Building-block approach for the straightforward incorporation of a new FRET (fluorescence-resonance-energy transfer) system into synthetic DNA

Article abstract of DOI:10.1002/hlca.200890008

The synthesis of the two new phosphoramidites 5 and 8 bearing a carbostyril (= quinolin-2(1H)-one) chromophore used as donor entity in our recently developed new FRET (fluorescence-resonance-energy transfer) system is described (Schemes 1 and 2) The high

Full text of DOI:10.1002/hlca.200890008

Helvetica Chimica Acta – Vol. 91 (2008)
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Building-Block Approach for the Straightforward Incorporation of a New
FRET (Fluorescence-Resonance-Energy Transfer) Systeminto Synthetic
DNA
by Lilia Clima 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)
The synthesis of the two new phosphoramidites 5 and 8 bearing a carbostyril (¼quinolin-2(1H)-one)
chromophore used as donor entity in our recently developed new FRET (fluorescence-resonance-energy
transfer) system is described (Schemes 1 and 2) The high stability of the chromophore to basic conditions
enables the incorporation of the phosphoramidites directly into DNA during solid-phase synthesis
(Schemes 3 and 4). Since this is also possible for the (bathophenanthroline)ruthenium(II) complex used
as acceptor (Scheme 4, Steps d and e), the whole labelling procedure to insert the FRET system into
synthetic DNA is straightforward and represents a major improvement to our previous strategy.
Introduction. – Fluorescently labelled nucleic acids are particularly important tools
in molecular biology, diagnostics, and structural studies [1][2]. In this context, DNA
with incorporated dyes representing fluorescence-resonance-energy transfer (FRET)
systems have proven to be important tools for the elucidation of distance-dependent
interactions on the molecular level and in real time mode. Any changes in donor–
acceptor distance will affect the rate of energy transfer which can be measured either
by steady-state or time-resolved fluorescence studies. The latter approach requires
labels with a long lifetime of their fluorescence, and this is usually achieved via the use
of lanthanide chelates as acceptor in FRET systems [3][4]. As alternative labels, we
have developed, some time ago, (bathophenanthroline)ruthenium(II) complexes
(bathophenanthroline ¼ 4,7-diphenyl-1,10-phenanthroline) which show, after excita-
tion of their metal-to-ligand charge transfer, a fluorescence lifetime in the low msec
range [5]. In combination with a quinolinone derivative as donor, this yielded a highly
efficient and robust new FRET system, of which the feasibility was first demonstrated
in peptides in the course of the development of a homogeneous assay for the serine
protease thrombin [6][7].
Later, we were also able to demonstrate the efficiency of the system in DNA [8]. In
this study, both constituents, the (bathophenanthroline)ruthenium(II) complex as well
as the carbostyril (¼quinolin-2(1H)-one) chromophore were attached to amino-
modified DNA after deprotection following solid-phase assembly of the oligonucleo-
tide chain. The described method was time-consuming. Hence, we sought for
possibilities to improve this labelling procedure.
ꢀ 2008 Verlag Helvetica Chimica Acta AG, Zürich

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In preliminary evaluations, we noted the outstanding chemical stability of both
dyes; concentrated ammonia at elevated temperature did not cause any harm to them.
Therefore, we surmized that their introduction into synthetic DNA should be possible
via the pertinent phosphoramidite building blocks, and the final deprotection with
conc. ammonia should have no influence on either chromophore.
Results and Discussions. – As possible attachment points for the carbostyril donor,
we envisaged the 8-position of adenine leading to the building block 5 and the 5-
position of uracil (¼a-position of thymine) leading to phosphoramidite 8.
For the synthesis of adenosine phosphoramidite 5 bearing the carbostyril moiety, 2’-
deoxyadenosine was first converted to 8-bromo-2’-deoxyadenosine (1) [9] (Scheme 1).
Scheme 1. Synthesis of Phosphoramidite 5
a) (MeO)2TrCl, pyridine, N,N-dimethylpyridin-4-amine (DMAP), Et3N, r.t., 4 h; 85%. b)
[Pd(PPh3)2Cl2], CuI, Et3N, 2,2,2-trifluoro-N-propynylacetamide, 508, 5 h; 85%. c) 25% aq. NH3 soln.,
MeOH, r.t., 16 h; quant. d) 3, TBTU, iPr2EtN, DMF, r.t, 16 h; 67% (over 2 steps). e) Me3SiCl, pyridine,
BzCl, 25% aq. NH3 soln.; 72%. f) (NCCH2CH2O)P(NiPr2)2, diisopropylammonium 1H-tetrazolide,
CH2Cl2, r.t., 2.5 h; 51%.

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After regioselective introduction of the 4,4’-dimethoxytrityl ((MeO)2Tr) group at the
5’-position, a Sonogashira coupling with 2,2,2-trifluoro-N-(prop-2-yn-1-yl)acetamide)
in the presence of [Pd(PPh3)2Cl2] as catalyst [10] and deprotection of the trifluoro-
acetyl group under basic conditions (aq. ammonia/MeOH) afforded compound 2 in
58% yield over 3 steps starting from 2’-deoxyadenosine. The chromophore 3, activated
with TBTU (TBTU ¼ O-(1H-benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium tetra-
fluoroborate [11]), was then coupled to the amino group of 2, which led to 4 in 67%
yield. This step was followed by protection of the 6-amino function via a three-step
protocol [12]. Finally, conversion to the pertinent phosphoramidite at the 3’-position
was performed with 2-cyanoethyl tetraisopropylphosphorodiamidite in the presence of
diisopropylammonium 1H-tetrazolide to yield the desired pure phosphoramidite 5
(51%) after purification by short-column chromatography, of which the 31P-NMR
revealed exclusively PIII signals.
For the synthesis of the building block 8, 2’-deoxy-5-iodouridine and 2,2,2-trifluoro-
N-(prop-2-yn-1-yl)acetamide) [13] were subjected to a Sonogashira reaction [14][15]
in the presence of Pd/C and Amberlite-IRA-67 resin which afforded the coupling
product in 76% yield (Scheme 2). This step was followed by regioselective introduction
of the 4,4’-dimethoxytrityl ((MeO)2Tr) group at the 5’-position. The trifluoroacetyl
group was cleaved off with aq. NH3 solution/MeOH to give amine 6. The reaction
between 6 and 3 was this time performed with TSTU (TSTU ¼ N,N,N’,N’-tetramethyl-
Scheme 2. Synthesis of Phosphoramidite 8
o
a) Pd/C, Amberlite IRA-67, CuI, DMF, 50 , 16 h; 76%. b) (MeO)2TrCl, DMAP, Et3N, pyridine, 4 h; 81%.
i
c) 25% aq. NH3 soln./MeOH; 92%. d) 3, TSTU, Pr2EtN, DMF, r.t., 16 h; 98%. e) (NCCH2CH2O)P-
(NiPr2)2, diisopropylammonium 1H-tetrazolide, CH2Cl2, r.t., 1.5 h; 54%.

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O-succinimidouronium tetrafluoroborate) [11]) as coupling reagent in DMFto give
product 7 in 98% yield. Compound 7 was converted to the pertinent phosphoramidite 8
at the 3’-position by phosphitylation with 2-cyanoethyl tetraisopropylphosphorodi-
amidite in the presence of diisopropylammonium 1H-tetrazolide (54%).
For the evaluation of the incorporation of the donor building block 5 during solid-
phase synthesis, DNA sequence 10 (Scheme 3) was chosen. The donor phosphorami-
dite 5 was inserted during a 1 mmol scale synthesis as base number eight. Then, the
synthesis was continued with standard phosphoramidite building blocks. As the last
building block, 5’-deoxy-5’-[(4-methoxytrityl)amino]thymidine 3’-(2-cyanoethyl N,N-
diisopropylphosphoramidite) was coupled, after which the (MeO)Tr was removed by
3% dichloroacetic acid to yield the 5’-unprotected amino terminus. Ammonia
treatment for the removal from the support and deprotection gave the crude DNA
fragment 9. After replacement of the NHþ4 ions by Kþ and desalting, a small amount
was analyzed by polyacrylamide-gel electrophoresis (PAGE) (Fig. 1, Lanes 4 and 8).
The rest was dissolved in a mixture of DMF/1,4-dioxane/H2O 1:1:1, and then iPr2EtN
together with an excess of (bathophenanthroline)ruthenium(II) complex activated as
ester with hydroxysuccinimide [5] were added and incubated for 24 h to yield fragment
10 having now both dyes of the FRET system incorporated. After removal of the
Scheme 3. Synthesis of 10
a) 25% aq. NH3 soln., 558, 4 h. b) (Bathophenanthroline)ruthenium(II) complex acceptor (as ester with
i
hydroxysuccinimide), DMF/1,4-dioxane/water 1:1:1, Pr2EtN, 24 h.

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Fig. 1. Polyacrylamide-gel electrophoresis (PAGE; 20%) of 9, 10, 12, and 13. At 366 nm: Lane 1, 12;
Lane 2, 13; Lane 3, 10; Lane 4, 9. Stained with 3,3’-diethyl-9-methyl-4,5,4’,5’-dibenzothiacarbocyanine
bromide soln.: Lane 5, 12; Lane 6, 13; Lane 7, 10; Lane 8, 9.
solvents, the excess Ru-complex was removed by CHCl3 extraction, and the crude
fragment 10 was purified by HPLC and analyzed by PAGE (Fig. 1, Lanes 3 and 7).
For the evaluation of donor-building block 8, we have chosen virtually the same
oligonucleotide sequence as for the incorporation of donor building block 5, except that
we inserted now building block 8 instead of 5 in position eight from the 3’-end
(Scheme 4). On the one hand, the continuation of the synthesis as well as the
attachment of the Ru-complex as acceptor to the crude fragment 12 yielding sequence
13 were performed in the same way as described for the preparation of sequence 10
(Scheme 4,a – c).
On the other hand, we tried as an alternative for the attachment of the
(bathophenanthroline)ruthenium(II) complex its solid-phase incorporation
(Scheme 4,d), according to a procedure described by Richert and co-workers [16]. If
successful, this would be a significant improvement compared to the tedious procedure
for the attachment of the Ru-complex in solution. For this purpose, a mixture of the
(bathophenanthroline)ruthenium(II) complex (as free acid), HBTU (HBTU ¼ O-
(1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate), HOBt
(HOBt ¼ 1-hydroxy-1H-benzotriazole), and iPr2EtN in DMFwere added to the solid-
phase-bound sequence 11 (Scheme 4,d). After coupling, the supernatant was removed
and the support washed several times. Cleavage with conc. ammonia (Scheme 4,e)
yielded then directly the desired fragment 13 with the incorporated FRET system.
A HPLC comparison of the crude DNA 13 obtained by the Ru-complex attachment
in solution (Fig. 2,a) and by the solid-phase coupling of the Ru-complex (Fig. 2,b))
revealed that high efficiency was achieved in both couplings but the latter approach
turned out to be far easier to perform.
To evaluate whether the type of base carrying the donor of the FRET system would
have any influence on the intensity of the FRET, sequences 10 and 13 were hybridized
to their pertinent complementary sequence according to Fig. 3,a and b. As can be seen
from Fig. 3,c, both FRET systems lead to a strong emission at 620 nm upon excitation
at 350 nm. There is no significant difference in the intensity of this emission, indicating
that both building blocks 5 and 8 carrying the donor are equally suited to our FRET
system in DNA.

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Scheme 4. Synthesis of 13
a) 2m NH4OH in EtOH, 30 min, 658. b) 7m MeNH2 in EtOH, 30 min, 658. c) (Bathophenanthroline)-
ruthenium(II) complex (as ester with hydroxysuccinimide), DMF/1,4-dioxane/H2O 1:1:1, iPr2EtN, 258,
i
24 h. d) (Bathophenanthroline)ruthenium(II) complex (as free acid), HOBt, HBTU, Pr2EtN, 24 h. e)
25% aq. NH3 soln., 408, 2 h.
Conclusions. – In summary, we have synthesized two new phosphoramidite building
blocks carrying the donor chromophore of our newly established FRET system that
allows their insertion into DNA during synthesis on solid support. Together with the
solid-phase coupling of the (bathophenanthroline)ruthenium(II) complex as acceptor,
this represents an easy and straightforward way to establish the FRET system in DNA.
Such labelled DNA is a valuable tool to monitor structural changes caused either by the
formation of secondary structures or by supramolecular interactions in real-time mode.
The authors would like to thank Prof. P. Gräber and R. Bienert for fluorescence-spectra
measurements and Dr. Moneesha DꢀCosta for help in the preparation of the manuscript.
Experimental Part
General. Oligonucleotide synthesis was carried out on an Expedite-6800 DNA synthesizer on a
1 mmol scale by means of standard phosphoramidite technique. Replacement of NHþ4 with Kþ was done
by co-evaporation (3Â) 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, A ¼ 0.1m Et3N ·
AcOH buffer at pH 7.0, B ¼ MeCN. UV Spectra: Perkin-Elmer Lambda-35 UV/VIS spectrometer.

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Fig. 2. HPLC Plots after the synthesis of 13: a) Crude 13 synthesized by the Steps a – c of Scheme 4), and
b) crude 13 synthesized by the Steps d and e of Scheme 4. HPLC conditions: reversed phase; 0 ! 90%
B/A within 40 min; A ¼ 0.1m Et3N · AcOH (pH 7), B ¼ MeCN.
Fluorescence spectra: Perkin-Elmer LS45 spectrometer. 1H-(400 MHz) and 13C-NMR (100 MHz)
Spectra: Bruker DRX-500 spectrometers; chemical shifts d in ppm rel. to Me4Si, referenced to residual
solvent signals; J in Hz. MS: LCQ Advantage (ESI) mass spectrometer.
Analytical Polyacrylamide-Gel Electrophoresis (PAGE). 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 Vand 4 mA.
Oligonucleotide bands were visualized at 366 nm or stained with a soln. of 3,3’-diethyl-9-methyl-4,5,4’,5’-
dibenzothiacarbocyanine bromide (¼ꢁStains-Allꢂ; Fluka).
8-Bromo-2’-deoxyadenosine (1) [9]. To a soln. of 2’-deoxyadenosine (1 g, 3.72 mmol) in 1,4-dioxane
(10 ml) and 0.5m aq. AcONa buffer (pH 4.7) (16 ml), a soln. of Br2 (0.24 ml, 4.64 mmol) in 1,4-dioxane
(20 ml) was added dropwise while stirring. The mixture was stirred overnight at r.t. To the mixture, conc.
Na2S2O5 soln. was added until the red color vanished. The soln. was neutralized to pH 7.0 with 0.5m
NaOH. The 1,4-dioxane was evaporated, when a white solid precipitated. The solid was filtered off,
washed with cold 1,4-dioxane, and dried under high vacuum: 1 g (82%) of 1. 1H-NMR (400 MHz,
(D6)DMSO): 2.15 – 2.21 (m, 1 HÀC(2’)); 3.20 – 3.27 (m, 1 HÀC(2’)); 3.46 – 3.49 (m, 1 HÀC(5’)); 3.62 –
3.66 (m, 1 HÀC(5’)); 3.86 – 3.89 (m, HÀC(4’)); 4.46 – 4.49 (m, HÀC(3’)); 5.28 (br. s, OHÀC(3’)); 5.35
(br. s, OHÀC(5’)); 6.28 (t, J ¼ 6.44, HÀC(1’)); 7.48 (br. s, NH2); 8.10 (s, HÀC(2)). 13C-NMR (100 MHz,
(D6)DMSO): 36.97; 62.10; 71.14; 86.34; 88.30; 119.68; 126.61; 149.91; 152.35; 155.06. ESI-MS: 214.2 (92,
C5H3BrNþ5 ), 216.2 (90, C5H3BrNþ5 ), 254.4 (40), 256.4 (43), 329.7 (100, [M þ 1]þ), 331.7 (97.5, [M þ 1]þ).
Anal. calc. for C10H12BrN5O3: C 36.38, H 3.66, N 21.21, found: C 36.38, H 3.73, N 21.07.

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Fig. 3. a) b) Hybridization of 10 and 13, respectively, to their complementary sequences (hybridization
conditions: 1:3 ratio of 10 or 13 to complementary sequence, 708, slow cooling to r.t.). c) Fluorescence
emission spectra of hybridized 10 (—) and hybridized 13 (---) (excitation at 350 nm)
(MeO)2Tr Protection of 1: 8-Bromo-5’-O-[bis(4-methoxyphenyl)phenylmethyl]-2’-deoxyadenosine.
The 8-bromo-2’-deoxyadenosine (1) [9] (2 g, 6 mmol) was co-evaporated with anh. pyridine and then
dissolved in anh. pyridine (30 ml). Et3N (1.7 ml, 12 mmol), DMAP (180 mg, 1.5 mmol), and (MeO)2TrCl
(3.3 g, 9.6 mmol) were added under Ar, and the mixture was stirred at r.t. for 4 h. Then, MeOH (5 ml)
was added and the mixture extracted with CH2Cl2. The org. phase was washed with aq. NaHCO3 soln.,
the combined org. layer dried (Na2SO4) and concentrated, and the residue purified by CC (silica gel, 2 !
5% MeOH/CH2Cl2): 3.14 g (85%) of product. Slightly yellow foam. 1H-NMR (400 MHz, CDCl3): 2.24 –
2.31 (m, 1 HÀC(2’)); 3.32 (m, CH2(5’)); 3.44 – 3.51 (m, 1 HÀC(2’)); 3.68 (s, MeO); 3.68 (s, MeO); 4.03 –
4.07 (m, HÀC(4’)); 4.85 – 4.89 (m, HÀC(3’)); 5.74 (s, NH2); 6.32 (t, J ¼ 6.19, HÀC(1’)); 6.65 – 6.70 (m, 4
arom. H); 7.06 – 7.30 (m, 9 arom. H); 7.98 (s, HÀC(2)). 13C-NMR (100 MHz, CDCl3): 36.85; 45.87; 55.25
(2 C); 63.84; 72.94; 86.07; 86.06; 86.34; 113.10 (2 C); 113.22; 120.44; 126.81; 127.79 (2 C); 127.89; 128.19
(2 C); 129.22; 130.06 (2 C); 130.09 (2 C); 135.99; 136.05; 144.8; 150.83; 152.61; 154.21; 158.50. ESI-MS:
289.2 (18), 303.2 (100, [(MeO)2Tr]þ), 631.6 (37, Mþ), 633.7 (38, Mþ).
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-2’-deoxy-8-{3-[(2,2,2-trifluoro-1-oxoethyl)amino]prop-
1-yn-1-yl}adenosine. Through the soln. of 5’-O-[bis(4-methoxyphenyl)phenylmethyl]-8-bromo-2’-de-

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oxyadenosine (510 mg, 2.82 mmol) in dry DMF(7 ml), Ar was bubbled for 10 min. [Pd(Ph 3P)2Cl2]
(57 mg, 0.08 mmol) was added, and Ar was bubbled through the soln. for another 5 min. Et3N (230 ml,
1.62 mmol) was added via syringe, followed by 2,2,2-trifluoro-N-(prop-2-yn-1-yl)acetamide (245 mg,
1.62 mmol) and CuI (31 mg, 0.16 mmol). The mixture was stirred at 508 for 5 h, the solvent evaporated,
and the residue purified by CC (silica gel, 2 ! 5% MeOH/CH2Cl2): 480 mg (85%) of product. Slightly
1
yellow foam. H-NMR (400 MHz, (D6)DMSO): 2.19 – 2.25 (m, 1 HÀC(2’)); 3.16 (m, CH2(5’)); 3.26 –
3.32 (m, 1 HÀC(2’)); 3.69 (s, MeO); 3.70 (s, MeO); 3.94 – 3.98 (m, HÀC(4’)); 4.36 (d, J ¼ 5.3, CH2NH);
4.61 – 4.67 (m, HÀC(3’)); 5.32 (br. s, OH); 6.40 (t, J ¼ 6.1, HÀC(1’)); 6.72 – 6.79 (m, 4 arom. H); 7.13 –
7.19 (m, 9 arom. H); 7.45 (br. s, NH2); 7.98 (s, HÀC(2)); 10.20 (t, J ¼ 5.4 Hz, NH). 13C-NMR (100 MHz,
CDCl3): 29.37; 36.71; 54.84 (2 C); 63.75; 70.75; 72.16; 84.50; 85.17; 85.84; 90.92; 112.88 (2 C); 112.93
(2 C); 114.27; 117.13; 126.40; 127.54 (2 C); 127.60 (2 C); 129.50; 129.63 (2 C); 132.88; 36.65 (2 C); 144.93;
148.39; 153.38; 155.86; 156.11; 156.48; 157.86; 157.92. ESI-MS: 303.2 (32, [(MeO)2Tr]þ), 587.1 (100, [M þ
1]þ).
8-(3-Aminoprop-1-yn-1-yl)-5’-O-[bis(4-methoxyphenyl)phenylmethyl]-2’-deoxyadenosine (2). To a
soln. of 5’-O-[bis(4-methoxyphenyl)phenylmethyl]-2’-deoxy-8-{3-[(2,2,2-trifluoro-1-oxoethyl)amino]-
prop-1-yn-1-yl}adenosine (320 mg, 0.45 mmol) in MeOH (3 ml), 25% aq. NH4OH soln. (9 ml) was
added, and the mixture was stirred overnight at r.t. The mixture was extracted with CH2Cl2/sat. NaHCO3
soln. and the org. layer dried (Na2SO4), and evaporated: 2 (275 mg, quant.). 1H-NMR (400 MHz,
(D6)DMSO): 2.19 – 2.26 (m, 1 HÀC(2’)); 3.18 (m, CH2(5’)); 3.23 – 3.29 (m, 1 HÀC(2’)); 3.62 (m, CH2);
3.70 (s, MeO); 3.71 (s, MeO); 3.95 – 3.99 (m, HÀC(4’)); 4.63 – 4.67 (m, HÀC(3’)); 5.33 (br. s, OH); 6.44
(t, J ¼ 6.9 Hz, HÀC(1’)); 6.73 – 7.79 (m, 4 arom. H); 7.12 – 7.38 (m, 9 arom. H); 7.95 (s, HÀC(2)).
13C-NMR (100 MHz, (D6)DMSO): 36.75; 40.17; 54.93 (2 C); 54.95 (2 C); 63.88; 70.87; 84.27; 85.19; 85.77;
112.90 (2 C); 112.95 (2 C); 119.11; 126.43; 127.56 (2 C); 127.61 (2 C); 129.55 (2 C); 129.64 (2 C); 133.67;
135.63; 135.69; 144.97; 148.48; 153.05; 155.73; 157.88; 157.93. LC/ESI-MS: 303.2 (9, [(MeO)2Tr]þ), 702.8
(100, [M þ 97 (CF3CO)]þ). APCI-MS (þ 90 V): 289.2 (47), 303.2 (100, [(MeO)2Tr]þ), 606.9 (37, Mþ),
702.6 (56, [M þ 97(CF3CO)]þ), 853.7(28).
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-2’-deoxy-8-{3-{{2-{4-{3-[6,7-dimethoxy-3-(4-methoxy-
phenyl)-2-oxoquinolin-1(2H)-yl]propyl}phenyl}acetyl}amino}prop-1-yn-1-yl}adenosine (4). To a soln. of
4-{3-[6,7-dimethoxy-3-(4-methoxyphenyl)-2-oxoquinolin-1(2H)-yl]propyl}benzeneacetic acid (3;
100 mg, 0.20 mmol) in DMF(4 ml), TBTU (72 mg, 0.22 mmol) and iPr2EtN (100 ml, 0.61 mmol) were
added under Ar. The mixture was stirred for 1 h, and 2 (150 mg, 0.24 mmol) was added, and the mixture
stirred overnight. After removal of DMF, the residue was purified by short CC (silica gel, 2! 10%
1
MeOH/CH2Cl2): 4 (145 mg, 67%). Slightly yellow foam. H-NMR (400 MHz, (D6)DMSO): 1.92 – 1.96
(m, CH2); 2.18 – 2.25 (m, 1 HÀC(2’)); 2.71 (t, J ¼ 7.2, C6H4CH2); 3.23 – 3.38 (m, CH2); 3.32 – 3.40 (m,
1 HÀC(2’)); 3.77 (s, MeO); 3.81 (s, MeO); 3.93 – 3.98 (m, HÀC(4’)); 4.20 (d, J ¼ 5.4 Hz, CH2NH); 4.25 –
4.28 (m, CH2N); 4.61 – 4.66 (m, CH2); 5.30 – 5.33 (m, 1 H); 6.39 (t, J ¼ 6.2, HÀC(1’)); 6.64 (s, 1 H); 6.71 –
7.12 (m, 24 arom. H); 7.66 (s, 1 H); 7.68 (s, 1 H); 8.16 (br. s, NH2); 8.68 (t, J ¼ 5.5, NH). 13C-NMR
(100 MHz, (D6)DMSO): 12.43 (2 C); 16.69 (2 C); 18.04 (2 C); 32.02; 39.75; 39.96; 40.17 (2 C); 53.55
(2 C); 54.90; 55.07; 55.68; 55.71; 85.17; 85.19; 93.22; 112.88; 112.93; 113.24 (2 C); 113.63; 119.16; 126.21;
126.41; 127.27; 127.55; 127.58; 127.60; 128.22; 128.26; 128.98; 129.03; 129.33; 129.49; 129.52; 129.58;
129.63; 129.80 (2 C); 133.14; 133.45; 133.62; 135.39; 135.55; 135.57; 135.64; 135.67; 139.39; 144.58; 144.95;
148.43; 151.62; 155.47; 155.81; 157.87; 157.92; 158.68; 160.06; 170.22. LC/ESI-MS: 1098.3 (100, [M þ
22]þ). Anal. calc. for C63H61N7O10 · 4 H2O: C 65.90, H 6.06, N 8.54, found: C 65.83, H 6.05, N 8.77.
N6-Benzoyl Protection of 4: N6-Benzoyl-5’-O-[bis(4-methoxyphenyl)phenylmethyl]-2’-deoxy-8-{3-
{{2-{4-{3-[6,7-dimethoxy-3-(4-methoxyphenyl)-2-oxoquinolin-1(2H)-yl]propyl}phenyl}acetyl}amino}-
prop-1-yn-1-yl}adenosine. Compound 4 (760 mg, 0.7 mmol) was dried 3 Â by co-evaporation with
pyridine and suspended in dry pyridine (6 ml). Trimethylchlorosilane (450 ml, 3.5 mmol) was added to the
ice-cooled mixture and stirred for 1.5 h at r.t. The mixture was cooled to 08, benzoyl chloride (410 ml,
3.5 mmol) added, and the mixture stirred at r.t. for another 3.5 h. It was then cooled in an ice bath, and
H2O (3 ml) was added. After 5 min, 25% aq. NH3 (1.5 ml) was added, and the mixture was stirred at r.t.
for 30 min. The mixture was concentrated and the residue purified by short CC (silica gel, 2 ! 5%
MeOH/CH2Cl2): 590 g (72%) of slightly yellow foam. 1H-NMR (400 MHz, (D6)DMSO): 1.90 – 1.96 (m,
CH2); 2.16 – 2.25 (m, 1 HÀC(2’)); 2.71 (t, J ¼ 7.0 Hz, C6H4CH2); 3.20 – 3.36 (m, CH2); 3.32 – 3.39 (m,

174
Helvetica Chimica Acta – Vol. 91 (2008)
CH2); 3.75 (s, 2 MeO); 3.80 (s, 3 MeO); 3.92 – 3.98 (m, HÀC(4’)); 4.10 (d, J ¼ 5.4 Hz, CH2NH); 4.24 –
4.27 (m, CH2N); 4.61 – 4.66 (m, CH2); 5.30 – 5.33 (m, 1 H); 6.40 (t, J ¼ 6.18, HÀC(1’)); 6.64 (s, 1 H);
6.71 – 7.12 (m, 28 arom. H); 7.64 (s, 1 H); 7.67 (s, 1 H); 8.66 (t, J ¼ 5.5, NH). LC/ESI-MS: 303.3 (18,
[(MeO)2Tr]þ), 796 (33), 1098.3 (23, [M À 106 (PhCO) þ 22]þ), 1202.3 (100, [M þ 22]þ).
N6-Benzoyl-5’-O-[bis(4-methoxyphenyl)phenylmethyl]-2’-deoxy-8-{3-{{2-{4-{3-[6,7-dimethoxy-3-(4-
methoxyphenyl)-2-oxoquinolin-1(2H)-yl]propyl}phenyl}acetyl}amino}prop-1-yn-1-yl}adenosine 3’-[2-
Cyanoethyl Bis(1-methylethyl)phosphoramidite] (5). The N6-benzoyl-protected adenosine obtained
from 4 (350 mg, 0.33 mmol) and diisopropylammonium 1H-tetrazolide (42 mg, 0.24 mmol) were co-
evaporated with anh. MeCN (3 Â 5 ml). They were then dissolved in dry CH2Cl2 (5 ml) and 2-cyanoethyl
N,N,N’,N’-tetraisopropylphosphordiamidite (217 ml, 0.72 mmol) was added under Ar. The mixture was
stirred for 2.5 h under Ar at r.t. Then, the mixture was poured into sat. NaHCO3 soln. and extracted with
CH2Cl2. The combined org. phase was dried (Na2SO4) and concentrated and the residue purified by short
CC (deactivated silica gel (0.1% Et3N), 2% MeOH/CH2Cl2: 5 (230 mg, 51%). Colorless foam. 31P-NMR
(120 MHz, CDCl3): 149.80; 150.00.
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-2’-deoxy-8-{3-{{2-{4-{3-[6,7-dimethoxy-3-(4-methoxy-
phenyl)-2-oxoquinolin-1(2H)-yl]propyl}phenyl}acetyl}amino}prop-1-yn-1-yl}uridine (7). As described
for 4, with 3 (100 mg, 0.20 mmol), DMF(6 ml), TSTU (76 mg, 0.26 mmol), iPr2EtN (100 ml, 0.6 mmol),
1
and amine 6 [17] (180 mg, 0.3 mmol): 7 (210 mg, 98%). Colorless foam. H-NMR (400 MHz, CDCl3):
2.05 – 2.13 (m, C6H4CH2); 2.19 – 2.28 (m, 1 HÀC(2’)); 2.39 – 2.43 (m, 1 HÀC(2’)); 2.68 – 2.69 (m, 1 H);
2.76 – 2.77 (m, CH2); 3.28 – 3.29 (m, CH2); 3.30 – 3.36 (m, CH2(5’)); 3.73 (s, MeO); 3.75 (s, MeO); 3.85 –
3.88 (m, CH2) 3.90 (s, MeO); 4.04 – 4.05 (m, HÀC(4’)); 4.3 – 4.31 (m, CH2N); 4.48 – 4.50 (m, HÀC(3’));
5.53 (t, J ¼ 5, 1 H); 6.25 (t, J ¼ 6, HÀC(1’)); 6.48 (s, 1 H); 6.76 – 7.78 (m, 4 arom. H); 6.86 – 6.89 (m, 3
arom. H); 7.15 (s, 4 arom. H); 7.20 – 7.37 (m, 9 arom. H.); 7.57 – 7.61 (m, 3 arom. H); 8.10 (s, HÀC(6));
8.76 (br. s, NH). 13C-NMR (100 MHz, CDCl3): 28.60; 30.15; 33.00; 38.69; 41.66; 42.73; 42.83; 55.33; 55.37;
55.42; 56.11; 56.27; 63.59; 72.20; 74.24; 77.30; 85.82; 86.59; 87.13; 89.33; 97.02; 99.43; 109.47; 113.25;
113.47 (2 C); 113.65 (2 C); 114.50; 127.09; 127.85; 127.92; 127.96; 128.16; 129.03; 129.21; 129.54; 129.60;
130.05; 130.10; 130.20 (2 C); 132.53; 134.09; 135.54; 135.59; 140.35; 143.04; 144.65; 145.17; 148.98; 151.86;
158.73; 158.75; 159.39; 161.36; 161.55; 162.62; 170.29. ESI-MS: 773 (12), 1075 (100, [M þ 22]þ). Anal.
calc. for C62H60N4O12 · MeOH · H2O: C 68.59, H 6.03, N 5.08, found: C 68.48, H 6.05, N 4.90.
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-2’-deoxy-8-{3-{{2-{4-{3-[6,7-dimethoxy-3-(4-methoxy-
phenyl)-2-oxoquinolin-1(2H)-yl]propyl}phenyl}acetyl}amino}prop-1-yn-1-yl}uridine 3’-[2-Cyanoethyl
Bis(1-methylethyl)phosphoramidite] (8). As described for 5, with 7 (500 mg, 0.47 mmol), diisopropyl-
ammonium 1H-tetrazolide (60 mg, 0.35 mmol), MeCN (3 Â 5 ml), CH2Cl2 (5 ml), and 2-cyanoethyl
N,N,N’,N’-tetraisopropylphosphordiamidite (390 ml, 1.22 mmol); reaction time 1.5 h: 8 (595 mg, 54%).
31P-NMR (120 MHz, CDCl3): 149.6; 150.1.
DNA d{(5’-Amino-5’-deoxy)T-C-C-A-A-C-T-C-{8-{3-{{2-{4-{3-[6,7-dimethoxy-3-(4-methoxyphen-
yl)-2-oxoquinolin-1(2H)-yl]propyl}phenyl}acetyl}amino}prop-1-yn-1-yl}A-A-C-A-C-C-A-C} 5’-Termi-
nal Amide with Bis (4,7-diphenyl-1,10-phenanthroline-kN1,kN10)[4-(7-phenyl-1,10-phenanthrolin-4-yl-
kN1,kN10)benzenepentanoic Acid]ruthenium (II) (10). The synthesis of 9 was carried out on a 1 mmol
scale. Modified building blocks 5 (100 mg) and 5’-deoxy-5’-{[(4-methoxyphenyl)diphenylmethyl]ami-
no}thymidine 3’-[2-cyanoethyl bis(1-methylethyl)phosphoramidite] [18] (60 mg) were incorporated
manually at specific sites. In the last cycle, the (MeO)Tr-protecting group at the 5’-end was cleaved off.
Cleavage from solid support was performed with 25% aq. NH3 at 558 for 4 h. After replacement of NH4þ
with Kþ and desalting with a NAP-10 column, 9 (26 OD, 157 nmol) was dissolved in DMF/1,4-dioxane/
H2O 1:1:1 (470 ml), and iPr2EtN (5 ml, 32 mmol) as well as (bathophenanthroline)ruthenium(II)
complex (as ester with hydroxysuccinimide) [5] (5 mg, 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 EtOH (3 Â 700 ml)
to remove the excess of the acceptor. The crude 10 was purified by reversed-phase HPLC EC-125/4-
Nucleosil-100-5 C18 column, 0 ! 90% B in 40 min (tR 27.23 min). After purification, 10 was analyzed by
PAGE (Fig. 1, Lanes 3 and 7).
DNA d{(5’-Amino-5’-deoxy)T-C-C-A-A-C-{2’-{[(1,1-dimethylethyl)dimethylsilyl]oxy}U-C-{a-{2-
{{2-{4-{3-[6,7-dimethoxy-3-(4-methoxyphenyl)-2-oxoquinolin-1(2H)-yl]propyl}phenyl}acetyl}amino}-
ethylidyne}}T-A-C-A-C-C-A-C} (12). The synthesis of 12 was carried out on a 1 mmol scale. Modified

Helvetica Chimica Acta – Vol. 91 (2008)
175
building blocks 8 (100 mg) and 5’-deoxy-5’-{[(4-methoxyphenyl)diphenylmethyl]amino}thymidine 3’-[2-
cyanoethyl bis(1-methylethyl)phosphoramidite] [18] (60 mg) were incorporated manually at specific
sites, by using 0.3m BMT (BMT¼ 5-(benzylmercapto)tetrazole ¼ 5-(benzylthio)-1H-tetrazole) as
activation reagent. In the last cycle, the (MeO)Tr-protecting group at the 5’-end was cleaved off 11.
For solid-phase coupling of the (bathophenanthroline)ruthenium(II) complex (see below), 10 mg of 11
(on solid support) was set aside. The rest of the material (11) was cleaved from the solid support with 2m
NH4OH in EtOH (500 ml) for 30 min at 658 and then treated with 7m MeNH2 in EtOH (500 ml) for 30 min
þ
at 658. After replacement of NH4 with Kþ and desalting with a NAP-10 column, 37 OD of 12 were
obtained.
DNA d{(5’-Amino-5’-deoxy)T-C-C-A-A-C-{2’-{[(1,1-dimethylethyl)dimetylsilyl]oxy}U-C-[a-{2-{{2-
{4-{3-[6,7-dimethoxy-3-(4-methoxyphenyl)-2-oxoquinolin-1(2H)-yl]propyl}phenyl}acetyl}amino}ethyl-
idyne}}T-A-C-A-C-C-A-C} 5’-Terminal Amide with Bis(4,7-diphenyl-1,10-phenanthroline-kN1,kN10)[4-
(7-phenyl-1,10-phenanthrolin-4-yl-kN1,kN10)benzenepentanoic Acid]ruthenium(II) (13). a) Coupling of
the (Bathophenanthroline)ruthenium(II) Complex as Ester to 12, in Solution. To soln. of 12 (28 OD,
i
1.72 nmol) in DMF/1,4-dioxane/H2O 1:1:1 (520 ml), Pr2EtN (6 ml, 34.5 mmol) and (bathophenanthro-
line)ruthenium(II) complex (as ester with hydroxysuccinimide); (6 mg, 4.3 mmol) were added. The
mixture was incubated at 258 in the dark for 24. After removal of the solvents, the residue was washed
with CHCl3 (3 Â 700 ml) to remove the excess of the acceptor. The crude 13 was purified by reversed-
phase HPLC (EC-125/4-Nucleosil-100-5- C18 column, 0 ! 90% B in 40 min; tR 27.88).
b) Coupling of (Bathophenanthroline)ruthenium(II) Complex as Free Acid to 11, on Solid Support.
A mixture of HOBt (15 mg, 100 mmol), HBTU (34 mg, 90 mmol), and (bathophenanthroline)ruthen-
i
ium(II) complex (as free acid; 119 mg, 100 mmol) was dissolved in dry DMF(600 ml). Pr2EtN (41 ml,
232 mmol) was added to this soln, followed by vortexing for 10 min. This resulting soln. was added to the
NH2-bearing solid support 11 (10 mg, ca. 0.35 mmol loading) in an Eppendorf tube. The soln. was slowly
vortexed for 24 h. After removal of the supernatant, the solid support was washed with DMF(2 Â 1 ml)
and MeCN (2 Â 1 ml). Cleavage from the solid support was performed with 25% aq. NH3 (500 ml) for 2 h
at 408: 13. The crude 13 was purified by reversed-phase HPLC (EC-125/4-Nucleosil-100-5-C18 column,
with a 0 ! 90% B in 40 min; tR 27.48 min).
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Received September 17, 2007