mmol
scale by means of standard phosphoramidite technique. Modified CPG1) was purchased from Proligo.
Replacement of NHþ4 with Kþ was done by co-evaporation (3ꢀ) of the oligonucleotide with KCl (10 mg)
in H2O (200 ml). Oligonucleotide samples were desalted on NAP-10 columns. Column chromatography
(CC): short column; Merck silica gel 60. HPLC: Merck/Hitachi system; reversed phase: SP-250/10-
Nucleosil-100-5-C18 or EC-125/4-Nucleosil-100-5-C18 columns, A ¼ 0.1m Et3NH(OAc) buffer at pH 7.0,
B ¼ MeCN; ion-exchange: 250/4 Dionex DNAPac-PA-100 column, A ¼ 20 mm KH2PO4 in MeCN/H2O
1:4 at pH 6.0, B ¼ 1m KCl, 20 mm KH2PO4 in MeCN/H2O 1:4 at pH 6.0. PAGE: polyacrylamide gels
were stained with a soln. of 3,3’-diethyl-9-methyl-4,5,4’,5’-dibenzothiacarbocyanine bromide (ꢁStains-

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Fig. 9. a) Structure of the molecular beacon and hybridization to the complimentary target sequence
(hybridization conditions: 5 mm phosphate buffer, 0.1 mm EDTA, 100 mm NaCl). b) Fluorescence
emission spectrum of MB (—), MB þ noncomplementary oligonucleotide 15 (· · · ), and MB 13 þ target
oligonucleotide 14 (- - -), excitation at 350 nm.
allꢂ; Fluka). UV Spectra: Perkin-Elmer-Lambda-35-UV/VIS spectrometer. Fluorescence spectra:
Perkin-Elmer LS45 spectrometer. Fluorescence lifetime decay: IBH Consultants Ltd. (Glasgow,
Scotland), N2 flash lamp as excitation source (microsecond range); FluoTime 200 from Picoquant with
a pulsed LED (nanosecond range). 1H- (300 MHz) and 13C-NMR (125 MHz) spectra: Varian Mercury-
VX-300, and Bruker DRX-500 spectrometers, resp.; chemical shifts d in ppm rel. to Me4Si, referenced to
residual solvent signals; J in Hz. MS: LCQ Advantage (ESI) mass spectrometer.
Analytical Gel Electrophoresis. Polyacrylamide gels (20%) of 0.4 mm thickness were used. Pre-
electrophoresis was performed for 2 h at 500 V with Tris borate running buffer. Oligonucleotide (1 ml,
0.1 OD units) and bromophenol blue/xylenecyanol soln. (3 ml) were heated to 908 for 2 min and rapidly
cooled to 08. Electrophoretic separation was performed for 2 h at 500 V and 4 mA. Oligonucleotide
bands were visualized at 366 nm or stained.
4-{3-[1,2-Dihydro-6,7-dimethoxy-3-(4-methoxyphenyl)-2-oxoquinolin-1-yl]prop-1-enyl}benzeneace-
tic Acid (5). A mixture of 6,7-dimethoxy-3-(4-methoxyphenyl)-1-(prop-2-enyl)-1H-quinolin-2-one (4;
500 mg, 1.42 mmol), 4-bromobenzeneacetic acid (458 mg, 2.13 mmol), Cs2CO3 (1.38 g, 4.26 mmol),
PPh3, (55.8 mg, 0.23 mmol), and [Pd(OAc)2] (15.9 mg, 0.07 mmol) was suspended in DMF/H2O 2 :1
(17 ml). After heating for 16 h at 1208, the solvent was evaporated and the residue dissolved in H2O
(50 ml) and extracted with Et2O. The aq. soln. was adjusted to pH 2.0 with 2n HCl and extracted with

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Fig. 10. a) Normalized fluorescence decay of the donor in fragment 11 (5 mm) in H2Oat 20 8 (pulse
excitation, 381 nm; detection, 420 nm). b) Normalized fluorescence decay of the acceptor in fragment 10
(5 mm) in H2Oat 20 8 (pulse excitation, 358 nm; detection, 620 nm).
AcOEt (3 ꢀ 30 ml). The org. layer was dried (Na2SO4) and concentrated and the crude product purified
by CC (silica gel, 2 ! 9% MeOH in CH2Cl2): 663 mg (99%) of 5. 1H-NMR (300 MHz, CDCl3): 3.50 (s,
CH2CO); 3.77 (s, MeO); 3.86 (s, MeO); 3.87 (s, MeO); 5.10 (d, J ¼ 5.0, CH2N); 6.21 – 6.28 (m,
CH¼CHC6H4CH2); 6.48 – 6.53 (m, CH¼CHC6H4CH2); 6.82 (s, HÀC(5)); 6.88 (d, J ¼ 9.0, HÀC(3’),
HÀC(5’)); 6.93 (s, HÀC(8)); 7.08 – 7.18 (m, arom. H); 7.63 (d, J ¼ 9.0, HÀC(2’); HÀC(6’)); 7.66 (s,
HÀC(4)). 13C-NMR (125 MHz, CDCl3): 39.96; 40.29; 55.08; 55.73; 55.91; 110.01; 113.27; 113.70; 124.18;

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126.12; 129.31; 129.61; 129.83; 131.71; 133.93; 134.46; 134.56; 144.72; 151.52; 158.72; 160.08; 172.51. ESI-
MS: 486.0 (100, [M þ H]þ).
4-{3-[1,2-Dihydro-6,7-dimethoxy-3-(4-methoxyphenyl)-2-oxoquinolin-1-yl]propyl}benzeneacetic
Acid (2b). Compound 5 (578 mg, 1.18 mmol) was hydrogenated in CH2Cl2/MeOH 1.5 :1 (35 ml), over
10% Pd/C (204 mg). After 20 h, more MeOH was added, and the catalyst was removed by filtration over
Celite. The solvent was evaporated: 580 mg (quant.) of 2b. 1H-NMR (300 MHz, CDCl3): 1.97 – 2.07 (m,
CH2); 2.73 (t, J ¼ 7.0, CH2C6H4CH2CO); 3.54 (s, CH2CO); 3.62 (s, MeO); 3.77 (s, MeO); 3.84 (s, MeO);
4.21 – 4.26 (m, CH2N); 6.34 (s, HÀC(5)); 6.86 – 6.89 (m, HÀC(3’), HÀC(5’), HÀC(8)); 7.15 (s, arom. H);
7.57 – 7.61 (m, HÀC(2’), HÀC(6’), HÀC(4)). 13C-NMR (125 MHz, (D6)DMSO): 28.24; 32.02; 39.96;
40.31; 55.08; 55.70; 97.05; 110.10; 113.25; 126.50; 127.29; 128.23; 129.33; 129.80; 132.59; 133.63; 135.38;
139.49; 144.58; 151.62; 158.68; 160.06; 172.73. ESI-MS: 488.2 (100, [M þ H]þ).
4-{3-[1,2-Dihydro-6,7-dimethoxy-3-(4-methoxyphenyl)-2-oxoquinolin-1-yl]propyl}benzeneacetic
Acid 2,5-Dioxopyrrolidin-1-yl Ester (6). To the soln. of 2b (130 mg, 0.26 mmol) in dry DMF(5 ml) under
i
Ar, TSTU (128.24 mg, 0.42 mmol) and Pr2EtN (114 ml, 0.66 mmol) were added under stirring. The
mixture was stirred for 3 h at r.t. After removal of DMF, the residue was purified by CC (silica gel, 1!
5% MeOH/CH2Cl2): 118 mg (79%) of 6. 1H-NMR (300 MHz, CDCl3): 2.03 – 2.08 (m, CH2); 2.73 – 2.78
(m, 3 CH2); 3.54 (s, CH2CO, 2 H); 3.62 (s, MeO); 3.77 (s, MeO); 3.84 (s, MeO); 4.21 – 4.26 (m, NCH2);
6.34 (s, HÀC(5)); 6.86 – 6.89 (m, HÀC(3’), HÀC(5’), HÀC(8)); 7.15 (s, arom. H); 7.57 – 7.61 (m,
HÀC(2’), HÀC(6’), HÀC(4)).
5’-Amino-5’-deoxythymidylyl-(3’ ! 5’)-2’-deoxycytidylyl-(3’ ! 5’)-2’-deoxycytidylyl-(3’ ! 5’)-2’-de-
oxyadenylyl-(3’ ! 5’)-2’-deoxyadenylyl-(3’ ! 5’)-2’-deoxycytidylyl-(3’ ! 5’)-uridylic Acid 5’-Terminal
Amide with Bis(1,7-diphenyl-1,10-phenanthroline-kN1,kN10)[4-(7-phenyl-1,10-phenanthrolin-4-yl-
kN1,kN10)benzenepentanoic Acid]ruthenium(II) (10). The synthesis of 10 was performed on a 1 mmol
scale by using CPG (controlled-pore glass) carrying a phosphate linker2) [24]. Modified building block 9
was incorporated manually (60 mg, 0.07 mmol) by using 0.3m of 5-(benzyl(thio)-1H-tetrazole (BMT,
700 ml) in MeCN as coupling reagent, and the coupling time was 6 min. In the last cycle, the MeOTr
protecting group at the 5’-end was cleaved under acidic conditions (3% CHCl2COOH in CH2Cl2).
Removal from the solid support and cleavage of the protecting groups was performed with dithioerythrol
(30 mg), 2m NH4OH in EtOH (500 ml), and 7m MeNH2 in EtOH (500 ml), at 658 for 30 min. The solvent
was removed by decantation, and the CPG was washed with EtOH/H2O 1:1. The combined solns. were
lyophilized in an Eppendorf concentrator. After replacement of NHþ4 with Kþ by co-evaporation with
KCl in H2O and desalting with a NAP-10 column, the crude oligonucleotide was lyophilized. The
oligonucleotide (6.2 OD, 87 nmol) was dissolved in DMF/dioxane/H2O 1:1:1 (261 ml), and iPr2EtN (3 ml,
17 mmol) and the activated (as hydroxysuccinimid-derived ester) acceptor 1b (2.9 mg, 2.18 mmol) were
added. The mixture was incubated at 258 in the dark for 24 h. After removal of the solvents, the pellets
were treated with CHCl3 (500 ml), vortexed, and centrifuged, and the CHCl3 was removed by decantation
(3ꢀ). Part of the crude product was purified by reversed-phase HPLC using (EC 125/4 Nucleosil 100 – 5
C18, 0 ! 60% B in 30 min; tR 15.23 min). The desired sequence 10 was analyzed by PAGE (Fig. 5,b) and
MS. ESI-MS: 3299.0 (100, [M – 2 Cl]2þ).
2’-Deoxythymidylyl-(3’ ! 5’)-2’-deoxy-{a-{2-{{{4-{3-[1,2-dihydro-6,7-dimethoxy-3-(4-methoxyphen-
yl)-2-oxoquinolin-1-yl]propyl}phenyl}acetyl}amino}ethylidyne}}thymidylyl-(3’ ! 5’)-2’-deoxyadenylyl-
(3’ ! 5’)-2’-deoxycytidylyl-(3’ ! 5’)-2’-deoxyadenylyl-(3’ ! 5’)-2’-deoxycytidylyl-(3’ ! 5’)-2’-deoxycyti-
dylyl-(3’ ! 5’)-2’-deoxyadenylyl-(3’ ! 5’)-2’-deoxycytidine (11). The synthesis of 11 was carried out on a
2
)

Helvetica Chimica Acta – Vol. 90 (2007)
1097
1 mmol scale. Modified building block 8 was incorporated manually (50 mg, 70 mmol) by using the
method described for 10. Removal from the solid support and cleavage of the protecting groups were
performed with 25% NH3 soln. at 558 overnight. After replacement of NHþ4 with Kþ and desalting, the
crude oligonucleotide (21.8 OD, 0.23 mmol) was dissolved in DMF/dioxane/H2O 1:1:1 (716 ml), and
iPr2EtN (8 ml, 47.7 mmol) and activated (as 6) donor 2b (3.5 mg, 5.9 mmol) were added. The mixture was
incubated at 258 in the dark for 16 h. After removal of the solvents, the residue was washed with EtOH
(3 ꢀ 500 ml) (as described for 10 with CHCl3) to remove the excess of the donor. Part of the crude product
was purified by reversed-phase HPLC (EC 125/4 Nucleosil 100 – 5 C18, 0 ! 100% B in 45 min; tR
35.63 min). The desired sequence 11 was analyzed by PAGE (Fig. 5,b) and MS. ESI-MS: 3135.0 (100,
[M þ 1]þ).
DNA d{(5’-Amino-5’-deoxy)T-C-C-A-A-C-(2’-hydroxy)U-C-{a-{2-{{{4-{3-[1,2-dihydro-6,7-dimeth-
oxy-3-(4-methoxyphenyl)-2-oxoquinolin-1-yl]propyl}phenyl}acetyl}amino}ethylidyne}}T-A-C-A-C-C-A-
C} 5’-Terminal Amide with Bis(1,7-diphenyl-1,10-phenanthroline-kN1,kN10)[4-(7-phenyl-1,10-phenan-
throlin-4-yl-kN1,kN10)benzenepentanoic Acid]ruthenium(II) (12). The synthesis of oligonucleotide 12
was carried out on a 1 mmol scale. Modified building blocks 8 and 9 were incorporated manually at
specific sites by using the method described for 10 and 11. In the last cycle, the MeOTr protecting group at
the 5’-end was not cleaved. After cleavage from the solid support with 2m NH4OH in EtOH (500 ml) for
30 min at 658, the oligonucleotide was treated with 7m MeNH2 in EtOH (500 ml) for 30 min at 658. After
replacement of NHþ4 with Kþ and desalting, the oligonucleotide (33 OD, 2.06 nmol) was dissolved in
DMF/dioxane/H2O 1:1:1 (618 ml), and iPr2EtN (7 ml, 41 mmol) and the activated (as 6) donor 2b (3 mg,
5.1 mmol), were added. The mixture was incubated at 258 in the dark for 16 h. After removal of the
solvents, the residue was washed with EtOH (3 ꢀ 500 ml) (as described for 10 with CHCl3). The product
was purified by reversed-phase HPLC (SP 250/10 Nucleosil 100-5 C18, 0 ! 60% B in 45 min; tR
23.68 min). The sequence was analyzed by PAGE. Afterwards, it was treated with 80% AcOH
(320 ml) for 20 min to remove the MeOTr protecting group, and the product was precipitated with 50 ml of
3m AcONa and 1 ml of iPr2OH and centrifuged at 11 000 r.p.m. for 20 min at 48. The solvent was removed
and the pellets dried in an Eppendorf concentrator at 458. The product (6.5 OD, 0.4 nmol) was dissolved
in DMF/dioxane/H2O 1 :1 :1 (121 ml), and iPr2EtN (1.38 ml, 8 mmol) as well as activated (as
hydroxysuccinimide-derived ester) acceptor 1b (1.3 mg, 1.0 mmol) were added. The mixture was
incubated at 258 in the dark for 24 h. After removal of the solvents, the residue was washed with CHCl3
(3 ꢀ 500 ml; by using the method described for 10) to remove the excess of the acceptor. Part of the crude
oligonucleotide 12 was purified by reversed-phase HPLC (EC 125/4 Nucleosil 100-5 C18, 0 ! 100% B in
45 min; tR 33.04 min). The product was analyzed by PAGE (Fig. 6).
DNA d{5’-Amino-5’-deoxy)T-A-C-A-A-T-C-C-T-T-T-A-T-T-G-T-{3’-O-{6-{{{4-{3-[1,2-dihydro-6,7-
dimethoxy-3-(4-methoxyphenyl)-2-oxoquinolin-1-yl]propyl}phenyl}acetyl}amino}hexyl}}A} 5’-Terminal
Amide with Bis(1,7-diphenyl-1,10-phenanthroline-kN1,kN10)[4-(7-phenyl-1,10-phenanthrolin-4-yl-
kN1,kN10)benzenepentanoic Acid]ruthenium(II) (13). Oligonucleotide 13 was synthesized in a similar
manner as 12 on a 1 mmol scale by using amino-ON CPG1). Modified building block 9 was incorporated
manually by using the method described for 10. The MeOTr protecting group at the 5’-end was not
cleaved. Removal from the solid support and cleavage of the protecting groups were performed with 25%
NH3 soln. at 558 for 2 h. After replacement of NH4þ with Kþ and desalting, the resulting oligonucleotide
(40 OD, 0.22 mmol) was dissolved in DMF/dioxane/H2O 1:1:1 (669 ml), and iPr2EtN (8 ml, 44 mmol) and
activated (as 6) donor 2b (3.2 mg, 5.5 mmol) were added. The mixture was incubated at 258 in the dark for
16 h. After removal of the solvents, the residue was washed with EtOH (3 ꢀ 500 ml) to remove the excess
of the donor. The product was analyzed by reversed-phase HPLC (EC 125/4 Nucleosil 100-5 C18, 0 !
60% B in 45 min; tR 27.67 min) and PAGE (Fig. 8). Afterwards, it was treated with 80% of AcOH
(320 ml) for 20 min to remove the MeOTr protecting group and the product precipitated by using the
method described for oligonucleotide 12. The pellets (39 OD, 0.2 mmol) were dissolved in DMF/dioxane/
i
H2O 1:1:1 (652 ml), and Pr2EtN (8 ml, 43 mmol) and activated (as hydroxysuccinimide-derived ester)
acceptor 1b (7.4 mg, 5.4 mmol) were added. The mixture was incubated at 258 in the dark for 24 h. After
removal of the solvents, the residue was washed with CHCl3 (3 ꢀ 500 ml) to remove the excess of 1b. Part
of the crude 13 was purified by reversed-phase HPLC (EC 125/4 Nucleosil 100-5 C18, 5 min 100% A,
then 0 ! 90% B in 45 min; tR 29.5 min). The product was analyzed by PAGE (Fig. 8).

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Helvetica Chimica Acta – Vol. 90 (2007)
Hybridization was carried out with 13/14 1:2 in 300 ml of 5 mm phosphate buffer pH 7.0, 100 mm
NaCl, and 0.1m EDTA. The probe was heated at 708 for 4 min and cooled down slowly to r.t.
Cleavage of Oligonucleotide 12. Oligonucleotide 12 (1.2 OD) was treated with 0.5m NaOH (50 ml)
and stirred for 5 h at r.t. The mixture was neutralized with AcOH (100 ml) and evaporated.
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Received January 19, 2007