Synthesis of 5-(2,2′-bipyridinyl and 2,2′-bipyridinediiumyl)-2′-deoxyuridine nucleosides: Precursors to metallo-DNA conjugates

Article abstract of DOI:10.1081/NCN-120015068

The synthesis of 2,2′-bipyridinyl-2′-deoxyuridine metal-chelator nucleosides (Bipy-dU) with either ethynyl or ethylenyl linkers was now been accomplished. These new nucleosides will permit the construction of a number of corresponding metallo-DNA conjugat

Full text of DOI:10.1081/NCN-120015068

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SYNTHESIS OF 5-(2,2′-BIPYRIDINYL AND 2,2′-
BIPYRIDINEDIIUMYL)-2′-DEOXYURIDINE NUCLEOSIDES:
PRECURSORS TO METALLO-DNA CONJUGATES
Samir T. Gaballah a , Charles E. Kerr a c , Bruce E. Eaton b & Thomas L. Netzel a
a Department of Chemistry , Georgia State University , Atlanta, Georgia, 30303, U.S.A.
b Department of Chemistry , North Carolina State University , Raleigh, North Carolina,
27695, U.S.A.
c Motorola Life Sciences , 757 S. Raymond Ave., Pasadena, CA, 91105, U.S.A.
Published online: 07 Feb 2007.
To cite this article: Samir T. Gaballah , Charles E. Kerr , Bruce E. Eaton & Thomas L. Netzel (2002) SYNTHESIS OF 5-(2,2′-
BIPYRIDINYL AND 2,2′-BIPYRIDINEDIIUMYL)-2′-DEOXYURIDINE NUCLEOSIDES: PRECURSORS TO METALLO-DNA CONJUGATES,
Nucleosides, Nucleotides and Nucleic Acids, 21:8-9, 547-560, DOI: 10.1081/NCN-120015068
To link to this article: http://dx.doi.org/10.1081/NCN-120015068
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MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 21, Nos. 8 & 9, pp. 547–560, 2002
SYNTHESIS OF 5-(2,20-BIPYRIDINYL AND
2,20-BIPYRIDINEDIIUMYL)-20-DEOXYURIDINE
NUCLEOSIDES: PRECURSORS TO
METALLO-DNA CONJUGATES
Samir T. Gaballah,1 Charles E. Kerr,1,* Bruce E. Eaton,2
and Thomas L. Netzel1,{
1Department of Chemistry, Georgia State University,
Atlanta, GA 30303
2Department of Chemistry, North Carolina State University,
Raleigh, NC 27695
ABSTRACT
The synthesis of 2,20-bipyridinyl-20-deoxyuridine metal-chelator nucleo-
sides (Bipy-dU) with either ethynyl or ethylenyl linkers was now been
accomplished. These new nucleosides will permit the construction of a
number of corresponding metallo-DNA conjugates where many types of
metals can be complexed to the 2,20-bipyridinyl chelator group and the
resulting metallo-dU conjugates incorporated into DNA oligonucleo-
tides. Additionally this paper also reports the synthesis of a di-N-alky-
lated bipyridinediiumyl-20-deoxyuridine nucleoside (Bipy2þ-dU) with an
ethylenyl linker. The Bipy2þ-dU nucleoside was found to decompose
under basic conditions precluding its use in standard automated DNA-
synthesis by the phosphoramidite method. No such restrictions apply to
the two Bipy-dU nucleosides reported here for use as metal chelators.
*Current address: Motorola Life Sciences, 757 S. Raymond Ave., Pasadena, CA 91105.
{Corresponding author. Fax: (404) 651-3129; E-mail: tnetzel@gsu.edu
547
DOI: 10.1081=NCN-120015068
Copyright # 2002 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

548
GABALLAH ET AL.
INTRODUCTION
Bipyridines have been widely used as ligands in coordination chemistry.
Because of its stability, ease of functionalization, and chelating metal-
coordination, 2,20-bipyridine, in particular, has been heavily used to link
metals to other molecular subunits.[1] Additionally, bipyridines have played
important roles in constructing supramolecular assemblies,[1–3] developing
pharmaceuticals[4–10] and sensors,[11,12] and mimicking photosynthetic light
conversion.[13] Recently, bipyridines have also been used to make functio-
nalized polymers[14–17] and photocatalysts.[18]
Two desires motivated our efforts to link 2,20-bipyridine to 20-
deoxyuridine. First we were interested in synthesizing new bipyridine-
deoxyuridine conjugates where the linker between the metal chelator and
uracil was short, two carbons, and could be either flexible or rigid. It was also
desired to prepare these new bipyridine-deoxyuridine conjugates so that they
could be incorporated into DNA as metal-chelators. A number of research
groups have constructed DNA duplexes with one or more covalently
attached metal complexes.[19–25] These groups were interested in the physical
properties of novel metallo-DNA constructs, in questions of the distance
dependence of the rates of energy and electron transfer between the attached
metallosubunits themselves and between the metallosubunits and particular
DNA bases, and in the development of DNA hybridization probes and
sensors. Thus routes to constructing new metallo-DNA conjugates appear to
be of broad interest. This work reports the synthesis of 2,20-bipyridinyl-dU
conjugates with either ethynyl (6, Sch. 2) or ethylenyl (1, Sch. 3) linkers that
will permit the construction of a number of corresponding metallo-DNA
conjugates, since many types of metals can be complexed to the 2,20-bipyr-
idinyl chelator group and the resulting metallo-dU conjugates can then be
incorporated into DNA oligonucleotides in a variety of ways.[26–30]
Secondarily, it was also of interest to convert these new conjugates into
methyl viologen analogs, 5-(2,20-bipyridinediiumyl)-20-deoxyuridine nucleo-
sides (Bipy2þ-dU). By analogy to 4,40-bipyridinediium (methyl viologen),[31,32]
we reasoned that Bipy2þ-dU nucleoside conjugates could function as rever-
sible electron acceptors when incorporated into DNA duplexes. Importantly,
singly reduced Bipy2þ radicals are known to have strong absorbances in the
UV-vis region and therefore should serve as good indicators of excess elec-
tron trapping in kinetics studies of electron transport in DNA.[33]
To date our efforts in the area of nucleoside conjugate chemistry have
produced a number of pyrenyl-dU (Py-dU) conjugates that are capable of
photoinjecting an excess electron into a DNA p-stack.[34–38] In these con-
jugates the lowest electronic, excited singlet-state of pyrene reduces the
attached uracil in less than 30 ps, and in methanol the resulting primary
electron transfer (ET) product, Pyꢀþ=dUꢀ ꢁ , lives from 6 ps to 2.1 ns,
depending upon the type of linker that joins the pyrenyl and uracil subunits.

PRECURSORS TO METALLO-DNA CONJUGATES
549
Scheme 1. (a) PdL4, toluene, reflux, overnight, (b) PdL2Cl2, CuI, Et3N, THF, TMSA, (c)
KF, THF, MeOH.
To further increase the lifetime of a photogenerated uracil anion in a DNA
duplex, we have also synthesized several dimethylanilino-dU conjugates
(DMA-dU) to serve as secondary electron donors.[39] In duplexes labeled
with both Py-dU and DMA-dU, secondary ET from DMA to Pyꢀþ=dUꢀ ꢁ
should produce the DMAꢀþ=Py=dUꢀ ꢁ ET product that is expected to be
Scheme 2. (a) PdL4, CuI, Et3N, THF.

550
GABALLAH ET AL.
Scheme 3. (a) H2 (50 psi), Pd=C, MeOH, (b) NH4OH, MeOH, overnight, (c) CF3COOH,
CH2Cl2, (d) BrCH2CH2Br, 100ꢂC, 24 h.
significantly longer lived than Pyꢀþ=dUꢀ ꢁ . Thus routes to producing elec-
tron-trap nucleosides are of current interest. In addition to the syntheses of
two kinds of Bipy-dU nucleosides (1 and 6), this paper also reports the
synthesis of a Bipy2þ-dU nucleoside with an ethylenyl linker (7, Sch. 3).
However, the nucleoside 7 was found to react rapidly with pyridine, and thus
will not survive the base deprotection conditions required after standard
automated DNA-strand synthesis by the phosphoramidite method. In con-
trast no such restrictions apply to the Bipy-dU nucleosides reported here for
use as metal chelators.
To make both Bipy-dU and Bipy2þ-dU conjugates, we focused on
connecting bipyridine via short alkynyl or alkyl linkers to the 5-position of
deoxyuridine as a direct way of positioning the chelating and electron
accepting subunits in the DNA major groove. These linkers were selected
both to provide strong electronic coupling between uracil and the poly-
pyridinyl subunits and to accurately locate the polypyridinyl subunits along
the surface of a DNA duplex. Additionally, locating the polypyridinyl sub-
units in the major groove was less likely to interfere with normal DNA base
pairing than would locating them in the minor groove. These considerations

PRECURSORS TO METALLO-DNA CONJUGATES
551
in turn made both ethynylbipyridine and ethynyl-dU attractive precursors to
final ethynyl- and ethylenyl-linked nucleoside conjugates.
Our synthetic scheme used a common bromobipyridine precursor (2,
Sch. 1), two Sonogashira couplings to effect conjugation of the bipyridine to
the deoxyuridine (Schs. 1 and 2), and a final alkyne reduction to convert 6 to
1 (Sch. 3). Several different methods of preparing bromobipyridines have
been reported.[40–45] After investigating several of these, we found that
adaptation of a pyridylstannane-based synthesis of 2,20:60,200-terpyridine[42]
produced 2 in good overall yield. Palladium catalyzed coupling of 2 to tri-
methylsilylacetylene (TMSA) followed by desilylation with KF was per-
formed successfully on a multi-gram scale (see Sch. 1).
With 6-ethynyl-2,20-bipyridine (4) in hand, the Sonogashira coupling
was performed, giving the ethynyl-linked Bipy-dU conjugate (6, Sch. 2). To
eliminate the p-conjugation between the deoxyuridine and the bipyridine
subunits, the alkyne linker was selectively reduced via catalytic hydrogena-
tion giving the ethylenyl-linked Bipy-dU conjugate (1, Sch. 3). Subsequent
quaternization of (1a) with 1,2-dibromoethane produced the ethylenyl-linked
Bipy2þ-dU conjugate (7, Sch. 3).
RESULTS AND DISCUSSION
A great variety of methods to prepare functionalized 2,20-bipyridines
have been described in the literature,[40,41,43,44,46–48] and forming 6-bromo-
2,20-bipyridine (2, Sch. 1) was key to synthesizing 6-ethynylbipyridine (4).
The key compound 2 had been synthesized previously using two different
approaches. One approach synthesized 2 from 6,60-dibromo-2,20-bipyri-
dine and phenyllithium.[40] A second approach used Stille-type cross-
coupling.[47,49,50] This latter method had been used to make terpyridines
and bipyridines using pyridyl stannane in conjunction with, respectively,
dibromopyridine and bromopyridine. It was found that reproducible
yields of 2 (67% isolated) could be obtained following the second approach
starting with 2-(trimethylstannanyl)pyridine and 2,6-dibromopyridine in
refluxing toluene in the presence of PdL4. Compound 3 was synthesized
from 2 and TMSA through a Pd(0)-cross coupling reaction.[51] The Pd-
catalysed reaction could be carried out using PdL4 (ca. 5 mol%) in dry tet-
rahydrofuran (THF) or dimethyformamide (DMF) in the presence of
CuI and triethylamine (Et3N) at 60ꢂC. Under these conditions using PdL4
catalyst, the observed yield was 68%. Also, the Pd-catalyst could be created
in situ from PdL2Cl2.[42] These latter conditions using PdL2Cl2 catalyst
gave the best yield of the desired coupling product (89%, see Sch. 1).
Removing the trimethylsilyl group of 3 with KF in THF=methanol (MeOH)
(1:1 by vol.) solution at room temperature produced compound 4 in 98%
yield.

552
GABALLAH ET AL.
The nucleoside 6c could be prepared directly by performing the Pd-
coupling with 4 and 5c (see Sch. 2). However, purification of the crude reaction
product was problematic. It was more convenient and reproducible to perform
the palladium coupling reaction and subsequent purifications on either the
50-O-DMT protected 6a or the 30-O, 50-O- diacetate protected 6b. Three steps
were employed to synthesize 1a in Sch. 3. First, the 50-OH of 5c was protected
with DMTCl in dry pyridine to afford 50-O-DMT-IdU, 5a.[52] Second, 5a was
coupled to 4 in dry THF or DMF at 60ꢂC using Pd(0)-cross coupling to form
6a. To produce 1a, compound 6a was hydrogenated on 10% Pd=C in dry
MeOH by stirring at room temperature for 24–48 h in a sealed glass vessel
pressurized with hydrogen (50 psi).[53] The glass vessel was sealed with either
stainless steel or brass fittings and topped with a pressure gauge. Before adding
6a, the Pd=C-catalyst in MeOH was activated by pressurizing the hydro-
genation vessel with hydrogen (50 psi) and stirring the slurry for 15–20 m.
We were also interested in synthesizing the diquat nucleoside conjugate
7 in Sch. 3, ethylenyl-linked Bipy2þ-dU. (Note that attempts to quanternize
6c, the alkynyl-linked Bipy-dU nucleoside, using similar reaction conditions
were not successful. This reaction was carried out in dry freshly distilled 1,2-
dibromoethane and a precipitate formed as in the quaternization of 1c.
However, NMR analysis showed that the precipitate was not the desired
diquat. Also, TLC analysis of this product showed multiple spots, and no
useful amount of the desired diquat was recovered.) The alkylation condi-
tions required forming the diquat conjugate 7 from the free nucleoside 1c.
Alkylation of the two nitrogen atoms of the bipyridine moiety was carried
out in dry freshly distilled 1,2-dibromoethane. Compound 7 was formed in
40% yield after refluxing 1c in a solution of 1,2-dibromoethane for 24 h.
While 7 was stable and readily isolated under neutral conditions, when it was
added to neat pyridine the resulting solution turned dark green within ten
minutes and eventually became black. TLC analysis of this black solution
showed numerous, uncharged degradation products. Related studies on the
stability of several model diquats, including methylviologen, to 10% diethyl
amine in MeOH and 0.05 M K2CO3 in MeOH showed that all of them
reacted rapidly to produce strongly colored solutions and uncharged
degradation products (as determined by TLC analysis).[54]
SUMMARY
Reproducible synthetic methods are now available to prepare bipyri-
dinyl-deoxyuridine conjugates with short, two-atom linkers that are either
stiff or flexible. These conjugates will locate the bipyridinyl subunits in the
major groove of DNA and will be valuable building blocks for creating
DNA structures for studies of the properties of metallo-DNA where precise
positioning of the bipyridinyl chelator is important. We have also shown

PRECURSORS TO METALLO-DNA CONJUGATES
553
that 7, a viologen analog, can be prepared via a straightforward dialkylation
of the 6c nucleoside with 1,2-dibromoethane, and is stable under neutral
conditions. Conjugate 7, however, is not stable under the standard base
deprotection conditions that are encountered in automated DNA synthesis.
Our current efforts continue to explore new methods for preparing viologen-
related deoxyuridine nucleosides that will survive automated DNA synthesis
and therefore will be able to serve as traps in studies of excess electron
transport in DNA.
EXPERIMENTAL
General Synthetic Procedures
All manipulations of tetrakis(triphenylphosphine)palladium(0) (PdL4)
and 4,40-dimethoxytrityl chloride (DMTCl) were performed in a Vacuum
Atmospheres M040–2 glove box pressurized with dry nitrogen gas. All other
reactions were carried out on the benchtop under a dry nitrogen atmosphere.
Chromatography was carried out on a Biotage Flash-40TM system using
either prepackaged KP-SilTM cartridges, or Flash-40TM cartridge housings
repacked with WhatmanTM flash silica (80 A pore, 230–400 mesh). Silica gel
used for pad filtrations and dry powder chromatography sample loading was
also WhatmanTM flash silica. PdL4 was prepared according to the litera-
ture[55] and stored in a glove-box freezer (ꢁ33ꢂC). The following solvents
were dried and redistilled in continuous circulation distillation apparati:
tetrahydrofuran (THF, dried with benzophenone =Na0), triethylamine (Et3N,
dried with CaH2), N,N-dimethylformamide (DMF, dried with CaH2), and
methanol (MeOH, dried with Mg turnings). Trimethylsilylacetylene (TMSA)
was obtained from GFS Chemicals and used after vacuum transfer from
CaH2. 1,2-dibromoethane was purchased from Aldrich and was distilled with
CaH2 after treating with 5% NaHCO3 and drying over anhydrous sodium
sulfate. All copper salts used in preparing bipyridines or palladium couplings
were obtained from Strem Chemicals. 5-Iodo-20-deoxyuridine (IdU) obtained
from Aldrich was used as received. Phenyllithium and butyllithium were
obtained from common sources and titrated prior to use with 2-butanol and
9,10-phenanthroline in hydrocarbon solution. Other reagents and solvents
were obtained from common suppliers and were usually used without further
purification. Mass spectrometry was performed at the Georgia Institute of
Technology. NMR Spectra were obtained from a Varian Unityþ300 NMR
spectrometer operating at either 75 or 300 MHz frequencies located at
Georgia State University.
6-Bromo-2,20-bipyridine, 2. 6-Bromo-2,20-bipyridine was prepared
from 2-trimethylstannylpyridine and 2,6-dibromopyridine by adaptation
of a literature method,[47,48] or less conveniently from 6,60-dibromo-2,20

554
GABALLAH ET AL.
bipyridine also by adaptation of a literature method.[40] From the stannane,
the procedure was as follows: 2-Trimethylstannylpyridine (44.35 g, 183 mmol),
2,6-dibromopyridine (45.60 g, 192 mmol), PdL4 (10.60 g, 9.2 mmol), and
500 mL dry toluene were combined and refluxed for 18 h. Toluene was
removed in vacuo. The resulting crude material was loaded onto a flash silica
column and chromatographed. Elution with hexanes then benzene afforded
both pure bromobipyridine (10.6 g as a white solid after recrystallization
from hexanes) and impure bromobipyridine. The impure material was
repurified by recrystallization from hexanes giving 18.4 g. The total yield of 2
was 67% based on 2,6-dibromopyridine and gave analytical data identical to
those previously reported.
6-(Trimethylsilanylethynyl)-2,20-bipyridine, 3. To a solution of 2
(1.82 g, 7.73 mmol) in THF (5 mL) were added, successively, PdL2Cl2 (0.27 g,
0.38 mmol), CuI (0.15 g, 0.77 mmol), Et3N (3 mL), THF (10 mL), and, finally,
TMSA (4.37 mL, 30.9 mmol) in a nitrogen atmosphere. The yellow solution
was heated at 60ꢂC until the starting material was completely consumed
(2–3 h). The solvent was evaporated to give a crude product that was purified
by pad filtration with ethyl acetate=CH2Cl2 (1:2 by vol.) then crystallized
from hexanes to give 0.99 g of 3 (89% yield).1H NMR (CDCl3, 300 MHz) d:
0.303 (9H, s, (CH3)3), 7.30 (1H, m, Ar), 7.49 (1H, dd, J ¼ 0.9, 7.5 Hz, Ar),
7.74–7.84 (2H, m, Ar), 8.36 (1H, dd, J ¼ 0.9, 8.1 Hz, Ar), 8.47 (1H, dt,
J ¼ 0.9, 8.1 Hz, Ar), 8.66 (1H, dq, J ¼ 0.9, 4.8 Hz, Ar).13C NMR (CDCl3, 75
MHz) d: ꢁ 0.21, 94.48, 103.98, 120.54, 121.62, 123.96, 127.54, 136.88, 136.95,
142.44, 149.04, 155.41, 156.44. HRMS (EI) m=z for C15H16N2Si (Mþ): calc’d.
252.1082, found 252.1075.
6-Ethynyl-2,20-bipyridine, 4. A mixture of 3 (0.985 g, 3.90 mmol) and
KF (0.27 g, 4.70 mmol) in MeOH=THF (50 mL) was stirred for 2 h at room
temperature. The crude product was purified using silica gel pad filtration
with ethyl acetate=CH2Cl2 (1:2 by vol.) to produce 0.68 g of 4 (98% yield).1H
NMR (CDCl3, 300 MHz) d: 3.22 (1H, s, ꢃ CH), 7.29 (1H, qd, J ¼ 0.9,
7.5 Hz, Ar), 7.49 (1H, dd, J ¼ 0.9, 7.5 Hz, Ar), 7.75–7.82 (2H, m, Ar), 8.40
(1H, dd, J ¼ 0.9, 7.9 Hz, Ar), 8.46 (1H, dt, J ¼ 0.9, 7.9 Hz, Ar), 8.65 (1H, dq,
J ¼ 0.9, 4.8 Hz, Ar).13C NMR (CDCl3, 75 MHz) d: 77.44, 82.93, 121.00,
121.34, 123.94, 127.33, 136.80, 136.97, 141.48, 148.92, 155.04, 156.36. HRMS
(EI) m=z for C12H8N2 (Mþ): calc’d. 180.0687, found 180.0674.
50 -O-(4,40 -Dimethoxytrityl)-5-((2,20-bipyridin-6-yl)-ethynyl)-20-deoxy-
uridine, 6a. To a solution of 5a (1.97 g, 3.0 mmol), PdL4 (0.173 g,
0.15 mmol), CuI (0.057 g, 0.3 mmol), and Et3N (2.09 mL, 15 mmol) in THF
(18 mL) was added 4 (0.702 g, 1.3 mmol) in a glove box. The reaction mixture
was stirred at 50–60ꢂC for 3 h. The reaction mixture was chromatographed

PRECURSORS TO METALLO-DNA CONJUGATES
555
on a silica gel column pre-equilibrated with 1% Et3N in CH2Cl2 (by vol.) and
eluted with 10:0.0, 9.9:0.1, 9.8:0.2, 9.7:0.3, and 9.6:0.4 CH2Cl2=MeOH (by
vol.). A yellow foam of 6a was obtained (1.80 g, 84% yield).1H NMR
0
0
(DMSO-d6, 300 MHz) d: 2.29–2.43 (2H, m, H2 b=H2 a), 3.22–3.33 (2H, m,
0 0 0 0
5 a=H5 b), 3.67 (6H, s, DMT-(CH3O)2), 4.03 (1H, m, H4 ), 4.37 (1H, m, H3 ),
H
0
0
5.42 (1H, d, J ¼ 4.5 Hz, OH3 ), 6.21 (1H, t, J ¼ 6.3 Hz, H1 ), 6.89 (4H, dd,
J ¼ 3.9, 8.94 Hz, Ar), 7.09 (1H, dd, J ¼ 0.9, 7.5 Hz, Ar), 7.19 (1H, t,
J ¼ 7.2 Hz, Ar), 7.31–7.38 (6H, m, Ar), 7.46–7.56 (3H, m, Ar), 7.89 (1H, t,
J ¼ 7.8 Hz, Ar), 7.97 (1H, dt, J ¼ 1.5, 7.65 Hz, Ar), 8.27 (1H, s, H6), 8.33–8.39
(2H, m, Ar), 8.75 (1H, m, Ar), 11.90 (1H, br, HN3).13C NMR (CDCl3, 75
MHz) d: 41.54, 55.07, 63.57, 72.23, 79.81, 86.11, 86.66, 86.96, 93.04, 99.57,
113.27, 120.18, 121.60, 123.89, 126.90, 127.43, 127.83, 127.97, 129.87, 129.96,
135.38, 135.52, 136.67, 136.87, 142.05, 143.43, 144.43, 148.88, 149.42, 155.31,
155.99, 158.6, 158.48, 161.42. HRMS (FAB) m=z for C42H37N4O7 (M þ H)þ:
calc’d. 709.2662, found 709.2679.
50 -O-(4,40 -Dimethoxytrityl)-5-((2,2’-bipyridin-6-yl)-ethylenyl)-20-deo-
xyuridine, 1a. Dry 6a (800 mg, 1.13 mmol) was dissolved in 300 mL of dry
MeOH and added to 30 mL MeOH solution containing 400 mg of Pd=C
previously activated by stirring under H2 (50 psi) for 20 m at room tem-
perature. The reaction mixture was further stirred under H2 (50 psi) at room
temperature until complete consumption of the starting materials (2 days).
The solution was then filtered, reduced in volume to 0.5 mL, and purified by
chromatography on a silica gel column pre-equilibrated with 1% Et3N (by
vol.) in CH2Cl2 and eluted with 10:0.0, 9.9:0.1, 9.8:0.2, 9.7:0.3, and 9.6:0.4
CH2Cl2=MeOH (v=v). A pale yellow foam of 1a was obtained (706 mg, 87%
yield).1H NMR (CD2Cl2, 300 MHz) d: 1.78 (1H, m, H2 b), 2.15 (1H, m, H2 a),
2.45 (2H, t, J ¼ 7.9 Hz, CH2), 2.83 (2H, t, J ¼ 7.9 Hz, CH2), 3.02 (1H, m,
0
0
0
0
0
H
5 b), 3.16 (1H, m, H5 a), 3.33 (1H, d, J ¼ 3 Hz, OH3 ), 3.61 (3H, s, DMT-
0
OCH3), 3.62 (3H, s, DMT-OCH3), 3.80 (1H, q, J ¼ 3.3 Hz, H4 ), 3.95 (1H, m,
0
0
H3 ), 6.15 (1H, t, J ¼ 6.3 Hz, H1 ), 6.68–6.82 (5H, m, Ar), 7.00 (1H, s, H6),
7.08–7.28 (10H, m, Ar), 7.54 (1H, t, J ¼ 7.8 Hz, Ar), 7.70 (1H, dt, J ¼ 1.8,
7.5 Hz, Ar), 8.45 (1H, m, Ar), 8.16 (1H, m, Ar), 8.47 (1H, m, Ar), 9.08 (1H,
br, HN3).13C (CD2Cl2, 75 MHz) d: 27.05, 36.88, 40.82, 46.34, 55.49, 63.58,
71.00, 84.33, 85.38, 86.76, 113.45, 114.37, 118.92, 121.88, 123.68, 123.99,
127.21, 128.22, 128.40, 130.34, 135.84, 135.87, 136.56, 137.29, 137.40, 144.91,
149.21, 150.66, 155.88, 156.81, 160.75, 163.71. HRMS (FAB) m=z for
C42H41N4O7 (M þ H)þ: calc’d. 713.2975, found 713.3003.
30,50 -Di-O-acetyl-5-((2,20-bipyridin-6-yl)-ethynyl)-20-deoxyuridine,
6b. In a glove box, 4 (2.80 g, 15.50 mmol), THF (44 mL), Et3N (11 mL),
PdL4 (1.79 g, 1.55 mmol), CuI (0.59 g, 3.10 mmol), and 5b (6.80 g,
15.50 mmol) were combined in a flask, and the flask sealed with a septum.
The flask was removed from the glove box, and the mixture was stirred and

556
GABALLAH ET AL.
heated to 60ꢂC for 3 h in an oil bath. The contents of the flask were dried
onto silica gel. A silica gel pad filtration was done to remove metals and
salts using CHCl3, 25% CHCl3 (by vol.) in ethyl acetate, and 5% MeOH
(by vol.) in ethyl acetate as eluents. The eluate was concentrated and loaded
onto a flash silica column. Elution using CH2Cl2, 50% ethyl acetate (by
vol.) in CH2Cl2, and ethyl acetate separated the desired product from the
bulk material. Concentration gave a foam that was recrystallized from
ethanol to give 6b (4.11 g, 8.38 mmol, 54% yield) as a white solid.1H NMR
(CDCl3, 300 MHz) d: 2.13 (3H, s, CH3), 2.26 (3H, s, CH3), 2.27 (1H, m,
0
0
H
2 b), 2.59 (1H, ddd, J ¼ 3.0, 6.0, 14.0 Hz, H2 a), 4.33 (1H, q, J ¼ 3.0 Hz,
0
0
0
0
H4 ), 4.40 (2H, m, H5 b=H5 a), 5.27 (1H, dt, J ¼ 3.0, 6.0 Hz, H3 ), 6.35 (1H,
0
dd, J ¼ 6.0, 8.0 Hz, H1 ), 7.33 (1H, m, Ar), 7.56 (1H, m, Ar), 7.82 (2H, m,
Ar), 8.09 (1H, s, H6), 8.41 (2H, m, Ar), 8.68 (1H, m, Ar), 10.41 (1H, br,
HN3).13C NMR (CDCl3, 75 MHz) d: 20.84, 20.93, 38.30, 63.78, 73.89,
79.60, 82.62, 85.51, 93.27, 100.12, 120.63, 121.34, 124.02, 127.33, 136.94,
137.07, 141.91, 142.71, 149.09, 149.12, 155.27, 156.46, 160.76, 170.27,
170.34. HRMS (FAB) m=z for C25H23N4O7 (M þ H)þ: calc’d. 491.15667,
found 491.15445.
30,50 -Di-O-acetyl-5-((2,20-bipyridin-6-yl)-ethylenyl)-20-deoxyuridine,
1b. Compound 6b
(545 mg, 1.11 mmol) and 10% Pd=C (254 mg) were
placed in a 250 mL pressure vessel, and the vessel was flushed with nitrogen.
MeOH (20 mL) was added, and the vessel was flushed repeatedly with
hydrogen before finally being pressurized to 50 psi. After stirring under
pressure for 2 d, the vessel was carefully depressurized, and the reaction
mixture was filtered. The retentate was washed with copious amounts of
chloroform and benzene. The filtrate was concentrated onto silica gel and
chromatographed using 0, 2.5 and 5% MeOH (by vol.) in CH2Cl2. The
product 1b was obtained as a white solid (414 mg, 0.84 mmol, 75.3%
yield).1H NMR (CDCl3, 300 MHz) d: 1.85 (1H, ddd, J ¼ 4, 6, 21 Hz, H2 b),
0
0
2.00 (3H, s, CH3), 2.05 (3H, s, CH3), 2.25 (1H, ddd, J ¼ 2, 6, 14 Hz, H2 a),
0
0
2.85 (2H, m, CH2), 3.09 (2H, t, J ¼ 7 Hz, CH2), 4.08 (2H, m, H5 b=H5 a), 4.18
0
0
0
(1H, m, H4 ), 4.99 (1H, m, H3 ), 6.22 (1H, dd, J ¼ 6, 9 Hz, H1 ), 7.11 (2H, m,
Ar), 7.27 (1H, ddd, J ¼ 1, 5, 8 Hz, Ar), 7.68 (1H, td, J ¼ 8, 2 Hz, Ar), 8.18
(1H, d, J ¼ 8 Hz, Ar), 8.38 (1H, d, J ¼ 8 Hz, Ar), 8.64 (1H, m, Ar), 9.83 (1H,
br, HN3).13C NMR (CDCl3, 75 MHz) d: 20.68, 20.77, 27.07, 36.27, 37.03,
63.69, 74.02, 81.92, 84.49, 114.60, 118.49, 120.93, 123.20, 123.57, 135.19,
136.77, 137.17, 149.09, 150.36, 155.40, 156.16, 159.76, 163.33, 169.99, 170.21.
HRMS (FAB) m=z for C25H27N4O7 (M þ H)þ: calc’d. 495.1879, found
495.1889.
5-((2,20-Bipyridin-6-yl)-ethynyl)-20-deoxyuridine, 6c. To a solution
of 5c (1.062 g, 3.00 mmol), PdL4 (0.173 g, 0.15 mmol), CuI (0.057 g,
0.30 mmol), and Et3N (2.1 mL, 15 mmol) in DMF (15 mL) was added 4

PRECURSORS TO METALLO-DNA CONJUGATES
557
(0.702 g, 1.30 mmol) in a glove box. The reaction mixture was stirred at 50–
60ꢂC for 4 h and then chromatographed on a silica gel column eluted with
10:0.0, 9.9:0.1, 9.8:0.2, 9.7:0.3, 9.6:0.4, 9.3:0.07, and 9.0:0.1 CH2Cl2=MeOH
(v=v). A yellow foam of 6c was obtained (1.12 g, 91% yield).1H NMR
0
0
(DMSO-d6, 300 MHz) d: 2.11–2.25 (2H, m, H2 b=H2 a), 3.55–3.70 (2H, m,
0
0
0
0
H
5 b=H5 a), 3.82 (1H, q, J ¼ 3.3 Hz, H4 ), 4.26 (1H, quintet, J ¼ 4.5 Hz, H3 ),
0
0
5.21 (1H, t, J ¼ 4.8 Hz, OH5 ), 5.28 (1H, d, J ¼ 4.5 Hz, OH3 ), 6.13
0
(1H, t, J ¼ 6.6 Hz, H1 ), 7.53 (1H, m, Ar), 7.6 (2H, d, J ¼ 7.5 Hz, Ar), 7.92–
8.0 (2H, m, Ar), 8.35 (2H, d, J ¼ 7.2, Ar), 8.50 (1H, s, H6), 8.75, (1H, d, J ¼
4.5 Hz, Ar), 11.77 (1H, s, HN3).13C NMR (DMSO-d6, 75 MHz) d: 60.81,
69.90, 82.28, 85.04, 87.66, 91.56, 97.30, 120.07, 120.74, 124.62, 127.47,
137.50, 138.05, 142.03, 145.13, 149.42, 149.44, 154.46, 155.76, 161.44. HRMS
(FAB) m=z for C21H19N4O5 (M þ H)þ: calc’d. 407.1355, found 407.1373.
5-((2,20-Bipyridin-6-yl)-ethylenyl)-20-deoxyuridine, 1c.
Dry 6c
(200 mg, 0.49 mmol) was dissolved in dry MeOH (100 mL) and added to
30 mL of MeOH solution containing 250 mg of 10% Pd=C previously acti-
vated by stirring under H2 (50 psi) for 20 m at room temperature. The
reaction mixture was further stirred under H2 (50 psi) at room temperature
until complete consumption of the starting material (2 days). The solution
was then filtered, reduced in volume to 0.5 mL, and chromatographed on a
silica gel column eluted with 10:0.0, 9.9:0.1, 9.8:0.2, 9.7:0.3, 9.6:0.4, 9.3:0.07,
and 9.0:0.1 CH2Cl2=MeOH (v=v). A pale yellow foam of 1c was obtained
(125 mg, 62% yield).1H NMR (DMSO-d6, 300 MHz) d: 1.88–1.95 (2H, m,
0
0
H
2 b=H2 a), 2.60–2.78 (2H, m, CH2), 2.99 (2H, t, J ¼ 7.8, CH2), 3.43–3.54
0
0
0
0
(2H, m, H5 b=H5 a), 3.711 (1H, q, J ¼ 2.7 Hz, H4 ), 4.14 (1H, m, H3 ), 5.01
0
0
(1H, t, J ¼ 4.8 Hz, OH5 ), 5.195 (1H, d, J ¼ 3.9 Hz, OH3 ), 6.10 (1H, t,
0
J ¼ 7.2 Hz, H1 ), 7.28 (1H, d, J ¼ 7.5 Hz, Ar), 7.42 (1H, m, Ar), 7.62 (1H, s,
H6), 7.82 (1H, t, J ¼ 7.5 Hz, Ar), 7.91 (1H, dt, J ¼ 1.2, 7.5 Hz, Ar), 8.37 (1H,
d, J ¼ 7.8, Ar), 8.65 (1H, d, J ¼ 6 Hz, Ar), 11.25 (1H, s, HN3).13C NMR
(DMSO-d6, 75 MHz) d: 26.26, 35.86, 61.32, 70.45, 83.80, 87.29, 112.86,
117.95, 120.50, 123.28, 124.10, 136.52, 137.24, 137.60, 149.26, 150.32, 154.60,
155.43, 160.21, 163.43. HRMS (FAB) m=z for C21H22N4O5 (M þ H)þ:
calc’d. 411.1668, found 411.1665.
5-[2-(4-(6,7-Dihydrodipyrido[1,2-a;20,10-c]pyrazinediylium))-ethyle-
nyl]- 20-deoxyuridine2þ, 7. In a Schlenk tube, 6c (100 mg, 0.024 mmol) was
co-evaporated with dry THF three times in vacuo. Freshly distilled 1,2-
dibromo-ethane (4 mL) was added to 6c in a glove box. The mixture was
degassed three times at –198ꢂC (2 x 10ꢁ4 torr) on vacuum manifold. The
solution was refluxed with stirring at 100ꢂC for 24 h. The yellow precipitate of
7 was filtered off, washed with acetone, CH2Cl2, and THF and dried on a
vacuum manifold (58 mg, 40% yield).1H NMR (DMSO-d6, 300 MHz) d: 2.09
0
0
(2H, t, J ¼ 6.0 Hz, H2 b=H2 a), 2.71–2.76 (2H, m, CH2), 3.49–3.60 (4H, m,

558
GABALLAH ET AL.
0
0
0
0
CH2= H5 b=H5 a), 3.76 (1H, q, J ¼ 3.6 Hz, H4 ), 4.21 (1H, m, H3 ), 4.96 (1H, t,
0
0
J ¼ 5.1 Hz, OH5 ), 5.22 (1H, d, J ¼ 4.2 Hz, OH3 ), 5.29 (4H, s, CH2CH2), 6.13
0
(1H, t, J ¼ 6.6 Hz, H1 ), 7.83 (1H, s, H6), 8.31 (1H, d,J ¼ 7.8 Hz, Ar), 8.45 (1H,
t, J ¼ 7.5 Hz, Ar), 8.83 (1H, t,J ¼ 8.1 Hz, Ar), 8.91–9.00 (2H, m, Ar), 9.06 (1H,
d, J ¼ 8.1 Hz, Ar), 9.38 (1H, d, J ¼ 5.7 Hz, Ar), 11.43 (1H, s, HN3).13C NMR
(CD3OD, 75 MHz) d: 26.31, 34.38, 41.36, 53.49, 62.72, 71.98, 86.56, 88.96,
111.77, 128.38, 129.75, 131.42, 133.79, 140.43, 142.30, 147.84, 148.01, 149.06,
151.99, 161.32, 165.90. HRMS (FAB) m=z for the singly reduced (Mꢀþ)
diquat (C23H26N4O5)2þ:[56,57] calc’d. 438.1903, found 438.1935.
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Received March 12, 2002
Accepted June 24, 2002