Phosphodiester cleavage by trivalent lanthanides in the presence of native cyclodextrins

Article abstract of DOI:10.1016/j.ica.2015.10.039

Testing phosphodiesterase activity of Eu(III) in the presence of native cyclodextrins revealed capacity of β-cyclodextrin (β-CD) to stabilize catalytically active metal hydroxocomplexes in mildly basic solutions. Kinetics of the hydrolysis of bis(4-nitrop

Full text of DOI:10.1016/j.ica.2015.10.039

Inorganica Chimica Acta 440 (2016) 9–15
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Phosphodiester cleavage by trivalent lanthanides in the presence
of native cyclodextrins
⇑
Mariel Ruiz Kubli, Anatoly K. Yatsimirsky
Facultad de Química, Universidad Nacional Autónoma de México, México D.F. 04510, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Testing phosphodiesterase activity of Eu(III) in the presence of native cyclodextrins revealed capacity of
b-cyclodextrin (b-CD) to stabilize catalytically active metal hydroxocomplexes in mildly basic solutions.
Kinetics of the hydrolysis of bis(4-nitrophenyl) phosphate (BNPP) and transesterification of 2-hydrox-
ypropyl 4-nitrophenyl phosphate (HPNP) as models of DNA and RNA respectively has been studied with
La(III), Pr(III), Nd(III), Eu(III), Gd(III) and Dy(III) cations in the presence of b-CD in the range of pH 7.0–9.0.
The overall catalytic effect with 2 mM lanthanide–b-CD complexes was up to 105 for HPNP and 108 for
BNPP at pH 8 demonstrating the highest catalytic activity among so far reported artificial phosphodi-
esterases. Analysis of concentration and pH-dependences of observed rate constants for different lan-
Received 20 September 2015
Received in revised form 18 October 2015
Accepted 26 October 2015
Available online 2 November 2015
Keywords:
Lanthanide
Cyclodextrin
Phosphate ester
Hydrolysis
Kinetics
2
thanides showed that active species are binuclear polyhydroxocomplexes of general type [M (b-CD)
6ꢀn
1
(OH)
n
]
with n = 3–5. The metal–b-CD and phosphodiester–b-CD interactions were studied by H
NMR spectroscopy. Mechanistic implications of much higher catalytic efficiency in BNPP hydrolysis as
compared to HPNP transesterification are discussed.
Catalysis
Ó 2015 Elsevier B.V. All rights reserved.
1
. Introduction
stable complexes with anhydrocyclodextrins lacking OH groups
[
4], which are principal binding groups for transition metal ions
Native cyclodextrins form complexes in water with metal ions
[1]. This confirms the original idea [3] that cyclodextrins behave
towards lanthanides essentially as crown ethers affording inclu-
sion complexes via ion–dipole contacts with neutral oxygen atoms.
Hydroxocomplexes of trivalent lanthanides constitute an
important group of most efficient catalysts for the hydrolysis of
phosphate esters including DNA, RNA and model compounds [5].
Development of these catalysts depends critically on the use of
appropriate stabilizing ligands, which must prevent precipitation
of metal hydroxide and at the same time allow for formation of
active hydroxocomplexes. Results of cited above studies point to
a possibility that CDs may act as such ligands. Metal-free cyclodex-
trins [6] as well as metal complexes of chemically modified
cyclodextrins [7] were studied as artificial phosphodiesterases,
but the potential of lanthanides stabilized by native CDs was not
explored yet. Previously we reported successful catalysis by lan-
thanides stabilized by neutral polyol ligands like bis-Tris propane
or Tris [8,9]. Like CDs these compounds act as neutral O-donor
ligands using non-dissociated hydroxyl groups as metal binding
sites [9,10]. They form rather weak complexes with lanthanide
aqua-ions with stability constants in the same range as with CDs,
but stabilize active binuclear hydroxocomplexes providing much
higher phosphoesterolytic activity than that observed with free
metal ions.
of different types including lanthanides [1]. Ability of cyclodextrins
to stabilize trivalent lanthanide ions against precipitation in basic
media was demonstrated long time ago by Komiyama and
co-workers [2]. The authors proposed formation of both 1:1 and
2
:1 metal cyclodextrin (CD) complexes on basis of solubility data,
demonstrated catalytic activity of the complexes in the hydrolysis
of a phosphate monoester but did not determine the stability
constants and did not extend the reactivity studies beyond testing
of just one phosphate monoester as a substrate. Fatin-Rouge and
Bünzli [3] reported a detailed study of lanthanide complexation
by different CDs both in acid and basic solutions. Stability
2
4
ꢀ1
constants for aqua-ions were in the range 10 –10 M and did
not vary significantly for -, b- and -CD. Spectroscopic data
a
c
confirmed inclusion of cations into CDs, which behaved like crown
ethers with anomeric oxygen atoms being possible binding sites. In
strongly basic solutions at pH > 12 complexation with deproto-
nated secondary OH groups was the predominant form of binding.
In a more recent study lanthanides were found to form equally
⇑
Corresponding author.
E-mail address: anatoli@unam.mx (A.K. Yatsimirsky).
http://dx.doi.org/10.1016/j.ica.2015.10.039
020-1693/Ó 2015 Elsevier B.V. All rights reserved.
0

1
0
M. Ruiz Kubli, A.K. Yatsimirsky / Inorganica Chimica Acta 440 (2016) 9–15
In this paper we demonstrate that similar effect is observed
all b-CD signals indicating stronger although less specific interac-
tion with hydroxo complexes of Nd(III). These observations con-
firm ability of b-CD to interact with potentially catalytically
active hydroxo complexes of lanthanides [5], which explains both
the stabilizing effect of b-CD and observed catalytic activity.
Potentiometric titrations of lanthanide salts (Eu(III), Nd(III)) and
b-CD were reproducible only at pH below 8 where the fraction of
hydroxo complexes was still very low and species assignment
with CDs, which act as somewhat less powerful stabilizing ligands,
but induce formation of significantly more active lanthanide
species at lower pH. The catalytic activity was studied with two
model phosphate diesters: bis(4-nitrophenyl) phosphate (BNPP),
which is considered as a model for DNA hydrolysis, and 2-hydrox-
ypropyl 4-nitrophenyl phosphate (HPNP), which is considered as a
model for RNA transesterification.
-
-
BNPP
HPNP
Comparison of results of this study with previously reported
results for other lanthanide-based artificial phosphodiesterases
shows that CD-stabilized complexes in mildly basic solution at
pH 8 reach the level of catalytic activity typical for the most active
systems and in some cases even surpass their activity making lan-
thanide/b-CD system a simple practically useful artificial phospho-
diesterase catalyst.
was very uncertain. Therefore a possible composition of active spe-
cies was inferred from kinetic results obtained at variable pH.
Besides interactions with metal ions b-CD may form inclusion
complexes with substrates possessing hydrophobic nitrophenyl
groups, which also may affect the observed reactivity. A complex-
ation of diphenyl phosphate with b-CD with the binding constant
ꢀ1
K = 200 M
previously [12]. Fig. 3(A) shows the course of H NMR titration of
b-CD by BNPP in D O. No shifts of the signals of external H-2 and
determined by fluorescence titration was reported
1
2
. Results and discussion
2
H-4 protons is observed, but the signals of internal H-3 and H-5
protons undergo strong up-field shifts consistent with inclusion
of aromatic groups of BNPP inside the CD cavity. The complexation
induced shifts in the signals of H-5 and H-3, which sit at opposite
sides of the cyclodextrin cavity, are similar by their magnitude
which means that BNPP can enter the cyclodextrin cavity from
both sides. The profile of the signal of H-3 proton vs. guest concen-
tration is shown in Fig. 3(B) and the fitting of this profile to a 1:1
binding isotherm gives the binding constant K = 320 ± 20 M for
BNPP. Similar experiment with HPNP showed much smaller com-
plexation-induced shifts in the signals and a linear profile (Fig. 3
(B)), which does not allow one to estimate a significantly smaller
binding constant in this case.
In preliminary experiments we observed that the catalytic
activity of lanthanides is significantly higher with b-CD than with
- or -CD (Fig. S1, Supplementary Material). All studies therefore
a
c
were performed with b-CD. In the concentration range of lan-
thanides from 0.5 to 2.5 mM addition of 1 equivalent of b-CD
was sufficient to prevent precipitation of metal hydroxide on
a
increase in pH approximately up to pK value of the respective
ꢀ
1
cation, e.g. up to pH 9 with less acidic La(III) and up to pH 8 with
most acidic Dy(III). In the absence of CD precipitation with 1 mM
lanthanides starts already at pH 7. An excess of CD did not improve
the stability. To control the reaction pH the 50 mM Tris/HCl buffer
was employed. The buffer by itself has some stabilizing effect but
the rate constants measured in the absence of b-CD were at least
one order of magnitude smaller than those in the presence of b-CD.
Attempts to characterize the interactions of lanthanides with
b-CD in more details as compared to those reported by Fatin-Rouge
and Bünzli [3], in particular establish the type of hydroxo com-
plexes, provided limited information. Lanthanides affect very little
Curiously, recent theoretical calculations predict external BNPP
binding to b-CD via H-bonding of the host OH groups to the phos-
phoryl group of the phosphodiester, [13] which obviously contra-
dicts the experimental results.
On basis of determined binding constant for BNPP one may con-
clude that with b-CD concentrations below 2.5 mM employed in
kinetic studies (see below) the degree of the substrate complexa-
tion by CD is less than 50% and for HPNP it should be even smaller.
In all kinetic experiments hydrolysis of BNPP proceeded though
intermediate formation of mono-4-nitrophenyl phosphate, which
further was hydrolyzed to the second 4-nitrophenolate anion and
inorganic phosphate but with a different smaller rate constant.
The details of kinetic analysis are given in the Section 4. The
observed first-order rate constants (kobs) for BNPP hydrolysis dis-
cussed below correspond to the first step of the reaction. In the case
of HPNP usually overlooked problem is that the compound pre-
pared by traditionally employed procedure [14] contains ca. 5% of
1-hydroxy-2-propyl isomer, which is ca. 10 times more reactive
than the main 2-hydroxypropyl isomer [15] (see also [16]). The con-
tribution from the hydrolysis of the more reactive isomer practi-
cally disappears after ca. 20% of hydrolysis and this initial part of
the reaction was excluded from the calculation of the rate constant.
1
1
the H NMR spectra of b-CD in acid solutions. Fig. 1 shows the H
NMR spectra of b-CD alone and in the presence of some lanthanide
cations at pH 5.5. A single noticeable metal-induced change is a
down-field shift of the signal of H-5 proton located in the interior
of the CD cavity [11]. The shift is very small for La(III), but larger for
more acidic Eu(III) and Nd(III) cations for which the signal overlaps
with more intense signal of H-6 protons and with increased con-
centration of Nd(III) it is shifted further to a more down-field posi-
tion (upper spectre in Fig. 1). These results confirm inclusion of
lanthanide cations into b-CD cavity.
More importantly the interaction between a lanthanide cation
1
and b-CD becomes stronger at higher pH values. Fig. 2 shows
H
NMR spectra of b-CD in the presence of Nd(III) recorded at
increased pH, which demonstrates that at pH 7 besides the signal
of H-5 the cation shifts also the position of H-3, another interior
proton of b-CD, and at pH 8 one observes a strong broadening of

M. Ruiz Kubli, A.K. Yatsimirsky / Inorganica Chimica Acta 440 (2016) 9–15
11
Fig. 1. The 1H NMR spectra of 5 mM b-CD alone and in the presence of some lanthanide cations at pH 5.5 (uncorrected pH value in D
2
O, 0.05 M MES buffer, the range between
3
.64 and 3.78 ppm containing the buffer signals is eliminated).
Fig. 2. 1H NMR spectra of 5 mM b-CD alone and in the presence of 4 mM Nd(III) at increased pH (0.05 M TRIS/HCl buffer, the range between 3.66 and 3.84 ppm containing the
buffer signal is eliminated; uncorrected pH values in D O are indicated).
2
Fig. 4A shows the profile of kobs vs. concentration of the equimo-
lar mixture of Eu(III) and b-CD at constant pH 8.0. Evidently at low
concentrations the profile is curved upward, but above 1 mM it
becomes linear. The same results in logarithmic coordinates
Such behavior agrees with a self-assembling process of catalyt-
ically inactive mononuclear metal–CD complexes into active binu-
clear complexes above 1 mM total metal concentration. In the
presence of 2 mM Eu(III) the reaction rate was similar with 1 or
2 mM b-CD, but with 2 mM b-CD reaction rate was proportional
to metal ion concentration above 1 mM. Such behavior agrees with
formation of catalytically active complexes of the composition
(Fig. 4B) demonstrate that initially the reaction rate is a quadratic
function of the concentration and above 1 mM it becomes a linear
function. Similar profile was obtained with HPNP as a substrate
(Fig. 2S, Supplementary Material).
2
M (b-CD). All further kinetic experiments were performed with

1
2
M. Ruiz Kubli, A.K. Yatsimirsky / Inorganica Chimica Acta 440 (2016) 9–15
3.94
3.92
3.90
3.88
3.86
3.84
HPNP
BNPP
0
1
2
3
4
5
[BNPP] or [HPNP], mM
(
A)
(B)
Fig. 3. (A) 1H NMR titration of 2 mM b-CD by BNPP in D
2 2
O. (B) Chemical shift of H-3 proton vs. concentration of BNPP or HPNP in D O.
-4
-4
-4
-4
-4
-4
6
5
4
3
2
1
.0x10
.0x10
.0x10
.0x10
.0x10
.0x10
-
-
3.2
3.4
(A)
(B)
slope = 1
-3.6
-
-
-
-
3.8
4.0
4.2
4.4
slope = 2
0
.0
-4.6
-3.4
0
.0
0.5
1.0
1.5
2.0
2.5
-3.3
-3.2
-3.1
-3.0
-2.9
-2.8
-2.7
-2.6
-2.5
[Eu(III)]=[β-CD], mM
log[Eu(III)]
Fig. 4. (A) Dependence of the observed first-order rate constant for the BNPP hydrolysis at pH 8.0 on the concentration of Eu(III) in the presence of 1 equivalent of b-CD. (B)
The same results in the logarithmic coordinates. Lines are drawn to visualize the trend and are not theoretical fits.
2
mM total metal when the reaction was first-order in the catalyst
which will be estimated on basis of pH-profiles of reaction rates
discussed below.
and although the presence of half of equivalent of b-CD was suffi-
cient to observe the maximum catalytic activity, 2 mM b-CD was
employed because of better stability and reproducibility with 1:1
metal:CD ratio.
Lanthanide cations are very prone to self-association through
hydroxide bridges [17]. In a recent detailed study of speciation of
Eu(III) by direct excitation luminescent spectroscopy formation of
Figs. 5 and 6 show the pH-profiles of the rates of catalytic
hydrolysis of BNPP and HPNP respectively at fixed 2 mM concen-
tration of different lanthanides in the presence of 2 mM b-CD.
With both substrates the reaction rate shows a very strong
dependence on pH. Plots of log kobs vs. pH are linear until pH is
a
approaching pK of the respective cation (9.33; 8.82; 8.70; 8.59;
4
+
binuclear [Eu
with aquo-ion [Eu(H
2
(l-OH)
2
(H
2
O)16
]
species was detected together
8.62; 8.37 for La(III), Pr(III), Nd(III), Eu(III), Gd(III), Dy(III) respec-
tively [19]), but at higher pH values solutions become unstable
and results are poorly reproducible. The slopes of the plots roughly
indicate the number of hydroxide anions bound to the metal to
form a reactive species. Alternatively they may reflect the number
of deprotonated OH groups of b-CD which may act as nucleophiles
towards BNPP in form of alkoxide anions [6]. In this case the phos-
phorylated b-CD should be the reaction product. To explore this
3
+
2 9
O) ]
and mono-hydroxide complex [Eu
2
+
(
OH)(H
2
O)
8
]
even at pH 6.5 in a micromolar concentration range
[
18]. One may expect therefore that the concentration profile in
Fig. 1 reflects transformation of mononuclear to binuclear hydrox-
ide bridged complexes of general type [Eu
Previous results for lanthanides stabilized by polyol ligands [8,9]
indicate that the bridging hydroxide ions lack the nucleophilic
reactivity and for this reason the active species must have an
additional number of non-bridging hydroxides, the number of
4ꢀn
2
(l
-OH)
2
(b-CD)(OH)
n
]
.
3
1
possibility the reaction was monitored by P NMR in the presence
of 2 mM b-CD, 2 mM La(III) and 2 mM BNPP at pH 9.0 until nearly

M. Ruiz Kubli, A.K. Yatsimirsky / Inorganica Chimica Acta 440 (2016) 9–15
13
-
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Table 1
Observed first-order rate constants for the cleavage of phosphodiesters by 2 mM
lanthanide cations in the presence of 2 mM b-CD at pH 8.0.
Nd(III)
-
BNPP
HPNP
Gd(III) Eu(III)
obs, s 1
ꢀ
k
obs/k
a
k
ꢀ1
obs, s
k
obs/k a
0
k
0
-
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
5
5
4
4
3
4
6
6
7
7
8
7
ꢀ4
ꢀ4
ꢀ3
ꢀ2
ꢀ2
ꢀ2
3
3
4
4
5
5
La(III)
Pr(III)
Nd(III)
Eu(III)
Gd(III)
Dy(III)
1.2 ꢂ 10
6.0 ꢂ 10
1.2 ꢂ 10
6.0 ꢂ 10
1.0 ꢂ 10
1.2 ꢂ 10
6.0 ꢂ 10
1.2 ꢂ 10
6.0 ꢂ 10
1.0 ꢂ 10
8.0 ꢂ 10
7.0 ꢂ 10
9.0 ꢂ 10
2.3 ꢂ 10
1.0 ꢂ 10
3.0 ꢂ 10
4.0 ꢂ 10
4.1 ꢂ 10
5.3 ꢂ 10
1.3 ꢂ 10
6.0 ꢂ 10
1.8 ꢂ 10
2.3 ꢂ 10
-
-
8.0 ꢂ 10ꢀ
a
Rate acceleration over the rate of spontaneous hydrolysis at pH 8.0; k
0
= 10–11
for BNPP and 1.7 ꢂ 10
-
ꢀ
1
ꢀ7 ꢀ1
s
s
for HPNP [5b].
-
Pr(III)
La(III)
hydroxide in power ‘‘n” where ‘‘n” is a number of hydroxide anions
bound to a catalytically active form of the complex. Assuming that
at lower pH with 2 mM metal and 2 mM b-CD the predominant
species are binuclear hydroxide bridged complexes of general com-
-
Dy(III)
.2
7
7.6
8.0
8.4
8.8
4
+
position [M
2 2
(l-OH) (b-CD)] the respective equilibrium can be
pH
expressed as the Eq. (1) with ‘‘n” ranging from 1 to 3.
Fig. 5. Observed first-order rate constants (sꢀ1) for the hydrolysis of BNPP in the
presence of 2 mM b-CD and 2 mM metal ion vs. pH.
4
þ
ꢀ
4ꢀn
½M
2
ðl
-OHÞ ðb-CDÞꢁ þ nOH ¢ ½M
2
ð
l
-OHÞ ðb-CDÞðOHÞ ꢁ
ð1Þ
2
2
n
The fractional values of ‘‘n” like e.g. 2.5 can be explained by sim-
ilar contributions of complexes with n = 2 and 3 to the reaction
rate. Such behavior agrees very well with previous results for phos-
phoesterolytic activity of lanthanides stabilized by polyol [8,9] and
amino acid ligands [20] where the binuclear hydroxo complexes of
similar type with ‘‘n” ranging from 1 to 3 were proved to be the
active species.
To illustrate the catalytic efficiency of cyclodextrin-stabilized
complexes Table 1 collects the observed rate constants determined
at a single pH 8.0 for different 2 mM metal ions. Let us consider
first the results for BNPP. The catalytic effect over the rate of spon-
Nd(III)
Dy(III) Gd(III)
La(III)
-1.5
-2.0
-2.5
-3.0
-3.5
Pr(III)
ꢀ
11 ꢀ1
taneous hydrolysis at pH 8 (k
0
= 1 ꢂ 10
s
[21]) reaches the fac-
8
tor of 10 (Table 1) which is among the largest for all reported so
far in aqueous solutions (larger acceleration factors were observed
in methanol [22] and 80% vol. aqueous DMSO [23]; accelerations of
the order 10 –10 in water were reported for Ce(IV) ions [24] with
BNPP, Zr(IV) complexes with dimethyl phosphate [25] and Lu(III)
with diribonucleotides [26]). Among lanthanide complexes of
bis-Tris propane at similar pH and metal ion concentration the
Eu(III)
7.6
8
9
7
.2
8.0
8.4
8.8
ꢀ
5 ꢀ1
most active is Pr(III) with kobs = 8.0 ꢂ 10
s
[9] while the
pH
cyclodextrin Gd(III) complex shows a one order of magnitude
ꢀ
3 ꢀ1
Fig. 6. Observed first-order rate constants (sꢀ1) for the hydrolysis of HPNP in the
presence of 2 mM b-CD and 2 mM metal ion vs. pH.
higher activity. The kobs values up to 2.5 ꢂ 10
s
were observed
for lanthanide complexes of 2(4)-imidazole carboxylic acid [20a],
which are the most active lanthanide based catalysts for BNPP
hydrolysis, but at pH above 9. As one can see from Fig. 5 similar
activity can be reached with b-CD Nd(III) complex at pH below 9.
The general trend of increased activity for heavier and more
acidic lanthanides reflects larger degree of formation of hydroxo
complete release of 4-nitrophenol measured spectrophotometri-
cally. The phosphorylated b-CD shows a characteristic signal at
.2 ppm [6], but only signals of BNPP (ꢀ11.28 ppm), mono(4-nitro-
phenyl) phosphate (ꢀ0.58 ppm) and inorganic phosphate
2.41 ppm) were observed during the reaction course. No phospho-
3
a
complexes at chosen pH below pK values for all cations.
(
Considering results for HPNP we observe the catalytic activity
also similar to that reported for complexes of 2(4)-imidazole
carboxylic acid [20a] and even a little bit higher than that for a
rylated b-CD was detected also with HPNP as a substrate.
With BNPP as a substrate the slopes are similar for all cations
besides Dy(III). The slope for Dy(III) is 2.70 ± 0.05 and for all other
cations it equals 2.1 ± 0.1. With HPNP as a substrate the reaction
rate in the presence of La(III), Pr(III) and Nd(III) initially is propor-
tional to free hydroxide concentration (slope 1 in the logarithmic
coordinates), but then the reaction rate sharply increases and log
obs becomes a linear function of pH with the slope 2.5 ± 0.2 for
all three cations. For reactions in the presence of Eu(III), Gd(III)
and Dy(III) only the slope 2.5 ± 0.2 is observed (Fig. 6).
ꢀ
2 ꢀ1
binuclear macrocyclic Eu(III) complex (kobs = 2.5 ꢂ 10
s ;
estimated from results of [27] at 2 mM Eu(III) and pH 8.0), which
is the best lanthanide-based catalyst for this substrate. We observe
similar general trend in activity of different lanthanide cations
increasing from La(III) to Dy(III), but much lower catalytic effect
over the rate of spontaneous hydrolysis of HPNP as compared with
effect in BNPP hydrolysis (Table 1, last column). By itself HPNP is
k
4
ca. 10 times more reactive than BNPP (see footnote to Table 1),
Linear plots in Figs. 5 and 6 without ‘‘saturation” indicate a
small degree of formation of hydroxo complexes when their
concentrations are still proportional to the concentration of free
but rate constants of catalytic hydrolysis are on average only 10
times higher for HPNP for than for BNPP. Previously similar effect
was noticed for lanthanide complexes of 2(4)-imidazole carboxylic

1
4
M. Ruiz Kubli, A.K. Yatsimirsky / Inorganica Chimica Acta 440 (2016) 9–15
OH-
-
H+
H+
+
-
-
-
-
(
A)
(B)
6 4 2
Scheme 1. (R = C H NO -p).
[
[
36]. The EM values for inclusion complexes generally are not high
37], but even a modest EM close to unity will provide a 1000-fold
OH-
Mn+
rate acceleration with reactants in mM concentration range
employed in this study.
-
-
-
3
. Conclusions
Mn+
In conclusion, using b-CD as a stabilizing ligand for hydroxo
(
A)
(B)
complexes of trivalent lanthanides provides a very simple means
of obtaining highly active phosphodiesterase catalysts operating
in mildly basic nearly neutral aqueous solutions. In the presence
of 2 mM Gd(III)–b-CD complex at pH 8 the half-life of BNPP is
reduced from 2000 years to 10 min and that of HPNP from 48 days
to just 20 s. The rate-concentration and the rate-pH profiles indi-
cate the binuclear polyhydroxo structure of the active complexes.
The system suffers to some extent from the difficulty to obtain
its full characterization, but it is compensated by simplicity of gen-
erating highly active catalytic species.
Scheme 2. Mechanisms of catalysis for the cleavage of a DNA model phosphodi-
ester (A) and an RNA model phosphodiester (B).
acid [20a]. Also the efficiency of HPNP cleavage is much smaller
than that of BNPP with Zn(II) complexes of ligands derived from
bis-2-pyridinylmethylamine, which bear additional hydroxyl or
aromatic amino groups [28], but efficiency of catalysis by Zn(II)
complexes of a series of simple tridentate polyamino ligands is
approximately similar for both substrates [29].
The generally more efficient catalysis in BNPP reaction can be
explained considering the difference in mechanisms of BNPP and
HPNP hydrolyses. The hydrolysis of BNPP proceeds as a direct
nucleophilic attack of hydroxide anion on the phosphoryl group
4. Experimental
4.1. Materials
[
30], but the cleavage of HPNP is a transesterification reaction,
2-Hydroxypropyl 4-nitophenyl phosphate (HPNP) was prepared
as the barium salt according to the literature procedure [14]. All
other reactants were commercial samples obtained from Sigma–
Aldrich. Bis(4-nitrophenyl) phosphate (BNPP) was recrystallized
from ethanol–water. Cyclodextrins, Trizma base and reagent-grade
lanthanide(III) perchlorates as 40 wt% aqueous solutions were used
as supplied. Distilled and deionized water (Barnstead Nanopure
system) was used for preparation of all solutions.
which both as a spontaneous [31] and as a catalytic process [32]
involves equilibrium predissociation of hydroxyl group with sub-
sequent nucleophilic attack of the alkoxide anion on the phospho-
diester group (Scheme 1a,b).
This means that the coordinated hydroxide ion of the catalyti-
cally active hydroxocomplex may act as a nucleophile towards
bound BNPP (Scheme 2a) with increased reactivity due to two fac-
ꢀ
tors: the proximity effect since OH now acts intramolecularly [33]
and the electrophilic assistance of the metal ion to the phosphoryl
group. In the case of HPNP the actual active form of the catalyst is
the respective aqua complex [32], the nucleophile already is
intramolecular and the catalytic effect is only due to electrophilic
assistance of the metal ion to the phosphoryl group (Scheme 2b).
Evidently the overall acceleration effect should be lower in the case
of HPNP and a most viable way to improve the catalysis towards
this substrate as well as to RNA is to provide additional elec-
trophilic assistance to the nucleophilic attack on the phosphoryl
group [34].
4.2. NMR spectroscopy
1H NMR spectra were recorded on a Varian Gemini 200 NMR
2
spectrometer in D O in TRIS or MES 50 mM buffers at appropriate
pH.
4.3. Kinetics
The course of BNPP hydrolysis and transesterification of HPNP
was monitored spectrophotometrically by the appearance of
4-nitrophenolate anion at 404 nm. Kinetic measurements were
performed on a Hewlett–Packard 8453 diode array spectropho-
tometer equipped with a thermostatted cell compartment at
25.0 ± 0.1 °C. Reaction solutions were prepared by combining
appropriate amounts of the metal salt in 50 mM Tris/HCl buffer
and b-CD. Reactions were initiated by adding an aliquot of the
substrate stock solution to final concentration 50–100 lM. The
observed first-order rate constants (kobs) were calculated by the
integral method or, for slow reactions, from initial rates.
With HPNP as a substrate the initial portion of the kinetic curve
corresponding to 20–30% of hydrolysis was excluded to avoid
interference with more reactive isomer [15,16]. With BNPP as a
The type of catalytically active hydroxocomplexes of lan-
6
ꢀn
thanides stabilized by b-CD namely M
2
(OH)
n
is the same as that
with simple polyol or amino acid ligands, but the reactivity is con-
siderably higher. This can be attributed to ability of b-CD to form at
least weak inclusion complexes with the substrates affording tern-
ary metal ion–CD–phosphodiester complexes. Inclusion of a phe-
nyl group into b-CD cavity leaves sufficient free space inside the
cavity and formation of ternary complexes of b-CDs with aromatic
guests and e.g. alcohols is well documented [35]. Formation of a
ternary complex will convert a bimolecular catalytic reaction into
a much faster intramolecular process the relative efficiency of
which is usually expressed in terms of Effective Molarity (EM)

M. Ruiz Kubli, A.K. Yatsimirsky / Inorganica Chimica Acta 440 (2016) 9–15
15
substrate more complex two-exponential kinetics was observed
and fitted to the Eq. (2).
(e) Y.-H. Zhou, M. Zhao, Z.-W. Mao, L.-N. Ji, Chem. -Eur. J. 14 (2008) 7193;
(
f) M. Zhao, S.-S. Xuea, X.-Q. Jianga, L. Zheng, L.-N. Jia, Z.-W. Mao, J. Mol. Catal.
A: Chem. 396 (2015) 346.
ꢀ
k2t
Þeꢀk1tÞ=ðk
[8] P. Gomez-Tagle, A.K. Yatsimirsky, J. Chem. Soc., Dalton Trans. (2001) 2663.
A ¼
e
NP½BNPPꢁ f2 þ ðk
1
e
þ ðk
1
ꢀ 2k
2
2
ꢀ k
1
Þg
ð2Þ
0
[
9] P. Gomez-Tagle, A.K. Yatsimirsky, Inorg. Chem. 40 (2001) 3786.
10] (a) S.J. Oh, Y.S. Choi, S. Hwangbo, S.C. Bae, J.K. Ku, J.W. Park, Chem. Commun.
1998) 2189;
(b) K.N. Nicholson, S.A. Wood, J. Solution Chem. 31 (2002) 703.
[
where A is the absorbance,
ity of p-nitrophenolate and initial substrate concentration respec-
tively, k and k are the first and the second steps of BNPP
hydrolysis to the monoester and inorganic phosphate respectively
9]. Given in the text kobs values correspond to k
0
eNP and [BNPP] are the molar absorptiv-
(
[11] A. Munoz de la Pena, T.T. Ndou, J.B. Zung, K.L. Greene, D.H. Live, I.M. Warner, J.
Am. Chem. Soc. 113 (1991) 1572.
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15] N.H. Williams, Pers. Commun.
1
2
[
[
[
[
[
[
1
.
Acknowledgment
16] D.O. Corona-Martínez, P. Gomez-Tagle, A.K. Yatsimirsky, J. Org. Chem. 77
(
2012) 9110.
Financial support by Dirección General de Asuntos del Personal
Académico, Universidad Nacional Autónoma de México (project IN
[17] C.F. Baes Jr., R.E. Mesmer, The Hydrolysis of Cations, Wiley, New York, 1976.
[
18] C.M. Andolina, R.A. Mathews, J.R. Morrow, Helv. Chim. Acta 92 (2009) 2330.
19] R.M. Smith, A.E. Martell, Critical Stability Constants, vol. 4, Plenum Press, New
York, 1976. 1989, vol. 6.
[
2
14514) is gratefully acknowledged.
[
20] (a) F. Aguilar-Pérez, P. Gómez-Tagle, E. Collado-Fregoso, A.K. Yatsimirsky,
Inorg. Chem. 45 (2006) 9502;
Appendix A. Supplementary material
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b) F. Medrano, A. Calderón, A.K. Yatsimirsky, Chem. Commun. (2003) 1968.
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21] N.H. Williams, B. Takasaki, M. Wall, J. Chin, Acc. Chem. Res. 32 (1999) 485.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ica.2015.10.039.
[22] (a) J.S.W. Tsang, A.A. Neverov, R.S. Brown, J. Am. Chem. Soc. 125 (2003) 1559;
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