Acid-base and metal-ion binding properties of the RNA dinucleotide uridylyl-(5′→3′)-[5′]uridylate (pUpU3-)

Article abstract of DOI:10.1002/chem.200500013

It is well known that Mg²⁺ and other divalent metal ions bind to the phosphate groups of nucleic acids. Subtle differences in the coordination properties of these metal ions to RNA, especially to ribozymes, determine whether they either promote

Full text of DOI:10.1002/chem.200500013

FULL PAPER
Acid–Base and Metal-Ion Binding Properties of the RNA Dinucleotide
Uridylyl-(5’!3’)-[5’]uridylate (pUpU3ꢀ)
Bernd Knobloch,[a] Danuta Suliga,[b] Andrzej Okruszek,[b] and Roland K. O. Sigel*[a]
Abstract: It is well known that Mg2+
and other divalent metal ions bind to
the phosphate groups of nucleic acids.
Subtle differences in the coordination
properties of these metal ions to RNA,
especially to ribozymes, determine
whether they either promote or inhibit
catalytic activity. The ability of metal
ions to coordinate simultaneously with
two neighboring phosphate groups is
important for ribozyme structure and
activity. However, such an interaction
has not yet been quantified. Here, we
have performed potentiometric pH ti-
trations to determine the acidity con-
stants of the protonated dinucleotide
H2(pUpU)ꢀ, as well as the binding
this dinucleotide by these metal ions.
Such a macrochelate is also possible in
an oligonucleotide, because the basic
structural units are the same, despite
the difference in charge. The formation
degrees of the macrochelated species
of [Zn(pUpU)]ꢀ and [Pb(pUpU)]ꢀ
amount to around 25 and 90%, respec-
tively. These findings are important in
the context of ribozyme and DNAzyme
catalysis, and explain, for example, why
the leadzyme could be selected in the
first place, and why this artificial ribo-
zyme is inhibited by other divalent
properties of pUpU3ꢀ towards Mg2+
,
Mn2+, Cd2+, Zn2+, and Pb2+. Whereas
Mg2+, Mn2+, and Cd2+ only bind to the
more basic 5’-terminal phosphate
group, Pb2+, and to a certain extent
also Zn2+
, show a remarkably en-
hanced stability of the [M(pUpU)]ꢀ
complex. This can be attributed to the
formation of a macrochelate by bridg-
ing the two phosphate groups within
Keywords: dinucleotides · magnesi-
um · metal-ion binding properties ·
ribozymes · RNA
metal ions, such as Mg2+
.
Introduction
trons by two consecutive transesterification reactions.[9–11]
The exact make-up of the spliceosomeꢀs active site is not
known; however, two of the RNA components of the spli-
ceosome (the small nuclear U2 and U6 RNAs) are consid-
ered to be the main candidates constituting the catalytic
center.[10] Furthermore, there is evidence for intrinsic metal-
ion binding in the intramolecular stem-loop of U6.[9,11,12]
Based on various experiments using thiophosphate deriva-
tives of U6, it was concluded that Mg2+ binds to the phos-
phate group of uridine 80 within the stem-loop of U6.[11,12]
For most of the nine classes of catalytic RNAs known
The observation that RNA molecules can perform enzymat-
ic functions,[1,2] once thought to be unique to proteins, has
stimulated interest in understanding the structure and mech-
anism used by these catalytic RNAs, that is, ribozymes.[3–6]
This interest has been further spurred by the discovery that
the ribosome is also a ribozyme, a consequence of the for-
merꢀs peptidyltransferase site being comprised exclusively of
RNA.[7,8] Another remarkable example is the spliceosome, a
huge ribonucleoprotein complex of more than 70 proteins
and five RNA molecules, which catalyzes the removal of in-
today, there is now considerable evidence for the require-
[13]
ment of divalent metal ions, especially Mg2+
,
in ribozymal
activity.[3,4,6,12–17] However, until now, very little quantitative
information concerning the binding strength of metal ions to
the individual sites of a nucleic acid has been reported.[18–20]
Evidently, the most frequently repeated individual site is the
[a] Dr. B. Knobloch, Prof. Dr. R. K. O. Sigel
Institute of Inorganic Chemistry, University of Zꢁrich
Winterthurerstrasse 190, 8057 Zꢁrich (Switzerland)
Fax :(+41)44-635-6802
ꢀ
E-mail: roland.sigel@aci.unizh.ch
phosphate–diester bridge, -O-P(O)2 -O-, in which the two
[b] D. Suliga, Prof. Dr. A. Okruszek
terminal oxygen atoms together carry one negative charge.
It is difficult to measure directly the metal-ion affinity of
such a site in a polymer or even in an oligonucleotide, such
as UpUpU2ꢀ. This is because no competition for binding
occurs between a metal ion and a proton within the experi-
Polish Academy of Sciences
Center of Molecular and Macromolecular Studies
Department of Bioorganic Chemistry, 90–363 Lꢂdz (Poland)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2005, 11, 4163 – 4170
DOI: 10.1002/chem.200500013
ꢃ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4163

mentally accessible pH range, as well as within the physio-
logical pH range, because the primary protons of phosphate
residues are released with pKa values of about one.
We measured the metal-ion affinity of such a phosphate–
diester unit by using a dinucleotide containing both a termi-
portantly, the metal-ion affinity of a carbonyl oxygen atom
in aqueous solution is too low for the formation of such a
macrochelate.[39] The second carbonyl group, (C2)O, cannot
be reached by a phosphate-bound metal ion for steric rea-
sons, because in the dominating anti conformation this ac-
ceptor group is directed away,[21,22] and the energy barrier
for a switch into the syn conformation is too high.[40] There-
fore, the metal-ion binding sites to be considered for
pUpU3ꢀ in aqueous solution are the terminal phosphate
group and the phosphate–diester bridge. Indeed, the stabili-
ty studies performed with Mg2+, Mn2+, Zn2+, Cd2+, and
Pb2+ presented here confirm this reasoning. In addition, our
experiments show that discrimination occurs; for example,
Mg2+ coordinates in only a monodentate fashion to the ter-
minal phosphate group, whereas Zn2+ forms a chelate that
also involves the phosphate–diester bridge.
nal 5’-phosphate group and
a
5’!3’-phosphate–diester
bridge. The main binding site in such a dinucleotide is the
terminal phosphate group; however, chelate formation with
the neighboring phosphate–diester bridge is at least theoret-
ically possible and, therefore, information can be gained
about the metal-ion affinity of this second site. Furthermore,
the following questions arise: Is there selectivity? Can all
biologically meaningful metal ions, for example, Mg2+ or
Zn2+, form such a chelate? Here, we address these ques-
tions and quantify the formation of macrochelates with dif-
ferent metal ions in dinucleotide complexes. Notably, the
steric demands of a dinucleotide are comparable to those of
neighboring phosphate groups in a larger oligomer.
To focus on the interaction between metal ions and the
phosphate–diester bridge, and to make the interpretation of
the results unequivocal, a dinucleotide with nucleobases of
low metal-ion affinity must be selected. We selected the di-
Results and Discussion
Acid–base properties of pUpU3ꢀ
:
This dinucleotide
(Figure 1) can accept a total of three protons at its phos-
phate groups; however, two of these protons are released at
a very low pH, and for one of these protons, a pKa value
can be estimated (see below). Therefore, the species H2-
(pUpU)ꢀ must be considered, as the two uracil residues can
also be deprotonated at their (N3)H sites. This then leads to
the following four deprotonation reactions:
nucleotide
uridylyl-(5’!3’)-[5’]uridylate
(pUpU3ꢀ,
Figure 1).[21,22] This molecule has previously been synthe-
H2ðpUpUÞꢀ Ð HðpUpUÞ2ꢀþHþ
ð1aÞ
ð1bÞ
ð2aÞ
ð2bÞ
ð3aÞ
ð3bÞ
ð4aÞ
ð4bÞ
KHH ðpUpUÞ ¼ ½HðpUpUÞ2ꢀꢁ½Hþꢁ=½H2ðpUpUÞꢀꢁ
2
2ꢀ
HðpUpUÞ Ð pUpU3ꢀþHþ
KHHðpUpUÞ ¼ ½pUpU3ꢀꢁ½Hþꢁ=½HðpUpUÞ2ꢀꢁ
Figure 1. Chemical structure of uridylyl-(5’!3’)-[5’]-uridylate (pUpU3ꢀ).
The two uridine units are shown in their dominating anti conforma-
tion.[21,22]
pUpU3ꢀ Ð ðpUpUꢀHÞ4ꢀþHþ
KHpUpU ¼ ½ðpUpUꢀHÞ4ꢀꢁ½Hþꢁ=½pUpU3ꢀꢁ
sized,[23–26] although no data are available regarding its
metal-ion binding properties,[27–29] and to the best of our
knowledge, its acid–base properties have also not yet been
studied.
4ꢀ
ðpUpUꢀHÞ Ð ðpUpUꢀ2 HÞ5ꢀþHþ
KHðpUpUꢀHÞ ¼ ½ðpUpUꢀ2 HÞ5ꢀꢁ½Hþꢁ=½ðpUpUꢀHÞ4ꢀꢁ
The uracil residue is known to be a poor ligating site, as
long as the (N3)H site is not deprotonated.[30] This does not
mean that carbonyl oxygen atoms cannot interact with
metal ions; indeed, such interactions in RNA molecules are
known for the solid state.[31–35] However, such an interaction
occurs only under sterically most favorable conditions, that
is, a suitable directing primary binding site must be close
by.[36–38] If such a neighboring binding site is not present, in
aqueous solution water molecules dominate the situation by
forming hydrogen bonds to the carbonyl oxygen atoms. For
pUpU3ꢀ, it seems feasable that a metal ion bound to the ter-
minal 5’-phosphate residue might reach (C4)O. However,
(C4)O is not in a sterically favored position, and more im-
Notably, in Equilibrium (3a), (pUpUꢀH)4ꢀ should be
read as “pUpU minus H”, meaning that one of the two
(N3)H sites has lost a proton, without defining which one.
Analogously, in the species (pUpUꢀ2H)5ꢀ, both (N3)H
sites are deprotonated.
For Equilibrium (1a), an acidity constant could be only
estimated (see below), whereas the values for the other
three equilibria were measured by performing potentiomet-
ric pH titrations. The results are listed in Table 1, and the
site attributions are evident from the given related data.[41–46]
The data show that, regarding the release of the final proton
from the phosphate groups, H(pUpU)2ꢀ is more basic than
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Chem. Eur. J. 2005, 11, 4163 – 4170

Metal-Ion Binding Properties of RNA Dinucleotides
FULL PAPER
Table 1. Negative logarithms of the acidity constants for the deprotona-
tion of the P(O)(OH)2 and (N3)H sites in H2(pUpU)ꢀ [Eqs. (1)–(4)], to-
gether with related data determined by performing potentiometric pH ti-
trations in aqueous solution (258C; I=0.1m, NaNO3).[a,b]
of the average acidity constant, pKa/av =9.31ꢂ0.09 (Table 1)
for the release of the (N3)H protons from pUpU3ꢀ with the
value for uridine itself (pKHUrd =9.18ꢂ0.02) reveals an inhib-
ition of the deprotonation reaction by DpKa =0.13ꢂ0.09
(3s), due to the three negative charges present in pUpU3ꢀ.
Nevertheless, this effect is relatively small compared to the
DpKa values between pairs of uridine and UMP2ꢀ, UDP3ꢀ,
or UTP4ꢀ, in which the differences amount to about 0.3 and
0.4 pK units (Table 1). To conclude, two neighboring uracil
residues show an increased acidity relative to that of “isolat-
ed” uracil groups, which is significant in the context of bio-
logical systems.
Acids[c]
pKa of the sites
Reference
P(O)(OH)2
P(O)2(OH)ꢀ
(N3)H
H2(pUpU)ꢀ
1.0ꢂ0.3[d]
6.44ꢂ0.02
6.24ꢂ0.01
8.99ꢂ0.03/
9.63ꢂ0.08
–
H(RibMP)ꢀ
uridine
H2(UMP)
H2(UDP)ꢀ
H2(UTP)2ꢀ
[43,44]
[30]
[43,44]
[45]
9.18ꢂ0.02
9.45ꢂ0.02
9.47ꢂ0.02
9.57ꢂ0.02
0.7ꢂ0.3
1.26ꢂ0.20
2.0ꢂ0.1[e]
6.15ꢂ0.01
6.38ꢂ0.02
6.48ꢂ0.02
[41]
[a] The errors given are three times the standard error of the mean value
or the sum of the probable systematic errors, whichever is larger. [b] So-
called practical, mixed, or Brønsted acidity constants[46] are listed (see
Stability constants of [M(pUpU)]ꢀ complexes: Stability con-
stants of several [M(pUpU)]ꢀcomplexes were determined
by performing potentiometric pH titrations. All experimen-
tal data can be perfectly explained by taking Equilibrium
(2a), as well as the following complex-forming Equilibrium
(5a) into account:
also Experimental Section). [c] RibMP2ꢀ
, ribose 5’-monophosphate;
UMP2ꢀ, UDP3ꢀ, or UTP4ꢀ, uridine 5’-mono-, 5’-di-, or 5’-triphosphate.
[d] Estimate based on the other values in this column (see also text).
[e] From reference [42].
H(UMP)ꢀ by around 0.3 pK units (6.44 compared to 6.15).
Because experience shows[47,48] that the same difference also
holds for the release of the primary phosphate proton, a
value for H2(pUpU)ꢀ could be estimated based on the data
M
2þþpUpU3ꢀ Ð ½MðpUpUÞꢁꢀ
ð5aÞ
ð5bÞ
KM
¼ ½½MðpUpUÞꢁꢀꢁ=ð½M2þꢁ½pUpU3ꢀꢁÞ
½MðpUpUÞꢁ
for H2(UMP), that is, pKHH ðpUpUÞ =0.7+0.3=1.0.
2
Our evaluation was not extended into the pH range in
which either hydroxo complexes are formed or (N3)H is de-
protonated. The pH range in which the formation of hy-
droxo complexes occurs was evident from the titrations in
the absence of ligand (see below).
The increased basicity of the terminal phosphate group of
pUpU3ꢀ compared to that of UMP2ꢀ is clearly a charge
effect, and is in accordance with the observations made for
UDP3ꢀ and UTP4ꢀ. Considering the competition between
H+ and Na+ ions for binding,[49] which has the effect of
slightly lowering the pKa in the case of di- and triphos-
phates,[49] the binding tendency of monophosphates[50]
toward Na+ ions is very low under the experimental condi-
tions.
The stabilities of the five metal-ion complexes studied are
listed in Table 2, along with the corresponding values of the
Table 2. Logarithms of the stability constants of [M(pUpU)]ꢀ complexes
[Eq. (5)] as determined by potentiometric pH titrations in aqueous so-
lution, in comparison with the corresponding values for [M(UMP)] and
[M(UDP)]ꢀ complexes determined under the same conditions (258C; I=
0.1m, NaNO3).[a]
Interestingly, the difference in acidity between the two
(N3)H sites in pUpU3ꢀ amounts to only DpKa =(9.63ꢂ
0.08)ꢀ(8.99ꢂ0.03)=0.64ꢂ0.09 (Table 1). Within the error
limits, this difference is identical to the one expected for
symmetrical diprotonic acids (H2L). In such instances, the
formation of HLꢀ is favored, because its formation by the
deprotonation of H2L can occur by two ways. Similarly,
there are two ways to protonate L2ꢀ to yield HLꢀ. As a con-
sequence, the formation of HLꢀ is doubly favored by a
factor of two, corresponding to DpKa =0.6.[51] The observa-
tion that the two uracil residues in pUpU3ꢀ have identical
acidic properties is somewhat surprising, as it could be ex-
pected that the uracil residue close to the 5’-terminal phos-
phate group, which carries two negative charges, is less
acidic. However, our result is in line with the recently pub-
M2+
logKM
logKM
logKM
½MðUMPÞꢁ
½MðpUpUÞꢁ
½MðUDPÞꢁ
Mg2+
Mn2+
Zn2+
Cd2+
Pb2+
1.56ꢂ0.02
2.11ꢂ0.02
2.02ꢂ0.07
2.38ꢂ0.04
2.80ꢂ0.04
1.84ꢂ0.04
2.49ꢂ0.05
2.57ꢂ0.03
2.75ꢂ0.03
4.45ꢂ0.25
3.32ꢂ0.05
4.07ꢂ0.05
4.07ꢂ0.05
4.22ꢂ0.05
5.30ꢂ0.15
[a] For the error limits, see footnote [a] of Table 1. The stability constants
of the [M(UMP)] complexes are from reference [43], except that for [Pb-
(UMP)], which is from reference [44]. The values for the [M(UDP)]ꢀ
species are from reference [45], the constant for [Pb(UDP)]ꢀ is an esti-
mate taken from reference [19].
1
lished[52] acidity constants determined by H NMR shift ex-
periments for two uridine derivatives carrying singly nega-
tively charged phosphate–ethyl ester groups at either the 3’
position or at both the 3’ and 5’ positions. The acidity con-
stants pKa(3’) =9.21ꢂ0.05 and pKa(3’/5’) =9.26ꢂ0.05[52] are
identical within their error limits, revealing that the effect of
a single negative charge in this case is small.
[M(UMP)] and [M(UDP)]ꢀ complexes for comparison. Evi-
dently, the stability of the [M(pUpU)]ꢀ complexes is gener-
ally closer to that of the [M(UMP)] species than to the [M-
(UDP)]ꢀ species. The only exceptions are the Pb2+ com-
plexes, in which the stability of [Pb(pUpU)]ꢀ is closer to
that of [Pb(UDP)]ꢀ than to that of [Pb(UMP)]. Therefore, a
more rigorous evaluation procedure is required to elucidate
the structures of the [M(pUpU)]ꢀ complexes in solution.
Nevertheless, these observations cannot be interpreted as
evidence that there are no charge effects at all: Comparison
Chem. Eur. J. 2005, 11, 4163 – 4170
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R. K. O. Sigel et al.
Comparison of the stabilities of [M(pUpU)]ꢀ complexes
with those of M[phosph(on)ate] species: For series of relat-
ed ligands,[53,54] straight lines are obtained if logKMMðLÞ is plot-
ted versus pKHHðLÞ. Such correlation lines are available[36,55]
for complexes formed between several divalent metal ions
(M2+) and simple phosphate monoester or phosphonate li-
gands (R–PO32ꢀ). The straight-line parameters for the five
metal ions studied here, along with Cu2+ (see Experimental
Section), are listed in Table 3.
Table 3. Straight-line correlations for M2+–phosphate monoester or
–phosphonate complex stabilities, and phosph(on)ate group basicities
(aqueous solutions; 258C; I=0.1m, NaNO3).[a]
M2+
m
b
SD
Mg2+
Mn2+
Zn2+
Cd2+
Pb2+
Cu2+
0.208ꢂ0.015
0.238ꢂ0.022
0.345ꢂ0.026
0.329ꢂ0.019
0.493ꢂ0.033
0.465ꢂ0.025
0.272ꢂ0.097
0.683ꢂ0.144
0.033
0.051
0.060
0.045
0.076
0.057
ꢀ0.017ꢂ0.171
0.399ꢂ0.127
ꢀ0.122ꢂ0.213
ꢀ0.015ꢂ0.164
[a] Slopes (m) and intercepts (b) for the straight-reference-line plots of
logKM
versus pKHHðRꢀPO (Figure 2), as calculated by using the
½MðRꢀPO3Þꢁ
3 Þ
least-squares procedure from the equilibrium constants for simple R–
PO32ꢀ/H+/M2+ systems (R=noncoordinating residue); the R–PO3 li-
2ꢀ
gands are listed in the legend of Figure 2. The straight-line equations are
defined by y=mx+b, in which x represents the pKHHðRꢀPO Þvalue of any
3
Figure 2. Evidence for an enhanced stability of the [Mg(pUpU)]ꢀ, [Zn-
monoprotonated phosph(on)ate group, and y the calculated stability con-
(pUpU)]ꢀ, and [Pb(pUpU)]ꢀ complexes ( ), based on the relationship
stant (logKM
) of the corresponding [M(R–PO3)] complex; the
*
½MðRꢀPO3Þꢁ
between logKM
and pKHHðRꢀPO for [M(R–PO3)] complexes of some
given errors of m and b correspond to one standard deviation (1s).[36,44,55]
Column 4 lists three times the standard deviations (SD), resulting from
the differences between the experimental and calculated values for the
½MðRꢀPO3Þꢁ
3Þ
2ꢀ
*
simple phosphate monoester and phosphonate ligands (R–PO3 ) ( ):
(from left to right) 4-nitrophenyl phosphate (NPhP2ꢀ), phenyl phosphate
(PhP2ꢀ), uridine 5’-monophosphate (UMP2ꢀ), d-ribose 5-monophosphate
(RibMP2ꢀ), thymidine [1-(2’-deoxy-b-d-ribofuranosyl)thymine] 5’-mono-
phosphate (dTMP2ꢀ), n-butyl phosphate (BuP2ꢀ), methanephosphonate
(MeP2ꢀ), and ethanephosphonate (EtP2ꢀ). The least-squares lines
2ꢀ
various R–PO3 ligands employed.[36,44,55] The above parameters are
taken from reference [55], except those for the Pb2+ systems, which are
from reference [44].
*
[Eq. (6)] are drawn through the corresponding eight data sets ( ) taken
from reference [43] for the phosphate monoesters, and from refer-
ence [55] for the phosphonates; the corresponding straight-line parame-
ters are listed in Table 3. The data points due to the M2+/H+/pUpU3ꢀ sys-
For the Mg2+, Zn2+, and Pb2+ systems, the data pairs of
the [M(R–PO3)] complexes, on which the parameters in
Table 3 are based, are shown in Figure 2, together with the
corresponding data points for the pUpU3ꢀ systems. For all
three [M(pUpU)]ꢀ complexes, an increased stability is ob-
served. However, this stability varies quite considerably
from metal ion to metal ion; for example, for [Pb(pUpU)]ꢀ,
the stability enhancement is clearly above one log unit!
A more quantitative evaluation was possible by applying
the parameters of Table 3 together with pKHHðpUpUÞ =6.44=
*
tems ( ) are based on the constants listed in Tables 1 and 2. The vertical
broken lines emphasize the stability differences from the reference lines,
logDM/pUpU, as defined in Equation (7) (see also Table 4, column 4). All
plotted equilibrium constants refer to aqueous solutions at 258C and I=
0.1m (NaNO3).
Table 4. Comparison of the stability constants of the [M(pUpU)]ꢀ com-
plexes between the measured stability constants [Eq. (5)] and the calcu-
lated stability constants for [M(R–PO3)] species, based on the basicity of
the terminal phosphate group of pUpU3ꢀ (pKHHðpUpUÞ =6.44) and the refer-
ence-line equation [Eq. (6)] defined in Table 3, together with the stability
pKHHðRꢀPO Þ (Table 1) to the straight-line Equation (6):
3
log KM
¼ pKHHðRꢀPO Þ ꢃ mþb
ð6Þ
½MðRꢀPO3Þꢁ
3
differences logDM/pUpU
,
as defined in Equation (7) (258C; I=0.1m,
The results of these calculations (Table 4) represent the
stability constants of [M(R–PO3)] complexes in which no ad-
ditional interaction occurs, that is, the metal ion is coordi-
nated only to a phosphate group that has the basicity of the
terminal phosphate group in pUpU3ꢀ. Comparison of these
data with the measured stability constants demonstrates an
enhanced stability for all five complexes studied.
NaNO3).[a]
M2+
logKM
logKM
logDM/pUpU
[b]
½MðpUpUÞꢁ
½MðRꢀPO3Þꢁ
Mg2+
Mn2+
Zn2+
Cd2+
Pb2+
1.84ꢂ0.04
2.49ꢂ0.05
2.57ꢂ0.03
2.75ꢂ0.03
4.45ꢂ0.25
1.61ꢂ0.03
2.22ꢂ0.05
2.20ꢂ0.06
2.52ꢂ0.05
3.05ꢂ0.08
0.23ꢂ0.05
0.27ꢂ0.07
0.37ꢂ0.07
0.23ꢂ0.05
1.40ꢂ0.26
[a] For the error limits, see footnote [a] of Table 1. The error limits (3s)
of the derived data in column 4 were calculated according to the error
propagation of Gauss. [b] From column 3 in Table 2.
Quantification of the enhanced stability of the [M(pUpU)]ꢀ
complexes and the extent of chelate formation: The stability
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Metal-Ion Binding Properties of RNA Dinucleotides
FULL PAPER
KI ¼ ½½MðpUpUÞclꢁꢀꢁ=½½MðpUpUÞopꢁꢀꢁ
ð10Þ
differences between the measured values for the [M-
(pUpU)]ꢀ complexes and the calculated values for the [M-
(R–PO3)] species were obtained by using Equation (7), and
are listed in Table 4.
As shown previously[36,53,54] the stability enhancement
logD* [Eq. (8)] is related to KI by Equation (11):
KI ¼ 10log D ꢀ1
log DM=pUpU ¼ log KM½MðpUpUÞꢁꢀlog KM
*
ð11Þ
ð7Þ
½MðRꢀPO3Þꢁ
Knowledge of KI facilitates calculation of the formation
degree of the closed species in Equilibrium (9) by using
Equation (12):
The stability enhancements are identical within the error
limits for the complexes of Mg2+, Mn2+, and Cd2+. Consid-
ering the different coordinating properties[56–58] of these
three metal ions, it becomes clear that this increased stabili-
ty is simply due to the charge effect by going from [M(R–
PO3)] to [M(pUpU)]ꢀ. In other words, the metal ion coordi-
nated to the terminal phosphate group in pUpU3ꢀ “feels”
the presence of the negative charge located on the neighbor-
ing phosphate–diester bridge.
% ½MðpUpUÞclꢁꢀ ¼ 100 ꢃ ½KI=ð1þKIÞꢁ
ð12Þ
The results for KI and % [M(pUpU)cl]ꢀ are listed in
Table 5.
As already stated, in the case of the [M(pUpU)]ꢀ systems
with Mg2+, Mn2+, and Cd2+, the values of logD* are zero
within the error limits. However, this means that within
these error limits, traces of chelated species might form; the
corresponding upper limits are given in parentheses in
Table 5. Indeed, for [Mn(pUpU)]ꢀ, it appears that a small
percentage of a closed species might occur. This is possible
because Mn2+ is known[45] for its relatively pronounced af-
finity for phosphate groups, and the given error limits corre-
spond to 3s. In any case, chelates for [Zn(pUpU)]ꢀ and [Pb-
(pUpU)]ꢀ definitely exist, with formation degrees of approx-
imately 25% and greater than 90%, respectively.
The average of the logDM/pUpU values for the Mg2+, Mn2+
,
and Cd2+ systems, logDM/pUpU/charge =0.24ꢂ0.04, represents
this charge effect. Hence, any further stability increase must
be attributed to an additional interaction of the metal ion al-
ready coordinated to the terminal phosphate group of
pUpU3ꢀ. This increase is defined by Equation (8):
*
log D ¼ log DM=pUpUꢀlog DM=pUpU=charge
ð8Þ
As discussed in the Introduction, the only other available
binding site in pUpU3ꢀ is the phosphate–diester bridge,
which allows the formation of a 10-membered chelate
(Figure 1) involving both phosphate groups.
The values for logD* according to Equation (8) are listed
in Table 5. As expected, these values are zero within the
Interestingly, our results are in good agreement with the
Stability Ruler proposed by Martin.[56–58] With regard to in-
teractions with oxygen donors, such as oxalate, Cd2+ is
placed significantly below Zn2+ and Pb2+, to give the order
Cd2+ !Zn2+ <Pb2+. In the case of pUpU3ꢀ discussed here,
Pb2+ may be especially favored due to its larger size, which
should facilitate binding of two neighboring phosphate
groups in a nucleic acid.
Table 5. Extent of chelate formation in [M(pUpU)]ꢀ complexes
[Eq. (9)], as calculated from the stability enhancement logD* [Eq. (8)]
and quantified by the dimensionless equilibrium constant KI [Eqs. (10)
and (11)], and the percentage of the chelated isomer [Eq. (12)] in aque-
ous solution (258C; I=0.1m, NaNO3).[a,b]
M2+
logDM/pUpU
logD*
KI
% [M(pUpU)cl]ꢀ
Mg2+
Mn2+
Zn2+
Cd2+
Pb2+
0.23ꢂ0.05
0.27ꢂ0.07
0.37ꢂ0.07
0.23ꢂ0.05
1.40ꢂ0.26
ꢀ0.01ꢂ0.06
0.03ꢂ0.08
0.13ꢂ0.08
ꢀ0.01ꢂ0.06
1.16ꢂ0.26
~0
~0
~0 (<11)
~0 (<22)
26ꢂ14
Conclusions
0.35ꢂ0.25
An important result from this study with regard to RNAs
and ribozymes is the observation that neighboring uracil res-
idues lead to a depression of the pKa value for the deproto-
nation of the (N3)H site by about half a log unit compared
to UMP2ꢀ. This deprotonation may be further promoted by
metal ions, such as Mg2+ and Ca2+, which have a relatively
selective affinity for the carbonyl oxygen atoms of nucleo-
bases, as shown recently[30,40] for the cytosine and uracil resi-
dues. As a consequence of such a facilitated deprotonation
of the (N3)H site, possibly further enhanced by the indicat-
ed coordination of alkaline earth ions, the pKa for this site
approaches the physiological pH range. Uracil residues are,
therefore, important factors to be considered in general
acid–base catalyzed reactions.
~0
~0 (<11)
93ꢂ4
13.45ꢂ8.65
[a] For the error limits, see footnote [a] of Table 4. [b] The values given
in column 2 are from column 4 in Table 4.
error limits for the [M(pUpU)]ꢀ complexes of Mg2+, Mn2+
,
and Cd2+. In contrast, for the logD* values of Zn2+ and
Pb2+, this is clearly not the case. The different stability en-
hancements for the [Zn(pUpU)]ꢀ and [Pb(pUpU)]ꢀ com-
plexes mean that the position of the intramolecular Equili-
brium (9), between an open (op) and a closed (cl) or chelat-
ed isomer, varies.
½MðpUpUÞopꢁꢀ Ð ½MðpUpUÞclꢁꢀ
ð9Þ
By considering the stability enhancement of the different
[M(pUpU)]ꢀ complexes, it is notable that Pb2+, and to a cer-
tain extent also Zn2+, differ considerably in their binding
properties towards phosphate groups. Firstly, the Pb2+–phos-
The position of Equilibrium (9) is defined by the dimen-
sionless intramolecular equilibrium constant KI [Eq. (10)]:
Chem. Eur. J. 2005, 11, 4163 – 4170
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ꢃ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4167

R. K. O. Sigel et al.
phate complexes have a rather high stability compared to,
for example, Mg2+, and secondly, Pb2+ is the only one of the
ions investigated here that shows a very pronounced ability
to interact strongly with two neighboring phosphate sites.
Therefore, it is not surprising that leadzymes,[59,60] as well as
Pb2+-dependent DNAzymes,[61] that is, RNA or DNA oligo-
nucleotides with a structural motif that selectively binds
Pb2+ to promote hydrolytic cleavage of a second nucleic
acid, could be selected by conducting in vitro selection ex-
periments. Our results also explain why some leadzymes are
strongly inhibited[62–65] by the presence of Mg2+ and other
divalent ions; such ions inhibit chelate formation of Pb2+. A
difference in RNA binding between Pb2+ and Mg2+ has also
been observed in hydrolytic cleavage experiments of a
group II intron ribozyme[15] and other large RNAs.[66] Fur-
thermore, despite its higher affinity, Pb2+ showed fewer
(and/or different) cleavage sites than was observed with
Mg2+ and Tb3+. These discrepancies in binding patterns are
explained by our results, namely that Pb2+ has a high ten-
dency to coordinate two neighboring phosphate groups on
its own, whereas Mg2+ can only do so if the binding is fur-
ther stabilized by surrounding nucleotides from other parts
of the nucleic acid.
Experimental Section
Synthesis of uridylyl-(5’!3’)-[5’]uridylic acid, H3(pUpU): Although salts
of pUpU3ꢀ have already been synthesized by using either the phospho-
diester[23,24] or phosphotriester[25,26] approach, we used a new method. The
trisodium salt of pUpU3ꢀ (1) was prepared by a multistep synthesis
(Scheme 1, Supporting Information) using the phosphoramidite method-
ology, with the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp) group
for protection of 2’-hydroxy functions.[77,78] 5’-O-dimethoxytrityl-2’-O-
Fpmp uridine-3’-O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite (2)
was prepared as previously described.[77] Compound 2 was then reacted
in CH2Cl2 solution with 2’,3’-di-O-acetyluridine in the presence of 1-H-
tetrazole to give, after subsequent I2/pyridine/H2O oxidation of the inter-
mediate phosphite, the fully protected dinucleosidephosphate (3). After
selective removal of the dimethoxytrityl (DMT) group with 2% dichloro-
acetic acid in CH2Cl2, compound 3 was 5’-O-phosphorylated by using the
bis-O,O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite reagent, and the
phosphite intermediate was oxidized with I2/pyridine/H2O.[79] The fully
protected 5’-O-phosphorylated dinucleotide (4) obtained was purified by
silica gel chromatography and subjected to stepwise deprotection by
heating to 558C for 16 h in the presence of 30% aqueous ammonia (re-
moval of 2-cyanoethyl and acetyl groups) followed by treatment with
0.01m HCl (pH 2.0) (12 h at RT; removal of the Fpmp group). The crude
product 1 was purified by ion-exchange chromatography using DEAE
Sephadex A-25 (elution with a linear gradient of triethylammonium bi-
carbonate from 0.1 to 0.6m). Purified 1 was then transformed into its tri-
sodium salt by passing it through Dowex 50Wx8 (Na+ form) and lyophyl-
ized to yield a white solid in 12% overall yield (based on 2’,3’-di-O-
acetyluridine). The structure of 1 was confirmed by using spectroscopic
methods: proton-decoupled 31P NMR (D2O) two singlets at d=ꢀ0.14
and 0.61 ppm [Bruker Avance, 200 MHz]; FAB MS: m/z: 629.2 (negative
ions) (Finnigan MAT 95) (calculated MW 630.4 for free acid). Analytical
RP-HPLC of product 1 showed a single peak.
In the context of Pb2+, two further points should be em-
[57,58,67]
phasized: Firstly, Pb2+ is a well-known mimic of Ca2+
,
and the alkaline earth metal ion itself is a potent inhibitor
of ribozyme activity.[68,69] One may, therefore, assume,
though this is still to be proven, that in a nucleic acid, Ca2+
can bind simultaneously, like Pb2+ as discussed above, to
two neighboring phosphate groups, whereas Mg2+ can not.
Secondly, the straight-line parameters in Table 3 for the
Pb2+ and Cu2+ systems are identical within their error
limits. Indeed, the stability constants calculated with a pKa =
6.44 for the two complexes [Pb(R–PO3)] and [Cu(R–PO3)],
that is, logKP½PbbðRꢀPO Þꢁ =3.05ꢂ0.08 and logKC½CuuðRꢀPO Þꢁ =2.98ꢂ
Materials: The disodium salt of ethylenediamine-N,N,N’,N’-tetraacetic
acid (Na2H2EDTA·2H2O), potassium hydrogen phthalate, nitric acid,
sodium hydroxide (Titrisol), and the nitrate salts of Na+, Mg2+, Mn2+
,
Zn2+, Cd2+, and Pb2+ (all pro analysi) were from Merck, Darmstadt,
Germany. The buffer solutions (pH 4, 7, 9), all based on the NBS scale
(now U.S. National Institute of Science and Technology, NIST), were
from Metrohm, Herisau, Switzerland. All solutions were prepared by
using deionized, ultrapure (Milli-Q185 Plus; from Millipore S.A., 67120
Molsheim, France) CO2-free water.
3
3
0.06, are also identical within the error limits. Based on this
observation and on the Stability Ruler of Martin,[56–58] it can
be concluded that the properties regarding stability and
structure of the [Pb(pUpU)]ꢀ and [Cu(pUpU)]ꢀ complexes
are similar.
The titer of the NaOH solution used for the titrations was established by
using potassium hydrogen phthalate, and the exact concentrations of the
stock solutions of Mg2+, Mn2+, Zn2+, Cd2+, and Pb2+ were determined
by performing potentiometric pH titrations using their EDTA complexes.
The aqueous stock solutions of pUpU were freshly prepared daily and
the pH of the solution was adjusted to 8.3 with sodium hydroxide. The
exact concentration of the ligand solutions was determined in each ex-
periment by evaluation of the corresponding titration pair, that is, the dif-
ferences in NaOH consumption between solutions with and without
ligand (see below).
The selective coordination of Mg2+ to only a single phos-
phate group is also revealing with regard to this metal ionꢀs
crucial role in ribozyme catalysis. Although structures are
known in which Mg2+ bridges several phosphate groups,[70,71]
this occurs in a structurally enforced way. Many more exam-
ples are known that involve a Mg2+ ion coordinated to a
single phosphate unit.[3,4,70–74] Among the latter group, Mg2+
binding to uridine 80 within U6 of the spliceosome,[11,12] or
to adenosine 2 within domain 5 of the group II intron
ai5g[16,75] are only two prominent examples. For cases in
which two or more neighboring phosphate groups bind to
Mg2+ ions, these then tend to cluster close together, as ob-
served in the loop E motif,[14] the active site of group I in-
trons,[73] as well as at a single nucleobase.[76] The findings
presented here are in good agreement with all of these ob-
servations, and furthermore, they assign the first quantitative
evaluation to these phenomena.
Potentiometric pH titrations: The pH titrations were conducted by using
a Metrohm E536 potentiograph connected to a Metrohm E665 dosimat
and a Metrohm 6.0253.100 Aquatrode-plus combined double-junction
macro glass electrode (all from Metrohm, Herisau, Switzerland). The
equipment was calibrated by using the buffer solutions mentioned above.
The direct pH meter readings were used in the calculations of the acidity
constants of H(pUpU)2ꢀ; thus, the constants determined at I=0.1m
(NaNO3) and 258C are so-called practical, mixed, or Brønsted con-
stants,[46] and may be converted into the corresponding concentration
constants by subtracting 0.02 from the listed pKa values.[46] This conver-
sion term includes both the junction potential of the glass electrode and
the hydrogen ion activity.[46,80] It should be emphasized that the ionic
product of water (KW) and the conversion term mentioned do not enter
into our calculation procedures, because the differences in NaOH con-
sumption between solutions with and without ligand (see below) are eval-
uated.[46,81] No conversion is necessary for the stability constants of the
4168
ꢃ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 4163 – 4170

Metal-Ion Binding Properties of RNA Dinucleotides
FULL PAPER
[M(pUpU)]ꢀ complexes because these are, as usual, concentration con-
stants.
The final results for the stability constants of all [M(pUpU)]ꢀ complexes,
KM½MðpUpUÞꢁ, are the averages of three independent titrations in the case of
the Mg2+, Mn2+, and Zn2+ systems, whereas for Cd2+ and Pb2+, two and
four independent pairs of titrations were performed, respectively. There
was no indication of a metal-ion-promoted hydrolysis of pUpU3ꢀ during
the time required for a titration experiment (about 30 min).
Determination of equilibrium constants: The acidity constants KHHðpUpUÞ
,
KðHpUpUÞ, and KHðpUpUꢀHÞ of H(pUpU)2ꢀ [Eqs. (2)–(4)] were determined by
titrating 30 mL of aqueous 0.5 mm HNO3 (258C; I=0.1m, NaNO3) under
N2 with up to 3.0 mL of 0.03m NaOH in the presence and absence of
0.27 mm H(pUpU)2ꢀ. Additional titrations were performed with a ligand
concentration of 0.18 mm, and in this case, 3.0 mL of 0.02m NaOH was
used. Notably, no difference between the two conditions was observed.
The experimental data were evaluated by employing a curve-fitting pro-
cedure using a Newton-Gauss non-linear least-squares program, in which
the difference in NaOH consumption between such a pair of titrations at
every 0.1 pH unit was used. The acidity constants of H(pUpU)2ꢀ were
calculated within the pH range 4.8 to 9.7, corresponding to 2% neutrali-
zation (initial) for the equilibrium H(pUpU)2ꢀ/(pUpU)3ꢀ and around
Acknowledgements
We thank Professor Helmut Sigel, University of Basel, for helpful sugges-
tions during the preparation of this manuscript. Financial support from
the Swiss National Science Foundation (SNF-Fçrderungsprofessur to
R.K.O.S., PP002–68733/1) and the Polish State Committee for Scientific
Research is gratefully acknowledged.
54% (final) for (pUpUꢀH)4ꢀ/(pUpUꢀ2H)5ꢀ
. The final results for
pKHHðpUpUÞ, pKðHpUpUÞ, and pKðHpUpUꢀHÞ are the averages of the values from
six independent pairs of titrations.
After each of these titrations, the solutions were adjusted to the initial
pH of around 3.3 by adding a small volume (about 1 mL) of 0.1m HNO3.
Subsequently, a comparatively small volume of a solution of M(NO3)2
(M2+ =Mn2+, Zn2+, Cd2+) was added and the titration was repeated.
From the data obtained in the presence of M2+ (with and without
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of the [M(pUpU)]ꢀ complexes
½MðpUpUÞꢁ
[Eq. (5)] were calculated. The total volume of these solutions was around
35 mL, and the ionic strength I varied between 0.1 and 0.13m. This small
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with Mg2+, NaNO3 was fully replaced by Mg(NO3)2. The metal-to-ligand
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65:1, and 63:1 for Mn2+; 65:1, 42:1, and 32:1 for Zn2+; 33:1 and 21:1 for
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The titration data, except those for the determination of KPb
of the
½PbðpUpUÞꢁ
[Pb(pUpU)]ꢀ complex with a very small metal-to-ligand ratio (2.4:1 and
1.8:1), were evaluated by employing a curve-fitting procedure using a
Newton-Gauss non-linear least-squares program for each titration pair
(i.e., with and without ligand), by calculating the apparent acidity con-
stant K0a. Depending on the metal ion under consideration, the evaluation
commenced at a formation degree of the [M(pUpU)]ꢀ species of about 2
to 10% (see below), and the upper limit was given by either the onset of
the hydrolysis of M(aq)2+, which was evident from the titrations without
ligand, or by the formation of the [M(pUpUꢀH)]2+ or [M2(pUpUꢀH)]
species, which was evident by the deviation of the experimental data
from the calculated curve. Representative examples for the pH ranges
employed in the case of the [M(pUpU)]ꢀ complexes are 4.6–6.8 (Mg2+),
4.2–6.3 (Mn2+), 3.9–5.6 (Zn2+), 4.1–5.8 (Cd2+), and 3.6–4.3 (Pb2+). These
pH ranges correspond to variations in the formation degrees of about 3–
56% for [Mg(pUpU)]ꢀ, 4–72% for [Mn(pUpU)]ꢀ, 2–39% for [Zn-
(pUpU)]ꢀ, 2–43% for [Cd(pUpU)]ꢀ, and 10–26% for [Pb(pUpU)]ꢀ. The
stability constants of the complexes were calculated as described previ-
ously.[30,53,82,83] Notably, the buffer depression DpKa =pKHHðpUpUÞꢀpK0a was
satisfactory in all titrations, i.e., DpKa ꢄ0.35.
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