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SCHEME 1
SCHEME 2
Structures of biocidic phosphate esters and related compounds with lethal dosis
LD50; model compound BNPP with approximate half-life time in water,
20oC, pH 7.0.
Simple phosphate esters like BNPP (Scheme 2) are not only convenient
for measuring kinetics by liberation of the colored p-nitrophenolate, but are
also model compounds for DNA and in particular for other biocidic esters
Ln-CATALYSED HYDROLYSIS OF PHOSPHATE ESTERS 373
which are partially too dangerous to work with under usual laboratory
conditions. Nerve gases such as soman or VX (Scheme 2) belong to the
most frightening inventions of synthetic chemistry; for obvious reasons
substantial effort and funding has been devoted to improved detoxification
of such warfare agents. Other phosphate esters such as parathion and their
degradation mechanisms are still of importance in view of their use as
insecticides.
Many natural nucleases contain metal ions such as zinc, calcium or
magnesium. In the active center they can both activate the phosphate ester,
stabilize the transition state bearing an additional negative charge and or the
leaving group, as well as activate water, delivering e.g. good nucleophiles
such as hydroxo anions at neutral pH (see Section 2.2). Lanthanide ions
are in principle much better suited to serve all these features due to their
high charge density. In addition, they exhibit high ligand exchange rates
necessary for efficient turnover. One can in fact speculate that nature might
have been using lanthanides in hydrolytic enzymes instead of the usually
encountered cations, if their bioavailability would have been different. The
high charge density of lanthanides, which is even higher in cerium(IV) or
zirconium(IV) cations, can accelerate the hydrolysis of phosphate esters
such as of BNPP with enzyme-like saturation kinetics [9] and by factors of
106, even in the absence of any cofactors (see Section 4). The presence of
even simple organic ligands such as Bis-Tris propane [10] (see Section 4.4)
can lead to total acceleration factors of 108. The role of the metal ions will
be discussed in detail in Sections 2.2 and 6, and that of functional amino
acid analogs in Section 7. In this context we will also analyze the action of
di- and polynuclear catalysts (Section 6), and illustrate further expansions
with other reactants such as hydrogen peroxide (Section 5). The combina-
tion of lanthanides and surfactants has - in analogy to earlier approaches
based on transition metal cations [11] - already led to promising activities,
also with respect to possible applications in biomembranes (see Section 8).
It is noteworthy that lanthanide catalysis appears to be efficient enough
for some biochemical applications besides RNA or DNA cleavage. Thus,
trivalent lanthanides were used to hydrolyze the hemoglobin-bound 2,3-
diphosphoglycerate, which induces conformational changes of globin and
affects its affinity to oxygen [12].
374 SCHNEIDER AND YATSIMIRSKY
The application of lanthanides for hydrolytic cleavage has only in the last
decades gained impressive momentum. However, as often in science there
are quite early discoveries which tend to be overlooked until new methods,
new mechanistic insights and in particular new possible applications lead to
new activity in a field. Bamann et al. have reported as early as in 1938 [13]
on large accelerations of RNA-type glycerol phosphate and in preliminary
studies on nucleic acid hydrolyses with lanthanides, and even contributed
an often forgotten review on this [14]. Bamann, who was a pharmaceutical
chemist, attributed the unusual efficiency of lanthanides to their tendency
to form gels; decades later it was shown that gel formation or aggregation
in fact leads to a strong reactivity decrease [15,16]. Westheimer et al.
[17] reported in 1955 on the hydrolysis of methoxyethyl phosphate by
lanthanum hydroxide gels, and demonstrated P-O cleavage using isotopes
(see Section 2.1). Blewett and Watts [18] showed in 1971 that yttrium salts
increase the cleavage rate of 4-nitrophenyl methyl phosphonate by a factor
of 106. After some scattered later studies [19] several groups [6,7,9,20-24]
started after 1990 to apply lanthanides for phosphate ester scission with
increasing success; their and other contributions will be discussed in the
following sections, and also in another chapter of the present monograph
concentrating on sequence-selective scission of nucleic acids [6].
(1)
(2)
The first step has a very small equilibrium constant of the order of 10–14;
therefore this mechanism requires a very large rate constant of the order of
107 M–1s–1 for the next step. However, this rate constant should be similar
to that for the alkaline hydrolysis of trimethylphosphate, which equals only
0.03 M–1s–1; for this reason Mechanism (3) was excluded [35]. Recently,
however, it was argued on the basis of free-energy profile calculations
that trimethylphosphate may not be an adequate model for the monoester
MeOPO3H2, and that the latter may react with OH– much faster [36]. A
similar mechanism as (3) may also be considered for monoester dianions:
Again, the rate constant for the second step must be very large and
should be similar to that for the alkaline hydrolysis of the respective diester.
It has been shown with 2,4-dinitrophenyl phosphate as a substrate that the
Ln-CATALYSED HYDROLYSIS OF PHOSPHATE ESTERS 377
rate constant for the second step in Equation (4) must be equal to 3.2×107
M–1 min–1, but the rate constant for the alkaline hydrolysis of methyl 2,4-
dinitrophenyl phosphate anion equals only 0.028 M–1 min–1 [37]. Moreover,
with nucleophiles other than OH–, such as F– or nicotinamide, for which
rates of interactions with ROPO3H– and ROPO3Me– can be measured
directly, it has been shown that the rate constants for these two substrates
are always similar, indicating that a phosphodiester serves as a good model
for the reactivity of a monoester monoanion [37]. This observation renders
the existence of mechanisms like (3) or (4) rather improbable.
Nucleophilic substitution in di- and triesters follows the addition-
elimination mechanism (Eq. (5)) with formation of a pentacoordinate
intermediate (or transition state) in which entering and leaving groups
occupy the axial positions [25-28].
(5)
TABLE 1
Percentage of P-O Cleavage in Some Phosphodiesters
378 SCHNEIDER AND YATSIMIRSKY
The hydrolytic stability of phosphate esters is so high, that one of the major
problems in the field is still the characterization of catalytic efficiency by
reference to the uncatalyzed reaction. Also for this reason, efforts to esti-
mate such rates have been the subject of several recent papers, mostly on
the basis of extrapolations. Diesters of phosphoric acid, with exclusion
of cyclic esters [28,42] and β-hydroxy esters (see below), are the least
reactive among all phosphates. For diaryl phosphates (ArO)2PO2– the rate
of spontaneous hydrolysis was studied as a function of the leaving group
basicity [43]. A linear correlation (6) was found between the logarithm of
the first-order rate constant k and the pKa of the ArOH leaving group (k in
min–1 at 100°C):
log k = 1.57 – 0.97 pKa (6)
Considerable efforts were paid to measure the rate of hydrolysis of
dimethyl phosphate, which also serves as an estimate for the rate of
uncatalyzed DNA hydrolysis. Hydrolysis of (MeO)2PO2– under neutral
conditions involves only C-O bond cleavage (Table 1) with a rate
constant of 1.6 × 10–13 s–1 at 25°C (extrapolated by using the activation
enthalpy 25.9 kcal/mol) [41]. The alkaline hydrolysis of (MeO)2PO2–
involves primarily C-O bond cleavage and the second-order rate constant
extrapolated to 25°C, based on the activation energy of 28.2 kcal/mol,
equals 3×10–11 M–1s–1 [44]. The fraction of P-O cleavage was estimated
to be about 10% (Table 1). On this basis the rate constant for the alkaline
hydrolysis at the phosphorus may be estimated as 3×10–12 M–1s–1 at 25°C.
Assuming the alkaline hydrolysis to be dominant at pH 7, one obtains
Ln-CATALYSED HYDROLYSIS OF PHOSPHATE ESTERS 379
2 3
380 SCHNEIDER AND YATSIMIRSKY
TABLE 2
Rate Constants and Activation Parameters (ΔH*, kJ/mol and ΔS* J/mol K) for
the Hydrolysis of Some Phosphate Esters
(7)
SCHEME 3
A simplified scheme of processes occurring in basic solutions of a dinucleoside
phosphate (adopted from [56] with permission of the publisher).
382 SCHNEIDER AND YATSIMIRSKY
TABLE 3
Rate and Equilibrium Parameters for the Hydrolysis of
Uridyl (3’,5’)uridine [56]
SCHEME 4
SCHEME 5
SCHEME 6
Possible mechanisms of catalysis for dinuclear metal complexes [4c].
in cis position to OH– is typically slow for Co(III) complexes, but in this
case the rate of hydrolysis is slower than that of the water substitution.
Therefore, the ligand structure affects the intramolecular phosphodiester
hydrolysis which may be explained only by assuming a nucleophilic
mechanism [51]. Mechanisms “e” and “f” in Scheme 5 illustrate possible
contributions of the general base and general acid assistance, which
probably is of minor importance for phosphodiesters. It should be noted,
that dissociation of the phosphate from the Co(III) complex with either
cyclen or trpn after the ester cleavage is rather slow, leading to poor
turnover properties of these otherwise very active Co(III) complexes.
Special attention was given to dinuclear metal complexes (see Section
6). Possible mechanisms are shown in Scheme 6 [4c]. They involve double
Lewis acid activation (“a”) and combinations of Lewis acid activation
with nucleophile activation (“b”) or leaving group activation (“c”).
The same divalent cations, as well as Cu2+ and Al3+ form 1:1
complexes with the phosphonate 8, but do not lead to catalysis; however,
La3+ has a very strong catalytic effect on the hydrolysis of 8 [61]. In
contrast to La3+, other cations form chelates with both quinoline groups
of 8 in a conformation which prevents metal binding to the phosphoryl
oxygens. It should be noted that the use of a phosphonate as a model
substrate is justified since phosphodiesters and phosphonate esters have
similar rates of hydrolysis if the leaving groups are the same.
Currently available data for different metal cations show that highest
activities in phosphodiester hydrolysis are observed with “hard” strongly
acidic cations like Zr(IV) [63-65], Th(IV) [66], and Ce(IV) [67,68],
(although the activity for UO22+ is low [69]), which are also able to hydro-
lyze non-activated substrates including dimethyl phosphate [65,68]. Such
tendencies (see also Section 4.3) indicate that the major contribution to
catalysis is provided by the electrophilic Lewis acid assistance. The problem
in applications of such cations is their strong tendency to form aggregates at
neutral conditions in water, which makes it difficult to obtain homogenous
solutions and reproducible kinetics. In particular, cleavage experiments
with plasmid DNA are carried out under conditions (see Section 4.1) where
it becomes difficult to control the homogeneity of mixtures.
FIG. 1. Scheme of a proposed catalytic mechanism of 5’-nucleotidase; (a) Michaelis complex, (b) transition state and
(c) primary product complex. Reproduced with permission of the publisher from [72].
SCHNEIDER AND YATSIMIRSKY
Ln-CATALYSED HYDROLYSIS OF PHOSPHATE ESTERS 391
TABLE 4
(8)
TABLE 5
Acidity of Lanthanide Aqua Ions and Stability Constants of Lanthanide
Complexes with some Monoanions and Macrocyclic Ligands at 25°C and Ionic
Strength I Indicated for Each Ligand [83,92]
K ≈ 3), but the binding of other simple inorganic monoanions like nitrate
or chloride is weak (log K ≈ 0). Carboxylates form more stable complexes
(see data in Table 5 for AcO–). Also binding of H2PO4–, which may be con-
sidered as a model for a phosphodiester complexation, is rather strong for
a monoanion (Table 5). In spite of a monotonic decrease in the cation size
in the lanthanide series, trends in stability constants often are not mono-
tonic [82a], as is the case for acetate complexation (Table 5) [83,92]. This
is a general phenomenon for which no clear explanation exists, although
396 SCHNEIDER AND YATSIMIRSKY
SCHEME 7
Logarithms of stability constants of lanthanide complexes with ligands used as
biological buffers.
EDTA and analogs like DOTA (13) are known to form very stable
complexes with lanthanide ions, developed mostly as imaging agents for
NMR tomography. They have been used for sequence selective cleavage
of nucleic acids [99], but at the same time have also shown to suppress
almost completely the activity of e.g. Eu(III) ions against RNA [20b] so
that the danger of a radical process is increased. Cleavage of oligonucle-
otides was reported to be efficient with the Ce(IV) complex of EDTA,
whereas dinucleotides were not hydrolyzed [100]. Similar inactivation of
lanthanides was found for BNPP hydrolysis, also with glucaric or gluconic
acid [9]. The rate retardations are not unexpected, as all mechanisms dis-
cussed above imply that negative charges around the metal cation should be
counterproductive. Interestingly, electroneutral analogs of DOTA bearing
no carboxylic but instead amide or alcohol groups 14-16 have been shown
to provide suitable functions for lanthanide complexation, because of the
significant kinetic stability of the pre-synthesized complexes (typical half-
Ln-CATALYSED HYDROLYSIS OF PHOSPHATE ESTERS 399
lives for dissociation in water are about 10 days) [101]. On the other hand,
neutral lanthanide complexes like 17 were found to be good catalysts for
the transesterification reaction with ester 4a [102].
Complexation of Ce(IV) represents a special problem. Weakly
bound ligands like biological buffers do not prevent the precipitation
of a metal hydroxide. Often studies with this cation are carried out in
heterogeneous conditions with Ce(IV) in the form of gel or some poorly
characterized complexes. Among ligands, which were employed for
Ce(IV) complexation as a homogeneous catalyst are several saccharides
(ribose, xylose, dextran, sugar alcohols, cyclodextrins, etc.) [103], EDTA
[99], 18 [104], and 19 [105].
Yb(III) for the sulfur ligand, indicating that steric strain in the first coordi-
nation sphere is largest for the smallest cation and for sulfur binding sites
[108]. An ab initio quantum chemical study was reported on the interac-
tion of M(III) cations (La(III), Eu(III), Yb(III)) with model ligands L (L =
amide, urea, thioamide, and thiourea derivatives). Trends in binding ener-
gies for ML3+ (urea > thiourea > amide > thioamide) were found to differ
from those of calculated protonation energies (thiourea > urea > thioamide
> amide). Adding counterions or increasing the coordination number may
also modify the relative affinities. The calculations revealed a striking dif-
ference in the binding mode of sulfur compared to oxygen ligands, and the
role of steric repulsions in the first coordination sphere, due to counterions
and increased coordination number [109]. Other computational studies
were performed for complexes with neutral phosphoryl ligands [110] and
for calixarenes [111] as well as on cation solvation [112].
FIG. 4. Saturation kinetics for BNPP hydrolysis in the presence of Eu(III), at dif-
ferent temperatures. Reproduced with permission of the publisher from [127].
TABLE 6
Kinetic Parameters for the Hydrolysis of 4a by Free and Complexed
Lanthanide Ions [21a,114]. Parameters for Free Lanthanides are at
pH 6.85 and for Complexes at a pH Above pKa, All at 37°C.
FIG. 5. Gel from plasmid DNA hydrolysis, showing the supercoiled form RF I,
the open form RF II as first cleavage product, and the linear form RF III. Example
from hydrolysis with Eu(III) and a co-reactant with additional amino acids (see
Schemes 13 and Section 7). Lane 1 and 9 are controls (the starting material
always contains some RF II); lane 2: + His; lane 3: + Asp; lane 4: + Ser; lane 5:
+ His + Asp; lane 6: + Asp + Ser; lane 7: + His + Ser; lane 8: + His + Asp + Ser).
Reproduced with permission of the publisher from [121].
Ln-CATALYSED HYDROLYSIS OF PHOSPHATE ESTERS 403
[8]. One can try to check for radical cleavage by the absence of products
other than the open circular form RF II, and by negligible effect of
oxidants such as hydrogen peroxide, or of radical scavengers like DMSO
etc. [120]. However, absence of such effects does not necessarily prove
the absence of the very potent radical cleavage mechanism, which often
goes undetected in plasmid DNA experiments.
FIG. 6. Saturation kinetics with plasmid DNA in the presence of Eu(III) and
coreactants (see Section 7 and Figure 5). Reproduced with permission of the
publisher from [121].
Hydrolytic scission yields intact ends in the products; this is the basis
of religation experiments, in which the ends of the broken nucleic acids
are reconnected after cleavage with lanthanides [121]. Although the com-
pleteness of religation could not be established due to unknown stainabil-
ity factors, the results demonstrated that at least significant parts of the
scission must have left the nucleic acids undegraded. Similarly, fragments
obtained from the cleavage of a DNA-22mer were successfully religated
Ln-CATALYSED HYDROLYSIS OF PHOSPHATE ESTERS 405
with the help of three different enzymes, although the hydrolysis yields
mixtures of 3’ and 5’ phosphates and the corresponding hydroxy termini
[122].
dinucleotide TpT, which in the case of radical cleavage should yield the
sugar-free nucleobase thymine itself, or its degradation products – these
were not found in significant degree after cleavage with lanthanides
[121,123]. The evolution of the products can be followed by gradient
HPLC (Figure 7), which, however, is not a convenient way to follow the
kinetics. With the much more labile RNA-type of nucleotides like ApA
the HPLC analysis has also been applied, showing mostly hydrolytic
products, with a remarkable decrease of a half-life time from about 130
years to about 10 minutes [124]. Similar positive evidence for hydrolytic
cleavage has been obtained for the scission of longer RNA-type oligo-
nucleotides with lanthanides [21b].
The problem with competition by radical cleavage was studied
with Ce(III) ions: only in the presence of oxygen cleavage of a
dideoxydinucleotide was found, but no products expected for the radical
attack were detected [125]. The unexpectedly high activity of Ce(III) was
attributed therefore to formation of Ce(IV). Other lanthanide(III) cations,
however, cannot undergo oxidation as easily as Ce(III) salts, for which
reason oxidative cleavage with these is less likely.
Cleavage experiments aiming at RNA are often done with dinucleotides,
which not necessarily correspond to the situation with native longer
sequences. Special techniques were developed using chimeric DNA/RNA
entities, which contain RNA nucleotides embedded in DNA sequences
[126]. The method allows kinetic assays and comparison of RNA and
DNA cleavage; it has been used with Pb(II), Ce(III), and Cu(II) salts,
leading exclusively to RNA scission.
yielded linear Eyring plots in each case, from which activation parameters
could be determined (Table 7) [127]. The observed change of the param-
eters with increasing catalyst concentration shows that the rate enhance-
ment by the lanthanide ion is mostly due a lowered activation enthalpy
ΔH*, with a decreasing disadvantage by the entropic term TΔS* (see
Fig. 8). The KM values decrease with increasing temperature to a smaller
degree compared to the increase in kcat.
TABLE 7
Temperature Effects on the BNPP Hydrolysis with Eu(III)Cl3
(a) Michaelis-Menten KM and kcat Values at Different Temperaturesa) [127] and
(b) Activation Parameters for kcat, KM , and the Uncatalyzed Reactionb)
(a)
(b)
b) Errors in ΔH : ±15%; in ΔS and TΔS: ±20% on the average; values for the uncatalyzed
reaction from the literature (see Section 2.1.2).
408 SCHNEIDER AND YATSIMIRSKY
FIG. 8. Activation parameters for the hydrolysis of BNPP with Eu(III) chloride;
activation enthalpy (ΔH*, in kJ/mol) and entropy (as –TΔS*, in kJ/mol ) as func-
tion of catalyst concentration at 323 K. (filled circles: ΔΗ*, open circles: –TΔS*).
Reproduced with permission of the publisher from [127].
TABLE 8
Salt Effects on the Eu(III)-Catalyzed Hydrolysis of BNPP [15].
Conditions: 50ºC, pH 7.0 in 0.01 M EPPS-buffer, [Eu3+]=5×10-3M.
(a)
(b)
FIG. 10. Rate constants (log k) for the Eu(III)-catalyzed hydrolysis of BNPP as
function of solvent polarity (ET(30) parameters: EtOH-H2O, circles; DMSO-
H2O, squares). Reproduced with permission of the publisher from [127].
412 SCHNEIDER AND YATSIMIRSKY
As pointed out in Section 2.2 one expects a catalytic activity increase with
increasing charge density of the metal ion with respect to several factors:
the more efficient activation of water, of the phosphoryl group, and/or
the leaving group as well as transition state stabilization. Indeed higher
efficiency was reported not only for cations with four charges like Ce(IV)
and Zr(IV) (see Section 2.2), but also for Yb(III) in comparison to Eu(III)
salts [133]. The systematic examination with all lanthanide ions showed,
however, some quite unexpected trends [15]. These are partially connected
to the strong tendency of the higher lanthanide ions to aggregate in aqueous
solution above pH 6.0 [134]. On the other hand, gels from lanthanides were
also reported to be active in cleavage [123,125]; as mentioned above,
earlier they were even believed to be the main reason for the particular
activity of lanthanides in phosphate ester cleavage [13]. In contrast, one
finds a regular increase of the rate of the lanthanide-catalyzed reaction
with increasing charge density only from La(III) to Er(III), and then an
FIG. 11. Dependence of the BNPP hydrolysis kcat-values on the ionic radii of
lanthanides. Reproduced with permission of the publisher from [15].
Ln-CATALYSED HYDROLYSIS OF PHOSPHATE ESTERS 413
TABLE 9
Kinetic Paramaters for the Hydrolysis of BNPP from Saturation Kinetics with
Different Lanthanide Ions and Ionic Radii of the Ionsa) [15].
12). Increasing aggregation can make the ions less potent. Only at low
mM concentration a similar dependence of cleavage rates with DNA is
observed as with BNPP (Figure 13).
TABLE 10
Kinetic Parameters for the Cleavage of Plasmid DNA with Different
Lanthanides [15].
FIG. 12. Inverse catalyst concentration profile for the cleavage of plasmid DNA
with Lu3+. Reproduced with permission of the publisher from [15].
In line with results for BNPP hydrolysis (Table 9), rate constants
for the cleavage of 4a by free lanthanide cations at pH 6.85 also show
an increase on going from La to Tb and then a decrease for Yb and Lu,
although the variation in reactivity is much smaller than in the case of
BNPP [21a]. It was argued, however, that the increase in reactivity on
going from La to smaller cations at pH near 7 may reflect only the higher
degree of formation of the reactive hydroxide complexes for more acidic
cations of lanthanides with larger atomic numbers [5b]. Indeed, pH-
dependencies of kobs for the hydrolysis of ApA by different lanthanides
demonstrate that below pH ca. 7.5 log kobs increases linearly with increase
in pH. The order of reactivities is La < Nd < Tb < Lu, but above pH 8
416 SCHNEIDER AND YATSIMIRSKY
FIG. 13. Cleavage rate [%RF I] of plasmid DNA with Ln(III) ions vs. ionic
radius. Reproduced with permission of the publisher from [15].
FIG. 14. Observed first-order rate constants for the hydrolysis of BNPP at 25°C
and the species distribution diagram for 2 mM Pr(III) and 20 mM BTP. Repro-
duced with permission of the publisher from [89].
25 26
cations. Thus, the second-order rate constant for the cleavage of 4a by the
alkoxide complex Eu(15H–1)2+ equals 0.072 M–1s–1 at 37°C (Table 6),
but for the structurally similar hydroxide complex Eu(20)OH2+ it is k =
3.7 M–1s–1 at the same temperature [137]. On the other hand, with BNPP
as the substrate the alkoxide complex Eu(15H–1)2+ and the hydroxo form
of the Eu(III) complex with the [2.2.1] cryptand show probably similar
reactivity: k = 0.19 M–1s–1 at 37°C for the former [151] and ≈0.4 M–1s–1
at 50°C for the latter [98].
Complexes with macrocyclic aminoalcohol ligands like 15 and 16
were studied in detail mainly with the RNA model substrate 4a and RNA
[114,151,155]. Some kinetic parameters are given in Table 6. The pH-rate
profiles in all cases indicate that the reactive forms are deprotonated spe-
cies (Section 4.4), which may be either alkoxide or hydroxide anions. It
should be noted that one cannot identify the type of deprotonated species
from potentiometric titration data or from the pH-rate profiles because
they are rapidly equilibrating isomers of the same stoichiometry: M(OH–
)(ROH) and M(H2O)(RO–). An obvious criterion is the product analysis:
for alkoxide complexes one expects formation of the phosphorylated
ligand via the nucleophilic attack of the coordinated alkoxo anion on the
phosphoester; this was indeed observed for a number of lanthanide com-
plexes with aminoalcohol ligands [151,155]. However, product analysis
fails if the intermediate product undergoes hydrolysis as fast or faster than
the nucleophilic attack, and this was indeed shown with some carbox-
ylic acid esters [4a]. Of course, in the case of substrates like 4 and RNA,
which undergo intramolecular nucleophilic substitution, coordinated alk-
oxide should provide a general base assistance. The reaction mechanism
proposed for the cleavage of phosphate esters of both types by alkoxide
complexes is illustrated in Scheme 8 [114,155].
In an important study phosphorylation of the hydroxy group of the
ligand in the Cu(II) complex 27 by bis(2,4-dinitrophenyl) phosphate is
observed, although the deprotonation site is believed to be the coordinated
water [156]. It was proposed that in this case the reactive form is the minor
alkoxo isomer. Thus, the thermodynamically predominant deprotonation
type corresponds not necessarily to the kinetically active species.
It has been shown recently that La(III) methoxide complexes are
readily formed in methanol solutions of La(III) triflate in a pH range 7-11
Ln-CATALYSED HYDROLYSIS OF PHOSPHATE ESTERS 423
SCHEME 8
Reaction mechanism for phosphate ester cleavage by alkoxide complexes illus-
trated by Ln(15)3+ species [114,155].
27
424 SCHNEIDER AND YATSIMIRSKY
6.1. Di- and Polynuclear Models with Metal Ions Other than Ln(III)
SCHEME 9
substrate 4a, e.g., with two Zn(II) ions a rate enhancement which was
calculated for the fully bound substrate to be around 200 compared with
the uncatalyzed process.
Several studies have addressed the use of polynuclear complexes also with
lanthanides [185]. Thus, with dinuclear lanthanide complexes quite large
enhancements of up to f = 72 in comparison to the mononuclear analogs
were observed if the distance enforced by the macrocyclic ligands were
large enough (Scheme 10, structure (b) vs (a)) [148]. The additional rate
acceleration by the second metal ion was significantly larger for the Pr(III)
complexes than for the also investigated Eu(III) catalysts; this applied both
for the hydrolysis of BNPP and plasmid DNA. Another dinuclear complex
contains two La(III) ions bound to the ligand 23 and has been character-
ized also with respect to the pKa values of coordinated water: for the dinu-
clear complex pKa = 7.75, but for the mononuclear complex pKa = 11.15.
This decrease in pKa by 3.4 units implies that a water molecule is bridged
between the metal ions [149]. The reaction with BNPP is very fast and
exhibits a rare third-order dependence with respect to the metal complex
(see Section 4.4); the fourth-order rate constant for the dinuclear dihy-
droxo complex equals 4×108 M–3s–1. The rate acceleration with only one
metal ion is 75-fold less, which has been attributed also to the decreased
ability to form hydroxo complexes with the mononuclear species [149b].
The contribution of the phenolic OH groups in the ligand has not been
clarified, but may well accelerate phosphoryl transfer in the way discussed
in Sections 4.4. and 7. Also dinuclear La(III) complexes bearing several
bis(imidazol-2-yl) groups were with a rate enhancement of 190 much
more efficient than Ni(II) (rate factor 4.3) or, e.g., Co(II) (factor 7.1) [164].
Analysis of plasmid DNA cleavage products with dicerium complexes
26 with 5-methyl-2-hydroxy-1,3-xylene-α,α-diamine-N,N,N’,N’-tetraace-
tate 19 as ligand show one double-strand cleavage per ten single-strand
cleavages, implying that linear DNA (RF III) cannot be the result of two
random single-strand breaks. Remarkably, DNA restriction fragments are
hydrolyzed with high regioselectivity (more than 90% 5’-OPO3 and 3’-
Ln-CATALYSED HYDROLYSIS OF PHOSPHATE ESTERS 429
SCHEME 10
Some ligands and efficiency factors for dinuclear lanthanide catalysts (f = kdinucl/
kmononucl relative to the corresponding mononuclear complexes¸ with DNA : %
cleavage) [148].
30
31
SCHEME 11
Cooperation of a lanthanide macrocyclic complex with Zn(II) in the cleavage of
the 5’-cap structure [191a].
Virtually all enzymes use functional groups of amino acids in the active
center for the transfer of reactants, or, e.g., for stabilizing a transition
state by specific non-covalent interactions. In the context of hydrolases
the best known example is the catalytic triad of serine proteases, where
the carboxylic group of an aspartate eases transfer of protons to and from
the imidazole of histidine, and the hydroxy group of a serine acts as
nucleophile attacking the ester bond. As discussed in Section 2.3 similar
432 SCHNEIDER AND YATSIMIRSKY
SCHEME 12
Hydrolytic Zn(II) complexes with pendent hydroxylalkyl groups.
Ln-CATALYSED HYDROLYSIS OF PHOSPHATE ESTERS 433
higher than that for the hydroxy anion reaction itself. Such Zn(II) ane-
complexes show also promise with respect to sequence-selective RNA
cleavage [193]. In particular the hydrolysis of RNA has been found to be
significantly accelerated by lanthanide complexes in which hydroxyalkyl
groups are present [114,151].
Polyhydroxy compounds can also support lanthanide-catalyzed
hydrolysis if they are not part of a well defined metal complex but only
added as cofactors. With such vicinal polyols formation of a cyclic
phosphate intermediate can accelerate cleavage (Scheme 13) [118],
in analogy to the therefore more reactive RNA-type substrates (see
Sections 1.1 and 2.1.2). After transfer of the phosphoryl group to the first
neighboring hydroxy group it can transfer to the next and finally liberate
inorganic phosphate. Due to weak associations between metal ion and
cosubstrate the observed rate effects on plasmid DNA cleavage with such
polyhydroxy compounds are not spectacular, but remarkably dependent
on the chosen polyol and lanthanide ion (Scheme 13).
Most ligands used for metal complexation contain several nitrogen
atoms, which by themselves can also act as nucleophiles. This has been
observed early with azacrown ethers which catalyze, e.g., hydrolysis of
anhydrides such as ATP by intermediate phosphoryl transfer to a nitrogen
center [194,195]. Furthermore, the neutral amine groups can cooperatively
promote general base catalysis and the protonated ammonium functions
general acid catalysis [181].
Carboxylic groups in the vicinity of the phosphodiester bond pro-
mote cleavage in the presence, but not in the absence of Zn(II) ions
[196,197]. With the complex shown in Scheme 14 a distinct dependence
on the distance of the carboxylate from the metal center was observed for
the hydrolysis of BNPP, whereas the cleavage of plasmid DNA was not
affected [198]. As expected (see Section 3), the pendent carboxylic side
groups enhance the stability of the Eu(III) complex, however, surprisingly
independent of the spacer length. Under the conditions used for BNPP
hydrolysis the complexation degree is invariably around 80%. Neverthe-
less, only with the shorter chains a rate decrease as result of the metal
protection and unfavorable electrostatic interactions of neighboring nega-
tive charges is observed in rate retardation in comparison to catalysis with
Eu(III) alone.
434 SCHNEIDER AND YATSIMIRSKY
SCHEME 13
Imidazole is a frequently used ligand for binding the metal ion in natu-
ral and artificial hydrolases [2-4,199,200]. In metal-free serine proteases
histidine serves to abstract a proton from the serine hydroxy group, which
in metalloenzymes is brought about by, e.g., a zinc ion. Model studies have
given evidence of general base catalysis by imidazole and the role of both
Im and ImH+ in ribonucleases [201,202]. In the few artificial metallophos-
phatases studied until now the imidazolyl residue plays essentially the role
Ln-CATALYSED HYDROLYSIS OF PHOSPHATE ESTERS 435
SCHEME 14
SCHEME 15
Imidazole-containing cofactors [203].
SCHEME 16
Assay used for screening a combinatorial library [210].
Ln-CATALYSED HYDROLYSIS OF PHOSPHATE ESTERS 437
32
Among the lanthanides similar activities were observed with Sm, Eu,
Tm, Yb, and Lu, lower ones with Ce and much lower ones with La and
Pr. The optimum chain length n is 5 and the reaction rate increases on
increase in pH indicating that the hydroxo complexes are the active forms.
Variation of Lu(III) concentration at fixed ligand concentration shows a
maximum in the reaction rate at a ligand-to-metal ratio of 0.5. This was
interpreted in terms of a mechanism, which involves interaction of the
DNA phosphodiester bond with two lanthanide cations: one free aqua ion
providing electrophilic Lewis acid assistance and another one coordinated
to intercalated hydroxamate and bearing the hydroxo nucleophile. Another
438 SCHNEIDER AND YATSIMIRSKY
33
34 35 36
37 38
440 SCHNEIDER AND YATSIMIRSKY
TABLE 11
Selected Kinetic Parameters for BNPP Cleavage by Lanthanide and Some Other
Metal Complex Catalysts
rate law is often unknown we included in this table the observed first-order
rate constants at given conditions of pH, temperature and concentration,
which may be used for empirical comparison.
Lines 1-10 refer mainly to “free” (that is complexed only with buffer
components and/or OH anions) cations. As was mentioned in Section
2.2.1 tetravalent cations show the highest activities. The strongly elec-
trophilic Co(III) was studied only in form of polyamine complexes and
its reactivity depends significantly on the type of ligand (Scheme 5). The
highest activity was observed with the trpn (= tris(3-aminopropyl)amine)
ligand (Table 11, line 4) and even in this case Co(III) is less active than
tetravalent cations (the rate constant of about 10–2 s–1 is observed at higher
metal concentration and temperature). Activity of free lanthanide cations
varies strongly within the series (see Section 4.3). It is always lower than
that for the most active Co(III) complex, but much higher than that for
divalent cations. However, it should be borne in mind, that turnover of
the formed Co(III) phosphate complexes is rather slow. Neutral ligands
(line 11), do not change significantly the activity of lanthanides unless
they bear special functional groups (see Section 7). In contrast, neutral
ligands increase very significantly the reactivity of Cu(II) (lines 12 and
13). Any low reactivity of these complexes is in part due to their tendency
to form inactive hydroxo dimers [229]. However, the dimeric Cu(II)
hydroxide complex with an alicyclic aminoalcohol shown in Scheme 9c
has a very large activity (line 14), which is of the same order of magnitude
as activities of lanthanide complexes with other acyclic aminoalcohols
(lines 15 and 16). The active species in all these systems are dinuclear
hydroxide complexes stabilized by relatively weakly bound ligands,
which probably do not decrease significantly the metal electrophilicity,
but prevent efficiently formation of inactive polymers and precipitation
of metal hydroxides. Although copper complexes can for the hydrolysis
of BNPP and related esters well compete with the efficiency of lanthanide
systems, it should be borne in mind that only the latter are free from the
problem of radical cleavage of nucleic acids.
The entries in lines 17 and 18 allow to compare a lanthanide and non-
lanthanide macrocyclic complexes which use the alkoxide nucleophile.
Obviously, lanthanides are much more efficient. The last examples, which
also include ligands 39 and 40, refer to dinuclear hydroxide complexes of
Ln-CATALYSED HYDROLYSIS OF PHOSPHATE ESTERS 443
Zn(II) and Pr(III), which again clearly show the advantage of lanthanide
cations.
39 40
ligands also allow the covalent attachment of additional units, which can
be oligonucleotides for achieving selectivity, or other functions enhanc-
ing reactivity. The problem to avoid with highly reactive cations such as
Ce(IV) the overly fast but undesired radical cleavage may require efforts to
stabilize one particular oxidation state.
To place several metal centers and functional groups in the optimal
geometric disposition found in metalloenzymes calls for a perfect
preorganization of a host stucture which from its beginning is the hallmark
of supramolecular chemistry [232]. The synthetic effort towards such
complex structures, in which all functions should be ideally pre-oriented,
may look scary. However, recent investigations with host-guest complexes
having a systematically increasing number of single bonds indicate that
the disadvantage of connecting functions by simply introducing flexible
bonds is smaller than expected until now [233]. One has to remember that
there is significant mobility in the active center of enzymes [234]. It has
been postulated that maximum selectivity in enzymatic reactions requires
high flexibility in the active center, at the expense of possibly lowered rate
enhancements [235].
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