Bimetallic Cu2+ complexes of bis-terpyridine ligands as catalysts of the cleavage of mRNA 5′-cap models. the effect of linker length and base moiety

Article abstract of DOI:10.1039/b821724f

Ligands, where two terpyridine units are linked via an alkyl chain of three to five methylene units, have been synthesized. Their Cu²⁺ complexes have been studied as catalysts for the hydrolysis of the triphosphate bridge of three different dinucleoside triphosphates. The results show that the bimetallic complexes are up to 600 times more efficient catalysts than monomeric Cu²⁺-TerPy, and up to 5 × 10⁵-fold rate enhancement in comparison to the uncatalysed reaction, is achieved. However, the catalytic activity strongly depends on the length of the linker and the base composition of the substrate. The differences can be attributed to interactions between the Cu²⁺-TerPy and nucleic acid base moieties as well as steric factors that may hinder the productive interaction between the substrate and the catalyst.

Full text of DOI:10.1039/b821724f

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/njc | New Journal of Chemistry
Bimetallic Cu2+ complexes of bis-terpyridine ligands as catalysts
of the cleavage of mRNA 50-cap models. The effect of linker length
and base moiety
¨
Leena Maanpaa, Vincent Luzet, Glemence Guillaume, Sharmin Taherpour,
Esa Maki and Satu Mikkola*
¨
¨
Received (in Montpellier, France) 3rd December 2008, Accepted 7th April 2009
First published as an Advance Article on the web 12th May 2009
DOI: 10.1039/b821724f
Ligands, where two terpyridine units are linked via an alkyl chain of three to five methylene units,
have been synthesized. Their Cu2+ complexes have been studied as catalysts for the hydrolysis of
the triphosphate bridge of three different dinucleoside triphosphates. The results show that the
bimetallic complexes are up to 600 times more efficient catalysts than monomeric Cu2+-TerPy,
and up to 5 ꢀ 105-fold rate enhancement in comparison to the uncatalysed reaction, is achieved.
However, the catalytic activity strongly depends on the length of the linker and the base
composition of the substrate. The differences can be attributed to interactions between the
Cu2+-TerPy and nucleic acid base moieties as well as steric factors that may hinder the
productive interaction between the substrate and the catalyst.
Metal ion catalysts, such as complexes of Cu2+ and
Introduction
trivalent lanthanide ions efficiently enhance the triphosphate
hydrolysis, and the most efficient catalysts reduce the half-life
of the reaction from hundreds of years to few hours at 37 1C.1,5
While the mechanism of the metal ion promoted hydrolysis of
internucleosidic phosphodiester bonds of RNA has been
extensively studied, and mechanisms are well established,6 less
is known about the hydrolysis of the triphosphate linkage of
dinucleoside triphosphates. Intramolecular transesterification
of phosphodiester bonds of RNA7 and the hydrolysis of the
phosphoanhydride linkage involving an external nucleophile2
are mechanistically different reactions, and hence the mechanisms
of the metal ion promoted reactions are also likely to be
different. In the case of dinucleoside triphosphates, a clear
second-order dependence on the concentration of metal ion
catalysts has been observed.5c,e This suggests that two metal
ions are involved, and it has been proposed that a water ligand
of one metal ion acts as a nucleophile while coordinated metal
ions facilitate the reaction by electrostatic interactions with the
triphosphate bridge.5 The second-order dependence observed
Hydrolysis of dinucleoside triphosphates has been studied1 to
understand the reactions of the 50-terminal cap structure
(Scheme 1) found in RNA polymerase I synthesized mRNA
molecules. While 50-cap containing the more labile N7-methyl
guanine base undergoes three different hydrolytic reactions,1
the only reaction of substrates such as diadenosine triphosphate
(1a) under neutral conditions, is the hydrolysis of the
triphosphate bridge.2 This reaction is very slow, as it involves
a nucleophilic attack of water molecule on a negatively
charged phosphate,2 and in the case of 50-cap the rate of
the hydrolysis of the triphosphate bridge is negligible in
comparison to the hydrolysis of N-glycosidic bond1,3 or the
cleavage of N7-methyl guanine base.1,4 Presumably the
spontaneous hydrolysis of the triphosphate bridge is independent
of the bases present in the substrate, since nucleoside moieties
are not involved in the hydrolysis of the triphosphate bridge.2
Information on the hydrolysis and reactivity of the triphosphate
of 50-cap structure can, hence, be obtained with substrates
such as 1a–c containing more stable base moieties.
University of Turku, Department of Chemistry, FIN-20014 Turku,
Finland
Scheme 1
ꢁc
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2009 New J. Chem., 2009, 33, 1853–1858 | 1853

View Article Online
suggests also that efficient catalysts can be obtained by joining
two metal ion complexes. Consistent with this, it has been
previously shown that bimetallic Cu2+ complexes of ligands
containing 1,4,7-triazacyclononane (2b) as the chelating units,
are approximately a hundred times as efficient catalysts as a
single Cu2+ 1,4,7-triazacyclononane complex.5c
The cleavage of dinucleoside triphosphates to a nucleoside
monophosphate and a nucleoside diphosphate was followed
by capillary zone electrophoresis (CZE). First-order rate
constants were calculated on the basis of the decrease of the
normalized signal area as a function of reaction time using the
integrated first-order rate law. Eight to twelve consecutive
aliquots were withdrawn at appropriate time intervals and
analyzed as described in the Experimental section. Aliquots
were kept on an ice-bath until the analysis. No other
quenching method was utilized due to practical problems
encountered. Adding EDTA or phosphate buffer to deactivate
the metal ions complexes resulted in formation of a precipitate
that prevented the analysis. Treatment with chelating agents,
such as Chelex, was not possible because of the small sample
volumes.
In our work we set out to study in more detail the catalysis
by bimetallic complexes, and the effect of the structure of
the substrate and of the catalyst has been investigated.
Cu2+-terpyridine (Cu2+-2a) was chosen as the catalytic unit,
since Cu2+-TerPy (TerPy = 2,20:60,200-terpyridine) is a robust
complex that has been extensively studied as a catalyst of
the cleavage of phosphoesters.8 Our previous studies5e
with Cu2+-terpyridine (Cu2+-2a) have shown that while
the complex enhances the hydrolysis of 1a only modestly,
the reaction shows a clear second-order dependence on the
complex concentration. Dimers of appropriate structure
could, hence, be expected to show clearly enhanced catalytic
activity in comparison to monomeric Cu2+-TerPy. Alkyl
linked bis-terpyridine ligands were a logical choice, since the
flexible alkyl linker could be expected to disturb the inter-
action between the metal ion chelates and the triphosphate as
little as possible. Furthermore, alkyl linked bis-terpyridine
ligands are well-known compounds and several reports on
their metal ion complexes have been published.9 A series of
terpyridine based catalysts (3a–c) was, therefore, synthesized
and the catalytic activity of their Cu2+ complexes in
hydrolysis of three different dinucleoside triphosphate
substrates (1a–c) was studied.
Analysis of complex samples required long analysis times
and extensive flushing of the capillary between each sample.
This caused problems with the analysis, since in the absence of
an efficient quenching method, the reactions tended to proceed
while the aliquots were awaiting the analysis, even when kept
on an ice-bath. Rate constants were therefore determined also
on the basis of individually prepared samples: the reaction
solution was prepared and reaction was allowed to proceed
while the capillary was being conditioned for the analysis.
Once the capillary was ready, the sample was injected
immediately. The procedure was repeated a few times varying
the reaction time. The mol fraction of the substrate remaining
was determined and the integrated first-order rate law was
applied to calculate a rate constant. Values given in Table 1
are mean values of two–five of such individual values. This
method was applied also for the reactions involving ligand 3c.
The 2Cu2+-3c complex tended to block the capillary after the
analysis of a few samples and therefore sequence analysis of
several samples was not possible.
In cases where both methods were applied to calculate the rate
constants, values obtained were fully consistent with each other.
Calculation of rate constants on the basis of a sequence of
samples is, in principle, the most reliable method, but in the
present case, suffers from a slight uncertainty because the
reaction could not be quenched entirely. Analysis of an
individual sample, as described above, is carried out immediately
after the sample is withdrawn and therefore shows the extent of
the reaction at that precise moment. Since both methods still
gave the same rate constants, it can be concluded that the
potentially insufficient quenching does not distort the results,
but the rate constants based on the analysis of sequences of
10–12 samples also reliably describe the progress of the reactions.
The cleavage of dinucleoside triphosphates yielded a nucleo-
side monophosphate and diphosphate as products as is shown
in Scheme 2. In the case of ApppU (1b), two pairs of products
were formed, AMP (4a) + UDP (4b) and UMP (4c) + ADP
(4d) (routes A and B, respectively). Products were identified
by spiking with authentic samples. The mole fractions of
adenosine and uridine nucleotides were normalized using the
molar adsorbtion coefficients reported for AMP and UMP.10
No other reaction other than the hydrolysis of the
triphosphate bridge was observed. Hydrolysis of a nucleoside
diphosphate to the corresponding monophosphate was not
observed to any significant extent either.
Results and discussion
The cleavage of the dinucleoside triphosphate substrates 1a–c
was studied at pH 7.0 in the presence of 1 mM Cu2+
complexes. The reaction solutions were prepared by mixing
appropriate volumes of buffer and metal complex stock
solutions. Reaction solutions containing 2Cu2+-3c were
slightly cloudy, particularly after the addition of the substrate.
Solubility was improved upon addition of acetonitrile (50% in
reaction solution), but this resulted in clear decrease of the
catalytic activity of 2Cu2+-3b and 2Cu2+-3c. The concentration
of the substrate as detected by CZE (capillary zone electro-
phoresis) analysis was, however, same in cloudy and clear
solutions, and therefore the kinetic experiments were carried
out without acetonitrile.
ꢁc
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2009
1854 | New J. Chem., 2009, 33, 1853–1858

View Article Online
Table 1 Rate constants of the hydrolysis of dinucleoside triphosphates 1a–c in the presence of Cu2+-TerPy catalysts at pH 7.0 and 60 1C
Catalyst
105k(1a)/sꢂ1
105k(1b)b/sꢂ1
105k(1c)/sꢂ1
2Cu2+-3a (1 mM)
4.7 ꢃ 0.6
150 ꢃ 10
60a
2.3 ꢃ 0.1 (55%)
420 ꢃ 20 (25%)
160a (34%)
1.46 ꢃ 0.03 (60%)
1.2 ꢃ 0.2
460 ꢃ 20
170a
2Cu2+-3b (1 mM)
2Cu2+-3c (1 mM)
Cu2+ -TerPy(2a) (2 mM)
Cu2+-BiPy(2c) (2 mM)c
1.28 ꢃ 0.03
0.78 ꢃ 0.03
16d
10.1 ꢃ 0.2 (63%)
Average of two–four individual rate constants calculated on the basis of single sample. Percentage of reaction route resulting in formation of
4.5 ꢃ 0.1
a
b
c
d
AMP + UDP (route A, Scheme 2). At pH 7.5 and 60 1C. From ref. 5e.
if the maximal rate constant referring to saturating concentration
of 2Cu2+-3b was known, but due to poor solubility of the
complex, results obtained at higher concentrations were
difficult to interpret, and hence such experiments were not
attempted. Using the same kind of reasoning as above, the
catalysis by 2Cu2+-3b can be estimated to be of the same order
as that obtained with Eu3+ -THED (THED = 1,4,7,10-
tetrakis(2-hydroxyethyl)-1,4,7,10-tetraazacyclododecane) complex
together with Zn2+aq, which is the most efficient catalyst for
triphosphate hydrolysis reported to date.5d
Scheme 2
Comparison to the rate of the uncatalysed reaction shows
that the rate-enhancement by the bimetallic complexes is
remarkable. Previously reported data2 allows a fairly reliable
estimate of 1 ꢀ 10ꢂ8 sꢂ1 for the rate constant for the hydro-
lysis of the triphosphate bridge of dinucleoside triphosphates
under neutral conditions at 60 1C. The rate constants collected
in Table 1 represent, hence, a 150 000 to 500 000-fold rate
enhancement by the complexes.
Rate constants of the hydrolysis of 1a–c in the presence of
Cu2+ complexes of TerPy (2a) and bis-TerPy ligands (3a–c)
are collected in Table 1. The results show that the bis-terpy
complexes of Cu2+ are significantly more efficient catalysts
than monomeric Cu2+-TerPy, as expected, but the rate-
enhancement achieved depends clearly on the length of the
linker joining the two terpyridine moieties. When the terpyridines
are joined with a propyl linker, the Cu2+ complex is only
marginally better a catalyst than a single Cu2+-TerPy complex.
Complexes of ligands 3b and 3c, with butyl and pentyl linkers
respectively, are, in contrast, clearly more efficient catalysts
than Cu2+-TerPy. The catalytic advantage achieved varies
also with different substrates. The hydrolysis of ApppA with
2Cu2+-3b is 120 times faster than with Cu2+-TerPy. With
ApppU and UpppU the differences are 290- and 590-fold,
respectively. 2Cu2+-3c appears to be a slightly less active
catalyst than 2Cu2+-3b with all substrates studied.
The proportion of the reaction route resulting in formation
of AMP and UDP (route A in Scheme 2) is also included in
Table 1, and this data shows that while the cleavage by the less
efficient catalysts Cu2+-TerPy and 2Cu2+-3a slightly favor
reaction route A, the catalysis by 2Cu2+-3b and 2Cu2+-3c
results predominantly in formation of UMP and ADP as
products (route B). A 1.5-fold excess of AMP over UMP is
observed in the former case, whereas the latter reactions
produce UMP in 3- to 4-fold excess.
The results obtained clearly show that two TerPy-moieties
can enhance the hydrolysis co-operatively provided that the
linker joining the metal complexes is long enough. While this is
clearly possible with complexes of ligands with butyl and
pentyl linkers, the propyl linker is too short to allow
the simultaneous interaction of the two complexes with the
phosphate group. Since the rate constants are nearly the
same, it seems that 2Cu2+-3a acts in the same way as a single
Cu2+-TerPy complex. The cleavage pattern of ApppU is also
similar to that of Cu2+-TerPy supporting the suggestion that
the catalysis mechanisms are the same.
The rate-enhancement obtained with the bis-Cu2+
complexes of ligands 3b and 3c is even higher than observed
with other bimetallic catalysts. Comparison between two
systems that have been studied under different conditions is
difficult, but in this case an extrapolation can be attempted on
the basis of available data. The catalysis by the bis-Cu2+
complexes based on 2b, reaches maximal efficiency at about
1 mM catalyst concentration, and the saturating rate constants
at pH 7.3 and 37 1C are 4–7 ꢀ 10ꢂ5 s
.
ꢂ1 5c At 25 1C and pH 7.0
the rate constants of the hydrolysis of 1a–c in the presence of
1 mM 2Cu2+-3b vary from 0.9 to 2.0 ꢀ 10ꢂ4 sꢂ1. It is known
that below pH 7.5 the Cu2+-TerPy reaction shows a first-order
dependence on hydroxide ion concentration.5e Furthermore,
an approximately three-fold rate enhancement could be
expected on going from 25 to 37 1C.2 Using this information
it can be estimated that the rate constants obtained at 1 mM
concentration of 2Cu2+-3b are approximately ten times larger
than the maximal values obtained with bimetallic catalysts
based on 2b. A more accurate comparison would be obtained
Co-operative action of two Cu2+-TerPy complexes
observed for 2Cu2+-3b and 2Cu2+-3c shows
a slight
preference for uracil containing substrates 1b and 1c over
diadenosine triphosphate 1a. In the presence of these catalysts,
the reaction of 1a is slower than that of 1b and 1c, and the
cleavage site closer to the uracil base is preferred. This is in
contrast to the situation with Cu2+-TerPy, Cu2+-BiPy and
2Cu2+-1a, which cleave adenine containing substrates faster,
and which prefer a cleavage site closer to the adenine base. A
likely explanation of these differences arises from interactions
ꢁc
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2009 New J. Chem., 2009, 33, 1853–1858 | 1855

View Article Online
between the ligands and nucleic acid bases. Since the smaller
catalysts (Cu2+-TerPy, Cu2+-BiPy) show a slight preference
for adenine containing substrates, it can be suggested that
TerPy and BiPy complexes tend to interact with adenine base,
either via direct coordination of the Cu2+ ion or via a
hydrophobic interaction between the aromatic rings of
adenine base and the ligands. The interaction brings the
catalyst closer to the substrate and thus facilitates the
cleavage. The bifunctional catalysis by 2Cu2+-3b and
2Cu2+-3c may, however, require a more ordered structure,
where two catalytic moieties interact with the phosphate
group. The linker will also have to be accommodated. The
interaction between the adenine base and the Cu2+ complex
may, in this case, interfere with the catalysis by steering the
catalyst away from the triphosphate bridge, and therefore
the adenine containing substrates react more slowly than
diuridine triphosphate. The interactions may also dictate the
predominant cleavage site in ApppU and steers the catalyst
closer to the uridine site of the molecule.
5 mm particle size). 50 mM phosphate buffer, pH 6.0, was used
as an eluent. UV-detection at 260 nm was employed.
Rate constants were calculated on the basis of the decrease
of the mole fraction of the starting material that was
calculated on the basis of the normalized signal areas. In case
of ApppU, the different molar absorption coefficients10 of
adenine and uracil bases were taken into account when
calculating the proportions of the two reaction routes. Signal
areas from CE analysis were normalized by dividing them by
the migration time. The integrated rate law of a first-order
reaction was applied to calculate the rate constants.
Synthetic procedures
Two different methods were used in synthesis of ligands 3a–c:
Sequential addition of two 2,6-bis(2-pyridyl)-4(1H)-pyridones
(5a) by a Mitsunobu reaction with a corresponding diol (route
A in Scheme 3) was carried out applying a procedure reported
in ref. 11. A more direct method utilized a nucleophilic
substitution reaction between 4-chloroterpyridine (5b) and a
diol (route B in Scheme 3), following a procedure reported12
for the synthesis of a corresponding ethyl linked bis-terpyridine
ligand. Ligand 3b was synthesized according to route A while
3a and 3c were prepared via route B, but in principle either
method could have been utilized in the synthesis of all bis-
terpyridine ligands. Yields obtained with the latter method
were clearly better (80% vs. 20%). Dinucleoside triphosphate
substrates 1b and 1c were synthesized according to procedure
reported in the literature.13 Both syntheses and characterization
of the products are described below. The diadenosine
compound 1a is commercially available from Sigma. All
synthesis products were characterised by 1H NMR spectro-
scopy. For dinucleoside triphosphates13 and propyl linked
bis-TerPy ligand 3a9d this was regarded sufficient as their
synthesis and characterization has been reported before. Butyl
and pentyl linked bis-TerPy ligands 3b and 3c were characterized
also by HR-MS and 13C NMR. To improve the solubility of
the ligands, they were treated with aqueous HCl and then
dissolved in D2O for 13C NMR analysis.
Conclusions
Efficient catalysts of the cleavage of dinucleoside triphosphates
can be obtained by joining two terpyridine moieties with
a simple alkyl linker. Co-operative catalysis of the two
complexes is, however, achieved only when the linker is long
enough. Interactions between the ligand and the nucleic acid
bases also influence the catalytic activity.
Experimental
Kinetic experiments
Reaction solutions were prepared in doubly-distilled water.
The pH was adjusted with a MOPSO buffer (3-morpholino-2-
hydroxypropanesulfonic acid; pKa 6.9 at 25 1C). The complex
was added as a 5 mM stock solution. The complex stock was
prepared by weighing an appropriate amount of the ligand,
suspending it in water and adding Cu(NO3)2 solution slowly
while vigorously mixing. The stock solutions were checked by
UV-spectrometry and the concentrations were found to be the
same. UV maxima were detected at 280, 315 and 325 nm.
Reactions were carried out in Eppendorf tubes. The
temperature was controlled by a water-bath that was adjusted
to 60.0 ꢃ 0.1 or 25.0 ꢃ 0.1 1C. Reactions were followed by
withdrawing aliquots at appropriate intervals and analyzing
them either by capillary electrophoresis of reversed-phase
HPLC. Aliquots were kept on an ice-bath until the analysis.
Samples from reactions carried out at 25 1C were analysed
immediately.
4-((4-Methoxyphenyl)diphenylmethoxy)butan-1-ol
(6).
Butane-1,4-diol (16.19 mmol, 1.43 mL), 1 eq. of monomethoxy-
trityl chloride and a catalytic amount of DMAP in 75 ml of
pyridine were stirred at room temperature for one day. After
evaporation, the crude product was purified on silica gel with
dichloromethane as eluent. dH (400 MHz, CDCl3): 1.69 (4H,
m, HOCH2CH2CH2CH2O–), 3.11 (2H, t, HOCH2CH2CH2-
CH2O–), 3.64 (2H, t, HOCH2CH2CH2CH2O–), 3.79 (3H, s,
CH3O–), 7.2–7.45 (14H, m, arom. H).
1-((4-Methoxyphenyl)diphenylmethoxy))-4-(2,20:60,200-
terpyridine-4-yloxy)butane (7). Compound 6 (2 mmol, 0.7 g),
0.8 eq. of 2,6-bis(pyridine-2-yl)pyridine-4(1H)-one (5a; 1.6
mmol, 0.41 g) and 1 eq. of triphenylphosphine (2 mmol, 0.52
g) were dissolved in dry THF (50 mL). 1 eq. of DIAD (2 mmol,
0.4 mL) was added dropwise and the mixture is stirred during
2 h at room temperature. The pale and clear pink mixture
obtained was purified on basic aluminium oxide using
dichloromethane as the eluent. Product 7 was obtained as a
white solid by precipitation from diethyl ether. dH (400 MHz,
Capillary electrophoresis analysis was carried out with HP
3D instrument utilizing a UV-detection at 260 nm. Fused silica
capillary (77 cm length, 50 mm i.d.) and a 50 mM phosphate
buffer, pH 6.0, were used for the separation of the starting
material and the products. HPLC analysis was carried
out with a Perkin Elmer instrument consisting of two
micropumps, autosampler and UV detector. The column
was a Supelco LC-18-T reversed-phase column (25 ꢀ 4.6 cm,
ꢁc
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2009
1856 | New J. Chem., 2009, 33, 1853–1858

View Article Online
Scheme 3
CDCl3); 1.9 (4 H, m, –OCH2CH2CH2CH2O–), 3.17 (2 H, t,
TrOCH2CH2CH2CH2O–TerPy), 3.79 (3H, s, CH3O–), 4.22
(2 H, t, TrOCH2CH2CH2CH2O–TerPy), 7.2–7.45 (24H, m,
arom. H).
remained neutral. The organic layer was evaporated to
dryness. The purification was done in two steps: first on
alumina eluting with chloroform and then on silica eluting
with CHCl3–CH2Cl2 (9.2 : 0.8). The 1H NMR spectrum
obtained is consistent with that reported.9d dH (500 MHz,
CDCl3); 2.45 (2 H, m, –OCH2CH2CH2O–), 4.51 (4 H, t,
–OCH2CH2CH2O–), 7.34 (4 H, m, H5, H500), 7.85 (4 H, m,
H4, H400), 8.09 (4 H, s, H30, H50), 8.64 (4 H, d, H6, H600), 8.71
(4 H, d, H3, H300).
4-(2,20:60,200-Terpyridine-4-yloxy)-butan-1-ol (8). Compound
7 was dissolved in 80% acetic acid in water–acetonitrile (1 : 1)
and stirred for 24 h. The product was purified on silica gel
CH2Cl2–MeOH, 95 : 5 as the eluent. dH (400 MHz, CDCl3):
1.69 (4 H, m, –OCH2CH2CH2CH2O–), 3.13 (2 H, t,
HOCH2CH2CH2CH2O–), 3.63 (2 H, t, HO–CH2CH2CH2-
CH2O–TerPy), 7.2–7.45 (10 H, m, arom. H).
1,5-Bis(2,20:60,200-terpyridine-4-yloxy)pentane (3c). 1,5-
Pentanediol (20.3 mL, 0.187 mmol) was mixed in 2 mL of
DMSO with 100 mg KOH. Separately 200 mg (0.740 mmol)
40-chloro-2,20:60,200-terpyridine was added in 6 cm3 of DMSO.
The details of the reaction and work-up are similar to those
described above. The solid residue was washed with cyclo-
hexane to remove an unknown by-product. The product was
purified by column chromatography on silica gel eluting with
CH2Cl2–MeOH (9.2 : 0.8). NMR analysis shows that there is
some pentanediol left in the product, which could not be fully
removed. This was taken into account and the concentration
of the complexes was checked by UV-spectrometry. dH
(500 MHz, CDCl3): 1.6–1.7 (6 H, m, –OCH2CH2CH2CH2-
CH2O–), 4.26 (4 H, m, –OCH2CH2CH2CH2CH2O–), 7.35
(4 H, t, H5, H500), 7.86 (4 H, t, H4, H400), 8.03 (4 H, s, H30,
H50), 8.64 (4 H, d, H6, H600), 8.71 (4 H, d, H3, H300); dC
(100 MHz, D2O): 27, 39, 70, 110, 125, 128, 142–149, 169; m/z
1,4-Bis(2,20:60,200-terpyridine-4-yloxy)butane (3b). Compound
8
(2.8 mmol, 0.9 g), 0.8 eq. of 2,6-bis(pyridine-2-yl)-
pyridine-4(1H)-one (2.2 mmol, 0.55 g) and 1 eq. of
triphenylphosphine (2.8 mmol, 0.74 g) were dissolved in (dry)
THF (50 mL). 1 eq. of DIAD (2.8 mmol, 0.55 mL) was
added dropwise and the mixture was stirred for 2 h at room
temperature. The pale and clear pink mixture obtained was
purified on basic aluminum oxide using dichloromethane as the
eluent followed by precipitation from diethyl ether to yield
compound 3b. dH (400 MHz, CD2Cl2): 2.16 (4 H, m,
–OCH2CH2CH2CH2O–), 4.28 (2 H, t, –OCH2CH2CH2-
CH2O–), 7.36 (4H, m, H5, H500), 7.86 (4H, m, H4, H400), 8.07
(4H, s, H30, H50), 8.63 (4H, d, H3, H300), 8.68 (4H, m, H6, H600).
dC (100 MHz, D2O): 30, 70, 112, 124, 127, 142-148, 168; m/z
(EI) 552.2272 (C34H28N6O2 requires 552.2274).
+
(ESI) 567.2563 (C35H31N6O2 requires 567.2508).
1,3-Bis(2,20:60,200-terpyridine-4-yloxy)propane (3a). 1,3-
Propanediol (13.8 mL, 0.187 mmol) was mixed in 2 mL of
DMSO with 100 mg KOH. Separately 158 mg (0.590 mmol)
40-chloro-2,20:60,200-terpyridine (5b) was added in 6 cm3 of
DMSO. The suspensions were stirred for 1 h at 70 1C after
which they were mixed. The mixture was heated for 27 h at
70 1C under nitrogen atmosphere and the reaction was
followed with CHCl3 as an eluent. After cooling to room
temperature, the orange mixture was poured into water
(25 cm3) and NaCl-saturated water (5 cm3). The milky
suspension was extracted with chloroform (3 ꢀ 15 cm3) and
the extracts washed with water (10 + 5 cm3) until the pH
Synthesis of dinucleoside triphosphates
A nucleoside monophosphate triethylammonium salt was first
converted to the corresponding imidazolidate in a mixture of
anhydrous DMF and triethylamine in the presence of
2,20-dipyridyl sulfide and triphenylphosphine. The product
was crystallized by adding anhydrous NaClO4 and acetone.
The nucleotide imidazolidate was coupled to appropriate
nucleoside 50-diphosphate triethylammonium salt in anhydrous
dimethylformamide using ZnCl2 as the catalyst. The product
ꢂ
was purified on DEAE–Sephadex in HCO3 form using a
gradient elution from 0 to 0.1 M ammonium bicarbonate
ꢁc
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2009 New J. Chem., 2009, 33, 1853–1858 | 1857

View Article Online
buffer, pH 7.4. In the case of the asymmetric substrate 1b,
J. Chem. Soc., Dalton Trans., 1998, 2961–2963; (c) K. P. McCue
and J. R. Morrow, Inorg. Chem., 1999, 38, 6136–6142;
(d) D. M. Epstein, L. L. Chappell, H. Khalili, H. R.
M. Supkowski, W. DeW. Horrocks, Jr and J. R. Morrow, Inorg.
Chem., 2000, 39, 2130–2134; (e) S. Valakoski, S. Heiskanen,
AMP-imidazolidate was coupled with UDP. The products were
1
analysed by H and 31P NMR spectroscopy. Spectra are fully
consistent with those reported previously.14 1b: dH (400 MHz,
D2O): 4.06 (4H, m, H50, H500), 4.15 (2H, m, H40), 4.26 (2H, m,
H30), 4.40 (2H, m, H20), 5.61 (1H, d, J = 8.0 Hz, H5; Ura), 5.70
(2H, d, J = 5.6 Hz, H10), 7.63 (1H, d, J = 8.4 Hz, H6; ura), 8.10
(1H, s, H2; ade), 8.42 (1H, s, H8; ade); dP (162 MHz, D2O):
–11,28, –22,59. 1c: dH (400 MHz, D2O): 4,12 (2H, m, H50,H500),
4,17 (1H, m, H40), 4,25 (1H, m, H30), 4,28 (1H, m, H20), 5,87
(1H, s, H10), 5,87 (1H, d, J = 7,2 Hz, H5), 7,86 (1H, d,
J = 8,4 Hz, H6). dP (162 MHz, D2O): ꢂ11,45, ꢂ22,86.
S. Andersson, M. Lahde and S. Mikkola, J. Chem. Soc., Perkin
¨
Trans. 2, 2002, 604–610.
6 Reviews on metal ion promoted hydrolysis of RNA: (a) S. Kuusela
and H. Lonnberg, Met. Ions Biol. Syst., 1996, 32, 271–300;
¨
(b) B. N. Trawick, A. T. daniher and J. A. Bashkin, Chem. Rev.,
1998, 98, 939–960; (c) J. A. Morrow and O. Iranzo, Curr. Opin.
Chem. Biol., 2004, 8, 192–200.
7 M. Oivanen, S. Kuusela and H. Lonnberg, Chem. Rev., 1998, 98,
961–1026.
¨
8 (a) S. Liu and A. D. Hamilton, Tetrahedron Lett., 1997, 38,
1107–1110; (b) L. A. Jenkins, J. K. Bashkin, J. D. Pennock,
J. Florian and A. Warshel, Inorg. Chem., 1999, 38, 3215–3222;
(c) B. N. Trawick, T. A. Osiek and J. A. Bashkin, Bioconjugate
Chem., 2001, 12, 900–905.
9 (a) U. S. Schubert, C. Eschbaumer, O. Hien and P. R. Andres,
Tetrahedron Lett., 2001, 42, 4705–4707; (b) D. D. Kurth,
N. Severin and J. P. Rabe, Angew. Chem., Int. Ed., 2002, 41,
3681–3683; (c) P. R. Andres and U. S. Schubert, Synthesis, 2004,
1229–1238; (d) P. Lehmann, C. Symietz, G. Brezesinski, H. Krass
and D. D. Kurth, Langmuir, 2005, 21, 5901–5906; (e) V. W.
W. Yam, K. H. Y. Chan, K. M. C. Wong and B. W. K. Chu,
Angew. Chem., Int. Ed., 2006, 45, 6169–6173.
References
1 S. Mikkola, S. Salomaki, Z. Zhang, E. Maki and H. Lonnberg,
¨ ¨
Curr. Org. Chem., 2005, 9, 999–1022.
2 S. Mikkola, Org. Biomol. Chem., 2004, 2, 770–776.
3 (a) J. A. Zoltewicz, D. F. Clark, T. W. Sharpless and G. Grahe,
J. Am. Chem. Soc., 1970, 92, 1741–1750; (b) M. Lahti,
M. H. Santa, E. Darzynkiewicz and H. Lonnberg, Acta Chem.
¨
Scand., 1990, 44, 636–638; (c) L. Hevesi, E. Wolfson-Davidson,
J. B. Nagy, O. B. Nagy and A. Bruylants, J. Am. Chem. Soc., 1972,
94, 4715–4720; (d) M. Oivanen, E. Darzynkiewicz and
H. Lonnberg, Acta Chem. Scand., Ser. B, 1988, 42, 250–253.
¨
4 (a) E. Darzynkiewicz, I. Labadi, D. Haber, K. Burger and
H. Lonnberg, Acta Chem. Scand., Ser. B, 1988, 42, 86–92;
¨
(b) S. Hendler, E. Furer and P. R. Srinivasan, Biochemistry,
¨
1970, 9, 4141–4153; (c) E. Darzynkiewicz, J. Stepinski,
10 CRC Handbook Chemistry and Physics, CRC Press Inc., Cleveland,
OH, 58th edn, 1977.
11 J. Hovinen, Tetrahedron Lett., 2004, 45, 5707–5709.
12 E. C. Constable, C. E. Housecroft, M. Neuburger, S. Schaffner and
C. B. Smith, Dalton Trans., 2005, 2259–2267.
13 J. Stepinski, C. Waddell, R. Stolarski, E. Darzynkiewicz and
R. E. Rhoads, RNA, 2001, 7, 1486–1495.
S. M. Tahara, R. Stolarski, I. Ekiel, D. Haber, K. Neuvonen,
P. Lehikoinen, I. Labadi and H. Lonnberg, Nucleosides Nucleotides,
¨
14 S. R. Shaver, J. L. Rideout, W. Pendergast, J. G. Douglas,
E. G. Brown, J. L. Boyer, R. I. Patel, C. C. Redick, A. C. Jones,
M. Picher and B. R. Yerxa, Purinergic Signalling, 2005, 1, 183–191.
1990, 9, 599–618.
5 (a) B. F. Baker, J. Am. Chem. Soc., 1993, 115, 3378–3379;
(b) K. P. McCue, D. A. Voss Jr., C. Marks and J. R. Morrow,
ꢁc
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2009
1858 | New J. Chem., 2009, 33, 1853–1858