2003 LN - Cat Hydrol Phosph E Nucleic A in MIBS Ed Sigel

Ln-CATALYSED HYDROLYSIS OF PHOSPHATE ESTERS 369
11
Lanthanide-Catalysed Hydrolysis of Phosphate

Esters and Nucleic Acids
Hans-Jörg Schneider1 and Anatoly K. Yatsimirsky2
1FR Organische Chemie, Universität des Saarlandes,

D-66041 Saarbrücken, Germany
2Facultad de Química, Universidad Nacional Autónoma de México
04510 México D.F., México
1. INTRODUCTION AND SCOPE 370

1.1. Historical Perspectives 374
2. MECHANISMS OF PHOSPHATE ESTER HYDROLYSIS 374
2.1. Non-catalytic Hydrolysis 374
2.1.1. Mechanisms of Nucleophilic Substitution in
Phosphate Esters 374
2.1.2. Hydrolysis Rates and Half Life Times
under “Physiological” Conditions 378
2.2. Role of Metal Ions 383
2.2.1. General Considerations 383
2.2.2. Studies with Model Compounds 385
2.3. A Look at Natural Enzymes 388
3. FORMATION OF LANTHANIDE COMPLEXES
IN WATER AND THEIR CATALYTIC USE 394
369
370 SCHNEIDER AND YATSIMIRSKY
4. KINETICS OF CATALYTIC PHOSPHATE

ESTER HYDROLYSIS 400
4.1. Saturation Kinetics with Model Esters, Nucleic
Acids, and Oligonucleotides 400
4.2. Temperature, Salt, and Solvent Effects 406
4.3. Activity Variation within the Lanthanide Series 412
4.4. Complexes with Metal-Bound Hydroxy Anions and
Rate Equations Different from Saturation Kinetics 418
5. LANTHANIDE ALKOXIDE AND HYDROPEROXIDE
COMPLEXES 421
6. CATALYSIS WITH DI- AND POLYNUCLEAR
COMPLEXES 425
6.1. Di- and Polynuclear Models with Metal Ions Other
than Ln(III) 425
6.2. Dinuclear Lanthanide Complexes 428
7. ANALOGS OF FUNCTIONAL AMINO ACIDS IN
CATALYTIC CENTERS 431
8. OTHER LANTHANIDE-BASED SYSTEMS 438
9. A FINAL PERFORMANCE COMPARISON OF
LANTHANIDES WITH OTHER METAL IONS 440
10. CONCLUSIONS AND OUTLOOK 443
ACKNOWLEDGMENTS 444
ABBREVIATIONS 444
REFERENCES 446
1. INTRODUCTION AND SCOPE
Phosphate ester bonds belong to the kinetically most stable binding

units in nature. This is of fundamental importance for the stability of the
genetic machinery based on DNA, but also for the control of processes
like transcription involving RNA. Hydrolysis of dialkylesters like in DNA
(cf. Scheme 1) has under physiological conditions a half-life time which
can only be estimated to amount to about 200 million years. Even RNA-
type esters, which due to the presence of the 2’-hydroxy group as internal
nucleophile react much faster, have half-life times of about 100 years, like
the glycerol phosphate model (Scheme 1). Their scission is controlled by

enzymes with a performance which is still beyond that of artificial nucle-
ases. Several reviews are available both on the mechanisms of phosphate
ester cleavage [1] as well as on typical enzymatic pathways [2]. Chemists
have for a long time taken up the challenge to develop catalysts for the
hydrolysis of such extremely inert ester bonds. With some exceptions [3]
most artificial nucleases contain metal ions. Much is already known about
hydrolytic artificial metalloenzymes based mostly on transition metals [4];
the particularly effective lanthanide-based catalysts have after some early
attempts (see Section 1.2) only recently obtained much attention [5].
SCHEME 1
Important phosphate diesters in nature (B1, B2: nucleobases), with estimated

half-life times t1/2 under “physiological” conditions (t1/2 data after [4c]).
The development of artificial restriction enzymes is of particular

promise as the natural enzymes are rather limited with respect to their
sequence recognition ability. The combination of active catalytic sites with
sequence-selective longer oligonucleotides can lead to recognition and
cleavage of longer sequences with the now well established antisense or
antigene strategies. Of medicinal interest is that selective nucleases can in

principle target gene transcription, particularly in tumors. Sequence-selec-
tive chemical nucleases based on lanthanides are in the focus of another
review within the present volume [6] and will therefore not be dealt with
here in more detail [7]. They hold much promise for applications in bio-
technology, as the hydrolytic reaction products are chemically intact and
can be re-ligated to other units, in contrast to the longer known cleavage
reagents based on radical processes. Such cleavage reagents can work even
with extremely small catalyst concentrations, using redox active metal ions
like copper or iron ions [8]; they can, however, not really be called nucle-
ases, neither with respect to their mechanism nor to the products. They
produce sugar ends, which are degraded and therefore cannot be used for
other than analytical purposes such as footprinting. Another problem with
such systems is the often indiscriminate damage of cell components occur-
ring with highly reactive hydroxyl and other radicals.
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
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].
1.1. Historical Perspectives
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].
2. MECHANISMS OF PHOSPHATE ESTER HYDROLYSIS
2.1. Non-catalytic Hydrolysis
2.1.1. Mechanisms of Nucleophilic Substitution in Phosphate Esters
Several reviews are available on mechanisms of nucleophilic substitution

in phosphate esters [25-28]. The hydrolysis of phosphate esters may
involve either P-O or C-O cleavage, which can be discriminated by
appearance of the 18O label either in phosphate or in the alcohol product
if the reaction is carried out in 18O-enriched water [25]. In biological
systems enzyme-catalyzed substitution always involves P-O cleavage,
but non-catalytic hydrolysis may occur by considerable or even dominant

C-O cleavage.
Nucleophilic substitution in phosphate monoesters was considered
for a long time as a dissociative SN1-like mechanism, Eq. (1), with the
rate-determining elimination of a planar unstable metaphosphate anion
[25,29].
(1)
However, a purely dissociative mechanism is rather improbable [30],

in particular, because the rate of the cleavage of monoesters shows a weak,
but detectable dependence on the nucleophile basicity [31], which indi-
cates some degree of bond formation with the nucleophile in the transition
state. In addition, the observation of the inversion of configuration at the
phosphorus during the substitution in chiral monoesters of type 1 instead
of the racemization expected for the SN1 mechanism speaks in favor of

the associative mechanism in water [32]. In non-aqueous solvents the dis-
sociative mechanism seems to be more probable since the instability of
the metaphosphate monoanion in comparison to the dianionic phosphate
monoester is expected to be less pronounced in a non-polar medium.
Indeed, racemization was observed in organic solvents [33]. Recent high-
level quantum mechanical calculations of the reaction free-energy profiles
for phosphate monoester hydrolysis lead to the conclusion that the activa-
tion barriers for dissociative and associative pathways are similar [34],
underlining the difficulty of their discrimination.
Hydrolysis of monoanions of phosphate monoesters ROPO3H–

probably involves the intramolecular proton transfer to the leaving group,
as shown in Eq. (2):
(2)
Some special mechanism for the monoanion hydrolysis is required in

order to explain the much higher reactivity of monoanions than of struc-
turally similar phosphodiesters, e.g. the hydrolysis of the methylphos-
phate monoanion is much faster than that of dimethylphosphate [35]. A
strong argument in favor of Mechanism (2) is the much lower sensitivity
of the hydrolysis rate to the leaving group basicity than in case of dian-
ions (slopes of the plots of log k vs. pKa of the leaving alcohol are –1.23
and –0.27 for dianions and monoanions, respectively) [29]. An alterna-
tive mechanism involves fast reversible proton transfer from water to the
monoester anion followed by the nucleophilic attack of hydroxide on the
neutral monoester, Eq. (3).
H2O + ROPO3H– OH– + ROPO3H2 → ROH + H2PO4– (3)
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:
H2O + ROPO32– OH– + ROPO3H– → ROH + HPO42– (4)
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
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
Generally, triesters of phosphoric acid are hydrolyzed in basic solutions

faster than diesters and monoesters. Reactions of triesters with OH– always
involve P-O bond cleavage, but the reaction with water probably involves
C-O cleavage for trialkyl esters [25]. Some results illustrating the degree
of P-O cleavage in phosphodiesters with different nucleophiles are given
in Table 1 [38-41]. In general, hydrolysis involves predominantly P-O
cleavage for aryl esters, but C-O cleavage for alkyl esters.
2.1.2. Hydrolysis Rates and Half Life Times under “Physiological”

Conditions
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
the observed first-order rate constant 3×10–19 M–1s–1 in neutral solution.

An estimate from Equation (6) and a rather approximate extrapolation to
room temperature predicts the rate constant for water hydrolysis with P-O
cleavage to be 1.9×10–19 s–1 at 25°C, corresponding to a half-life time of
about 1011 (100 billion) years proposed as an estimate of DNA half-life
under “physiological” conditions [45]. For comparison, the also quite
stable peptide bonds have under similar conditions a half life time of about
300 years [46].
Recently the kinetics of the alkaline hydrolysis were studied with a
specially designed substrate 2 in which C-O bond cleavage is prevented
by steric effects [47]. Results obtained in the range 160-260°C give the
activation enthalpy 129 kJ/mol and the second-order rate constant of 10–15
M–1s–1 extrapolated to 25°C. The expected apparent first-order rate constant
for the hydrolysis of 2 at pH 7, assuming the base catalysis to be dominant
at this pH, is only 10–22 s–1. If this number is taken as an estimate of the rate
of spontaneous DNA hydrolysis, the efficiency of staphylococcal nuclease,
which has a turnover number of 95 s–1 (see Section 2.3) would be about
1024. However, the recently reported rate for the alkaline hydrolysis of the
dideoxynucleoside phosphate, thymidyl(3’,5’)thymidine (3) appeared to
be rather fast: the observed first-order rate constant was equal to 3.3×10–5
s–1 at 80°C and pH 14 [48]. This rate constant is even larger than that for
the hydrolysis of the activated substrate diphenyl phosphate under similar
conditions: 7×10–6 s–1 at 75°C in 1.0 M NaOH [49].
2 3
TABLE 2
Rate Constants and Activation Parameters (ΔH*, kJ/mol and ΔS* J/mol K) for
the Hydrolysis of Some Phosphate Esters
a Observed rate constant at pH 8.0.

a Observed rate constant at pH 8.0.
b Calculated from the results of [49] at 75 and 100°C.
c See text.
Rate constants and (when available) activation parameters for the

hydrolysis of several phosphate esters which are commonly used as
substrates in kinetic studies are collected in Table 2 [25,29,39,49-54].
A special type of phosphodiester substrates are β-hydroxyalkyl phos-
phate esters like 4 employed as models for dinucleoside phosphate, RNA
and glycerol phosphate hydrolysis, Eq. (7) [54,55]. Cyclization proceeds
faster than subsequent ring-opening for aryl esters, but slower for alkyl
esters. These reactions, which involve intramolecular nucleophilic attack
by the deprotonated β-hydroxyl group, are much faster than those with
phosphodiesters lacking the β-hydroxyl group. The rate constant for the
alkaline hydrolysis of 4a is given in Table 2.
(7)
The kinetics of hydrolysis of dinucleoside phosphates were studied in

much detail [1c,56]. A simplified scheme of processes occurring in basic
solutions of a dinucleoside phosphate is shown in Scheme 3, adopted from
[56]. It involves the interconversion of 3’,5’ and 2’,5’ isomers, deprot-
onation of a ribose hydroxyl and hydrolysis of deprotonated (dianionic)
and initial (monoanionic) species via formation of the relatively unstable
cyclic 2’,3’ monophosphate. Rate parameters for UpU as a substrate are
collected in Table 3.
SCHEME 3
A simplified scheme of processes occurring in basic solutions of a dinucleoside
phosphate (adopted from [56] with permission of the publisher).
In neutral solution the dominant route is the alkaline hydrolysis [56].

By using an estimate for kOH at 25°C (given in Table 3) one obtains at
pH 7 the observed first-order rate constant 7.3×10–13 s–1 and a half-life
of ca. 3×104 years, which drops to just 6 min in 1 M base when a ribose
hydroxyl becomes fully deprotonated (calculated from kg at 25°C, Table
3). The stability of RNA-type esters is thus much less than that of DNA
phosphate esters, but is also a considerable challenge for the development
of catalysts working under physiological conditions. It should be borne in
mind that hydrolysis of the polymeric phosphate esters present in double-
stranded DNA and RNA also requires additional steps, like unfolding of
the double helix.
TABLE 3
Rate and Equilibrium Parameters for the Hydrolysis of
Uridyl (3’,5’)uridine [56]
a Calculated from data at 333.2 and 363.2 K.

b Second-order rate constant for the alkaline hydrolysis
kOH = kgKa/Kw
2.2. Role of Metal Ions
2.2.1. General Considerations
General aspects of metal catalysis in phosphate ester hydrolysis are

discussed in several reviews [1a,4,57]. Scheme 4 illustrates the most
common types of mechanisms of catalysis.
Mechanism “a” involves metal coordination to the phosphoryl oxygen,
which leads to electrophilic Lewis acid activation of the substrate, i.e.
to an increase in the positive charge on the phosphorus, as well as to a
decreased charge repulsion with the nucleophile. Coordination to the
SCHEME 4
Common types of mechanisms of metal ion catalysis in phosphate

ester hydrolysis.
leaving group (mechanism “b”) is equivalent to a decrease in the leav-

ing group basicity. As one can see from Eq. (6), this effect may provide a
considerable acceleration of the hydrolysis. Activation of the nucleophile
(mechanism “c”), i.e. lowering pKa of its conjugated acid as a result of
coordination to the metal ion, increases the nucleophile concentration
in neutral solutions. With water as nucleophile this is often referred to
as water activation. Thus, the pKa value of the first water coordinated to
Co(III) in complexes shown in Scheme 5 is lowered by about 10 units

compared to free water molecules [58]. This mechanism, of course, can
cooperate with mechanisms “a” and “b”, as is illustrated in mechanism
“d”. With kinetically labile metal cations, like lanthanides, mechanisms
“a”-“d” are indistinguishable, but with kinetically inert Ir(III) [59] and
Co(III) [51,58] complexes one can easily discriminate between free
substrate, or substrate and nucleophile bound to metal. Such studies
show that mechanism “d” is the most effective one with the nucleophile
and the phosphate ester in cis positions. Interestingly, the rate constants
for the phosphodiester cleavage by di-aqua Co(III) complexes are very
sensitive to the structure of the polyamine ligand (Scheme 5). The low
reactivity of the bis(ethylenediamine) complex is explicable by its cis-
trans isomerization leading to formation of the unreactive trans isomer.
This isomerization does not occur, however, with rigid tetradentate ligand
complexes which nevertheless differ considerably in their reactivities.
Ligand substitution required to coordinate the substrate in place of water
SCHEME 5
Cleavage of BNPP by some Co(III) di-aqua complexes (0.01 M complex at

50°C, pH 7) [51].
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”).
2.2.2. Studies with Model Compounds
Kinetic studies with substrates bearing coordinating groups provide useful

mechanistic information for kinetically labile metal cations including
lanthanides [1a]. Thus, it was found that the intramolecular cleavage
of bis(8-hydroxyquinoline) phosphate 5 is catalyzed by divalent metal
cations (Ni2+, Co2+ and Zn2+) much more efficiently than by protonation
of the leaving group [60]. This was attributed to the ability of cations
to stabilize the leaving hydroxyquinolinate group by chelation to the
departing oxygen, as shown with 6, while the protonation of the nitrogen
atom of the leaving group (7) has only a smaller inductive effect.
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.
Hydrolysis in the presence of La3+ can be first-, second- and third-

order in metal [61]. Structure 9 illustrates the binding mode proposed for
the complex involving two metal cations. Here one cation is bound to the
phosphoryl group and to the alcohol leaving group, providing Lewis acid
and leaving group activation; the other cation provides an additional Lewis
acid and nucleophile activation. Coordinated water has a low pKa value
of 7.19. Thus, already at pH 8 it becomes nearly fully deprotonated and
the resulting OH– anion occupies the required axial position with respect
to the leaving group. The first-order rate constant for the hydrolysis of the
hydroxide complex (8)La2(OH)2+ equals 1.36×10–3 s–1 at 30°C, which
represents an acceleration factor of 1013 over the rate of spontaneous
hydrolysis of 8 at pH 8. The features of La3+ which explain its ability to

form complexes like 9 are its large ligation bond lengths of ca. 2.5 Å, the
nonrigid stereochemistry of ligation, and a high coordination number of
over 8. The unusual third-order in the metal reaction is interpreted as the
interaction of 9 with La(OH)2+ with the second-order rate constant 0.262
M–1s–1, as illustrated in the structure 10.
The phosphate diesters 11 have been used in modeling the metal ion
catalysis of RNA cleavage [62]. All of the metal ions tested promoted
intramolecular transesterification of 11, but with quite different rate
enhancements: 105 with Zn2+, 103 with Mg2+, 105 with Cu2+, and 109 with
La3+. Molecular modeling of the 1:1 complexes of 11 with these metal
ions shows that only La3+ can simultaneously interact with both negative
oxygens of O=P–O– and, also, with the oxygen of the leaving group due
to the greater La-O bond length and a lack of directionality of binding by
La3+ [1a].
Although other lanthanides were not studied with these substrates,

conclusions from the studies described above certainly apply to lanthanide
catalysis in general. One important point is that in contrast to d-transition
metal complexes where a significant contribution of covalent binding
imparts strong directional characteristics and produces a well-defined
geometry such as square planar or octahedral, lanthanides have nonrigid
stereochemistry of ligation and high coordination numbers. The flexibility
of the coordination sphere of lanthanides allows them to bind strongly
both to the ground and to transition states in catalysis [1a].
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.
2.3. A Look at Natural Enzymes
Many enzymes which catalyze the hydrolysis of phosphate esters are

activated by metal ions [2]. Often two or more metal ions are required for
the enzyme activity, although in some cases the presence of multiple metal
binding sites inferred from the crystal structures is an artifact resulting
from the use of the high salt concentration to soak the crystals, and the
use of Mn2+ or Co2+ instead of Mg2+ to identify metal binding sites [2a,b].
Another problem can be the distinction of functional and structural metal
ions, the latter not participating in the catalytic cycle but contributing
to the special protein conformation. There are also many enzymes, e.g.
staphylococcal nuclease (see below), which are activated by only one
metal cation. Enzymes for the hydrolysis of phosphodiesters possessing
the β-hydroxyl group (RNA [1c,2e] and of phosphatidylinositols [70])
often do not require metal cations at all.
Mechanisms of hydrolysis by two-metal active centers has attracted
much attention [2b,d]. Typically two metal ions are separated by a dis-
tance of 3-4 Å and provide Lewis acid, leaving group and nucleophile
activation, as well as enhancement of the affinity of the enzyme to an
anionic substrate. Metals which participate in such centers are most often
Zn and Mg, and also Fe, Mn, Co, and Ni. The hard magnesium ion with
the high charge density (Zi/ri) of 3.0 A–1 is particularly suited; Zn(II) ion
has a lower density of 2.7 A–1, yet it still is a stronger Lewis acid than
Mg(II) [2b,4f]. In most hydrolytic metalloenzymes the metal ions are

bridged by carboxylic groups from corresponding amino acid residues,
or by water or hydroxyl anions, respectively. In di- or polynuclear metal
enzymes the coordination number can also vary. Spectroscopic studies
of a ternary complex between the acid phosphatase uteroferrin substrate
and fluoride suggest that substrate binding leads to a shift of a hydroxide
bridge towards one of the metal ions, which then would be a good nucleo-
phile for an in-line backside attack at the phosphorous center [71].
A recent example for the mechanism of hydrolysis of a phosphate
monoester by the dimetal center which illustrates typical aspects of such
mechanisms is shown in Fig. 1 [72]. E. coli 5’-nucleotidase is a zinc-con-
taining enzyme, which belongs to a large superfamily of dinuclear metallo-
phosphatases including protein phosphatases and purple acid phosphatase.
The enzyme catalyzes the hydrolysis of uridine diphosphate glucose to
uridine, glucoso 1-phosphate and phosphate as well as of ATP to ADP,
AMP and adenosine. Crystal structures of the enzyme and its complexes
with the substrate analog inhibitor α,β-methylene-ADP and with the
reaction products, adenosine and phosphate, were obtained. The dimetal
center contains two metal cations separated by a distance of ca. 3.5 Å with
a bridging water molecule (or a OH– anion) and the carboxylate group of
aspartic acid (the structure was obtained for the Mn2 form). The model of
the Michaelis complex (Fig.1a) is proposed on the basis of the structure
of the enzyme-inhibitor complex. The (Zn-1)-bound water or hydroxide is
perfectly positioned for a nucleophilic attack from the side opposite to the
leaving group, and the phosphoryl group is activated electrophilically by
coordination to the second metal ion (Zn-2) and by electrostatic binding to
His117 and Arg410. The transition state, Fig.1b, is stabilized by binding to
both metal cations and to Asn116, Arg410, and His117. The model of the
product complex, Fig.1c, is based on the structure of the enzyme complex
with phosphate and adenosine.
The most thoroughly studied enzyme of this class is alkaline
phosphatase from E. coli [2d]. It utilizes the serine hydroxyl group instead
of water as a nucleophile. The distance between two zinc ions of the active
site (Fig. 2a) is 4.1 Å and a phosphomonoester substrate is coordinated
to both zinc ions (Fig. 2b) in such a way that Zn-1 activates the leaving
alcohol group and Zn-2 facilitates the deprotonation of the serine hydroxyl
390
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
group [2d]. An additional electrophilic activation is provided by Arg166.

After the phosphorylated enzyme is formed (Fig. 2d), Zn-1 coordinates a
water molecule and activates it by deprotonation (Fig. 2,e). Then the same
steps are repeated, but with hydroxide instead of the alkoxide nucleophile
leading to the final hydrolysis of a phosphomonoester.
The structural similarity of the active sites of two-metal enzymes
which catalyze the hydrolysis of very different substrates, including
phosphate mono-, di- and triesters, as well as peptides [2d,f] suggests
the possibility that a single active site may be able to catalyze the
hydrolysis of all of these substrates [73]. Indeed, it was demonstrated
[73] that a phosphomonoesterase alkaline phosphatase has a detectable
phosphodiesterase activity with a non-specific BNPP substrate (Table
4). In line with this observation, it was shown that dimetal derivatives
of a di-Zn aminopeptidase from Streptomyces griseus exhibit rather high
phosphodiesterase activities towards BNPP [74] (Table 4). It should be
noted, however, that although the catalytic activities of these systems
surpass those of many synthetic metal complexes, they are far below
activities reported for true phosphodiesterase enzymes [75] (Table 4).
TABLE 4
Kinetic Parameters for BNPP Hydrolysis by Some Biological Catalysts
a) Estimated from reported Vmax = 24 μmol min–1 mg–1, assuming the

molecular weight of the enzyme to be 108000.
Staphylococcal nuclease is an example of a single-metal phospho-

diesterase. For this enzyme extensive structural, kinetic, spectroscopic
and mutational data are available [76-80]. Staphylococcal nuclease can
cleave either DNA or RNA at the 5’-phosphate bond to produce 3’-
phosphomononucleosides. Studies with synthetic substrates show that the
absolute structural requirement is the presence of a nucleotide fragment,
FIG. 2. Mechanism of the hydrolysis of a phosphomonoester by alkaline phos-

phatase. Reproduced with permission of the publisher from [2d].
(8)
which however needs not to be part of a polynucleotide chain; simple

phosphodiesters like BNPP are not hydrolyzed by this enzyme [79a]. The
specificity for 5’-phosphate bond cleavage is so high that the enzymatic
hydrolysis of 12 proceeds only at this bond (Eq.(8)), without any detect-
able cleavage of the much more labile para-nitrophenyl phosphate bond
[77].
Staphylococcal nuclease requires for its activity only one Ca2+ cation.
Kinetic parameters with denatured calf thymus DNA as a substrate at
saturating Ca2+ concentration, pH 7.4 and 23.5°C are kcat = 95 s–1 and
FIG. 3. Mechanism of staphylococcal nuclease. Adapted with permission of the

publisher from [80c].
KM = 3.5 μg/mL [80a,b]. An enzyme in which Ca2+ is substituted by Sr2+

retains 60% of activity with DNA as substrate, but substitution with other
alkaline earth metals as well as with lanthanides yields a completely inac-
tive enzyme [80b]. Among transition metal cations only with Cu2+ and
Fe2+ some activity was retained (16% and 6%, respectively) [80b].
The mechanism of hydrolysis by staphylococcal nuclease is shown
schematically in Fig. 3. Water as nucleophile is coordinated to Ca2+ and is
further activated by hydrogen bonding to the carboxylate group of Glu43.
In the transition state phosphoryl and leaving group oxygens are stabilized
by Ca2+ and by two cationic arginine residues. Evidently, similar factors
operate in catalysis by two-metal and single-metal enzymes. This
conclusion is also supported by computer modeling studies [81].
3. FORMATION OF LANTHANIDE COMPLEXES IN WATER

AND THEIR CATALYTIC USE
The development of thermodynamically and even kinetically stable ligands

is crucial for many possible applications. If one wants to apply lanthanides
e.g. for antigen or antisense strategies, and generally for sequence selec-
tive cleavage of nucleic acids, suitable ligands must be available, which
at the same time do at least not lower significantly the catalytic efficiency.
Many of the lanthanide complexes described in this section were equipped
with additional binding units such as oligonucleotides; this application is
discussed in detail in Chapter 12 of the present volume.
Coordination properties of lanthanide cations are discussed in several
reviews [82]. Cations of trivalent lanthanides behave as “hard” Lewis
acids with predominantly electrostatic non-directional ligand binding.
Consequently, they have higher affinities to “hard” ligands with oxygen
donor atoms and to negatively charged groups. High coordination
numbers up to 12 are typical for lanthanide complexes. In the series of
lanthanides the ionic radius decreases with increasing atomic numbers. It
should be noted, however, that a significant dependence of ionic radii on
the coordination number, which often is unknown and may be variable,
renders correlations with lanthanide ionic radii rather uncertain [82c].
Lanthanide cations in water have no detectable affinity to ammonia
and acyclic polyamine ligands. They bind strongly “hard” F– anions (log
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
it was proposed that it may be related to a complex pattern of dehydration

of cations with different size upon complexation [82a].
Acidity of lanthanide aqua ions increases on going to heavier cations.
From the pKa values given in Table 5 one can see that a significant
formation of metal hydroxide complexes occurs at pH above 7. The
hydroxide complexes tend to aggregate affording di- and polynuclear
species such as M2(OH)24+ and M5(OH)96+ [84]. Formation of a dinuclear
complex La2(OH)5+ was reported in basic diluted (2 mM) solution of LaCl3
[85]. The very acidic cation Ce4+ (pKa = 0.7 [83]) is strongly hydrolyzed
already in acid solutions, affording polymeric species and hydroxo gels in
neutral solutions; this can make it difficult to obtain reproducible results.
Weakly complexing “biological buffers” often employed in studies
with lanthanides form detectable complexes (Scheme 7), which are espe-
cially stable with Bis-Tris. For the latter ligand the crystal structure of the
[La(Bis-Tris)2]Cl3 complex was obtained, which indicated coordination of
La(III) with nitrogen and four hydroxyl groups of each ligand molecule
[86]. Surprisingly, stability constants for complexation of lanthanides from
La(III) to Eu(III) with sulfonate buffers (MES, MOPSO, ACES, HEPES)
appeared to be practically the same for all metals and ligands studied
(Scheme 7) [86-90]. Similar stability constants were reported for amino
acids as ligands (see e.g. data for glycine in Table 5).
Additions of a base to solutions of lanthanides containing such weakly
coordinating ligands often leads to formation of polynuclear mixed
hydroxide complexes, some of which were proven to have high phos-
phoesterolytic activities [86-89]. Potentiometric titrations of lanthanides
(La, Pr, Nd, Eu, Gd and Dy) in the presence of Bis-Tris Propane (BTP)
revealed formation of a series of dinuclear complexes M2L2(OH)n6–n
where n = 2, 4, 5 or 6 in the pH range 7-9 [89]. Formation of similar com-
plexes of slightly different stoichiometry (M2L(OH)n6–n) was observed
for Y(III) [88]. Dimeric mixed hydroxide complexes M2L2OH2 (M = La,
Ce, Pr or Eu, L = MES, MOPSO, ACES, HEPES, AMP, CMP or GMP)
were reported [90]. Interesting tetranuclear complexes with a cubane-like
[Ln4(μ3-OH)4]8+ core stabilized by amino acids were isolated and charac-
terized by X-ray structure determination [91].
SCHEME 7
Logarithms of stability constants of lanthanide complexes with ligands used as
biological buffers.
Although lanthanides do not form complexes with acyclic polyamines

in water, nitrogen donor atoms do coordinate to lanthanides when they are
part of a polydentate ligand, e.g. glycinate complexes are substantially
more stable than acetate complexes (Table 5). With macrocyclic ligands
binding of lanthanides to neutral nitrogen is stronger than to neutral
polyether oxygen [82c,d]. Thus, 18-crown-6 does not form detectable
lanthanide complexes in water, but [18]aneN6 forms very stable
lanthanide complexes [92] (Table 5). For many macrocyclic complexes
the crystal structures were determined [82c,d]. They are often used
to interpret their solution behavior, although only in one case for the
lanthanum(III) complex with 1,9-diaza-18-crown-6 the NMR spectra
established the identity of solution and solid state structures [93]. In the
solid state structure the La cation is coordinated by four oxygens and
two nitrogens of the ligand and by six oxygens of three bidentate nitrate
groups, exhibiting a total coordination number of twelve.
Obvious ligands for neutral stable lanthanide complexes in water
seem to be available in the form of cryptands. The [2.2.2] and [2.2.1]
cryptands bind lanthanides in water with stability constants around 106

M–1 with practically no selectivity [94] (Table 5). An X-ray analysis of the
[2.2.2]-Eu(III) complex shows the metal ion in the center of the cavity of
the cryptand [95]. The catalytic activity of the europium(III) complex with
the [2.2.2]cryptand was reported to be almost the same as that of the “free”
cation [9]. However, later NMR investigations [96] showed that the metal
ion moves out of the cavity rapidly after dissolution in water. The complex
between the smaller [2.2.1] cryptand and Eu(III) does not disintegrate in
water, and still retains its full activity against BNPP, however not against
plasmid DNA [97]. The crystal structure of the complex of LaCl3 with
[2.2.1]cryptand shows that the metal ion is nine-coordinated and forms a
bowl-shaped structure with two coordinated chlorides located on the open
face with a cis geometry [98].
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-
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].
Considerable progress has been reached in molecular modeling of

f-block metal complexes [106]. A consistent set of Lennard-Jones param-
eters for La3+, Eu3+ and Lu3+ cations has been derived from molecular
dynamics and free energy calculations. It reproduces quite well experimen-
tal differences in hydration free energies, lanthanide-water distances and
coordination numbers in water. The calculated binding selectivities nicely
reproduce experimental trends in relative stabilities with EDTA4– in the
cation series: Lu(III) > Eu(III) > La(III) [107]. On the basis of quantum-
mechanical calculations on lanthanide complexes in a series of complexes
(M = La, Eu, and Yb) with negatively charged phosphoryl ligands L– =
(MeO)2PO2– and Me2PS2– the binding energies follow the order Yb(III) >
Eu(III) > La(III) for a given ligand, and (MeO)2PO2– > Me2PS2– for a given
cation. However, adding a neutral LH ligand to [ML3] changes the order
to Eu(III) > Yb(III) > La(III) for the oxygen ligand and La(III) > Eu(IIII) >
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].
4. KINETICS OF CATALYTIC PHOSPHATE ESTER

HYDROLYSIS
4.1. Saturation Kinetics with Model Esters, Nucleic Acids and
Oligonucleotides
Model compounds like structures 5 or 11 in which a metal-binding unit

is part of the phosphate ester substrate itself offer the advantage of better
defined geometric arrangements and also of large rate accelerations, but
can of course not show enzyme-like turnover. As a first step in analyzing
the catalytic performance of metal complexes one usually studies the rate
dependence as function of the catalyst concentration, which is also of
practical importance for any application. Since the activity of artificial
catalysts is usually beyond that of enzymes, one cannot neglect the
uncatalyzed reaction and use the catalyst and not the substrate in excess.
As illustrated in Figure 4, one observes, e.g., with Eu(III) salts and BNPP
as substrate a Michaelis-Menten-like saturation profile, which yields at
50oC kcat = 2.6×10–4 s–1, corresponding to an acceleration factor of about
106, and a dissociation constant for the catalyst-substrate complex KM
= 2.9×10–3 M [9]; a value which compares favorably with pK values of
lanthanides and phosphate around 3 [113] (at higher ionic strength like 0.2
M the log K is in the range of 1.5-2, see data in Table 5). The underlying
product-time profiles at fixed catalyst concentration always show good

first order kinetics.
It is often argued that hydrolysis rates with activated nitrophenyl esters
may be not typical for alkyl esters, due to a possible change in mechanism.
For this reason the rates were also measured with the diphenyl ester
analog of BNPP, which in spite of the absence of activating nitro groups
showed even a slightly higher rate enhancement [9]. Turnover without
consumption of the catalyst was demonstrated by experiments using
similar concentrations of substrate and catalyst.
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].
Michaelis-Menten type saturation kinetics was observed also for the

cleavage of the RNA model substrate 4a in the presence of both free lan-
thanide cations and their macrocyclic complexes (Table 6) [21a,114]. The
dissociation constants for the catalyst-substrate complexes are larger for
this substrate than for BNPP (cf. Table 9 in Section 4.3). One can also see
from these results that binding of lanthanide cations to neutral macrocy-
clic ligands does not change their affinities to the substrate.
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].
Cleavage of natural nucleic acids is usually followed by plasmid

DNA nicking experiments, based on the different electrophoretic mobility
of native supercoiled DNA (the so-called form RF I) and open circular
DNA (called RF II); further cleavage leads to a linear form RF III [115];
a corresponding gel is shown in Figure 5. Exact kinetic analyses are
more difficult here than with model esters. One needs exact control of
concentration, temperature and homogeneity in non-transparent micro-
tubes, as well as reliable hard- and software for the densitometry of the
electropherograms. The RF I, RF II, and RF III forms are quantified by
adding dyes such as ethidium bromide, and one needs to account for the
different stainability of the two forms [116]. Care has to be taken also for
the absence of nucleases by proper sterilization. Another problem can be
interference of radical cleavage, yielding the same product RF II, although
one observes then often further cleavage products, such as the linear RF
III arising from a double strand break, and more typically smaller pieces
characterized by smearing on the gels. In the presence of additional
complexing ligands (see sections below) like polyamines the mobility
of the DNA suffers too much, and special ion exchange techniques have
to be used to remove them before electrophoresis [117]. Using such
precautions, clean pseudo-first-order kinetics are observed with europium
chloride in large excess over the plasmid DNA at neutral pH at usually
37oC; with such rate constants saturation curves can be derived (Figure
6), yielding kcat = 0.6×10–3 s–1, and KM = 0.58×10–4 M [118]. The KM
value is in line with the formation of several salt bridges to neighboring
groove phosphates. A larger KM value of 5.9×10–4 M was reported for TpT
cleavage by Ce(IV) at pH 2 [123]. The cleavage velocity at 37oC seems
to be high for a dialkylester as present in DNA, but it must be borne in
mind that it refers to the cleavage of one single out of about 2000 ester
bonds present in plasmid DNA, which is sufficient to convert it from the
supercoiled to the open circular form. Cleavage of RNA sequences usually
is investigated either with short di- or oligonucleotides and HPLC analysis
(see below), or technically more demanding electrophoretic scission
product identification with 32P-labeled longer sequences like, e.g., 36-
mers (see e.g. [119]).
Traces of redox systems are sufficient to cleave supercoiled DNA¸ the
sugar moieties are destroyed by these earlier so-called chemical nucleases
[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
with the help of three different enzymes, although the hydrolysis yields
mixtures of 3’ and 5’ phosphates and the corresponding hydroxy termini
[122].
FIG. 7. HPLC chromatograms of TpT hydrolysis with 5 mM Eu(III)Cl3 at 70oC,

pH 7.0 in 0.01 M EPPS buffer; incubation times from 0, 2, 4, 7, 10, 12 and 14
days. Whole chromatograms (top) and enlarged cutouts (bottom). The compounds
are from left to right as follows: isonicotinic acid (reference); 5’-TMP; 3’-TMP;
3’,5’cTMP; thymidine; TpT. Reproduced with permission of the publisher from
[121].
Another way to establish a hydrolytic pathway is to identify the

cleavage products from oligonucleotides. This was done with, e.g., the
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.
4.2. Temperature, Salt, and Solvent Effects
Temperature and solvent effects on hydrolysis rates are of obvious practi-

cal importance, as one is often forced to use different reaction conditions
for cleavage of the extremely stable esters. Ligands which are less water
soluble often need admixture of organic solvents. Clearly, native DNA
or RNA is not stable enough to cover a desirable large range of reaction
conditions without side effects; in contrast, the hydrolysis of model esters
such as BNPP poses no problems. Variation of reaction temperature from
303 to 343 K at Eu(III) chloride concentrations from 0.4 mM to 10.0 mM
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)
a) From saturation curves; R: linear regression

coefficient; kcat in (s–1)×105 ; KM in ×103 M units
(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).
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].
One can also obtain saturation profiles at different temperatures from

the same data, and from these the ΔH and TΔS parameters (Figure 4, Table
7 [127]), derived with the Eyring equation for kcat and with the van t’Hoff
equation for KM. Comparison to the uncatalyzed reaction is difficult due
to the quite different reaction mechanism; it is, however, obvious that the
major advantage of the lanthanide catalysis is due to the enthalpic factor
for both the transition state and the catalyst-substrate complex, counter-
balanced to a large degree by a TΔS disadvantage. In contrast, the activa-
tion enthalpy of the uncatalyzed reaction of secondary phosphate esters
amounts to up to 130 kJ/mol [128] (for other values see Table 2), the
activation entropy is also quite negative, other than in monoesters [129].
Salt effects must also be considered in view of often used buffer sys-
tems, they also can shed light on the catalytic mechanism. As expected,
monovalent salts such as NaCl have less influence on the hydrolysis rates
of BNPP with Eu(III) ions than, e.g., MgCl2 (Table 8 [15]). The effects
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.
can be correlated with the ionic strength I by the extended Debye-Hückel

equation (Figure 9a), with rather similar slopes. These are in the range
expected for ion pair competition [130], and in line with the necessary
association between the lanthanide cation and the phosphate anion. Some
(a)
(b)
FIG. 9. Debye-Hückel correlation of salt effects on (a) the Eu(III)-catalyzed

BNPP hydrolysis rate constants, and (b) plasmid DNA cleavage;  : Na+,
 : Mg2+). Reproduced with permission of the publisher from [15].
polyamine ligands can lead to accelerated hydrolysis rates with transition

metal ions [131]; the opposite is seen with, e.g., the effect of Eu(III) ions
on BNPP cleavage (Table 8). Obviously, these ammonium salts have the
same retarding effect as inorganic salts, as expected increasing with the
number of charges in these ligands. The effect of NaCl and MgCl2 on
the Eu(III)-catalyzed plasmid DNA cleavage is similar to that observed
with BNPP, with again linear Debye-Hückel correlations of similar slope
(Figure 9b). It should be noted that magnesium chloride at concentrations
as low as 5×10–5 M already decreases the efficiency of a mM Eu(III) solu-
tion by at least 10%.
Solvent effects on the uncatalyzed monophosphate ester hydrolysis
are known to show an increase with increasing amount of organic solvent
[128,129,132]; a similar trend is seen with ethanol in the Eu(III)-catalyzed
reaction of BNPP (Figure 10) [127]. With added DMSO instead of ethanol
one finds surprisingly an opposite behavior. In both cases the solvent effect
correlates linearly with the solvent polarity parameter ET (Figure 10).
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].
4.3. Activity Variation within the Lanthanide Series
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].
TABLE 9
Kinetic Paramaters for the Hydrolysis of BNPP from Saturation Kinetics with
Different Lanthanide Ions and Ionic Radii of the Ionsa) [15].
a) Rates measured at 50 ºC in 0.01 M EPPS buffer, pH 7.0.
unexpected decrease for Yb(III) and Lu(III). The decrease is reminiscent of

the parallel changes in stability constants discussed in Section 3. From the
saturation kinetics observed with all lanthanides on the BNPP cleavage the
corresponding kcat and KM (Table 9) show a strong and regular decrease
with the ionic radii only for the kcat values (Figure 11); the stability of the
catalyst-substrate complex is less affected [15]. Similar, although smaller
rate increases with catalysts from La(III) to Er(III) are observed with the
cleavage of the monophosphate ester NPP. The reason for the rate decrease
observed for Lu(III) and Yb(III) is likely the increased aggregation of
these ions, although this is not visible by eye. This is in line with the
very unusual behavior of these ions in experiments with plasmid DNA
(Table 10). With the higher lanthanides Tm(III), Yb(III), and Lu(III) one
observes a rate decrease with increasing catalyst concentration (Figure
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].
a)Relative to the estimated rate k = 1.0 × 10–11 s–1 for the

uncatalyzed reaction, measurements with [LnCl3] = 1.0 mM at 37°C.
The activity differences between the lanthanides against double-

stranded DNA may also be related to conformational changes prior
to cleavage, which in an extreme case could even be the slow step of
hydrolysis. The native right-handed B-form of plasmid supercoiled DNA
can undergo such changes [135], and it was found that Ln(III) ions also
induce such phase transitions [15]. With Eu(III) they can be fitted to a
first-order rate equation with, e.g., k = 1.2×10–3 s–1 for a concentration

of 5×10–3 M at 37oC of both Eu(III) and Yb(III). Thus, these DNA phase
transitions occur much faster than cleavage, and show no significant
difference between Eu(III) and Yb(III).
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
the dependencies for all lanthanides are leveling off at approximately

the same value of kobs; La(III) becomes even more reactive than other
cations [5b]. Noticeably, Ce(IV) catalysis has been found to be rather
selective for phosphate monoesters in comparison to diesters [136].
FIG. 13. Cleavage rate [%RF I] of plasmid DNA with Ln(III) ions vs. ionic
radius. Reproduced with permission of the publisher from [15].
For complexes with macrocyclic ligands an inverse order of reactiv-

ity is observed, see Table 6 for Ln(16). A similar trend was observed for
Ln(14): only the La(III) complex was active, complexes with Eu(III) and
Dy(III) did not have measurable activity [101d]. This was explained by a
decrease in the coordination numbers for smaller cations, which therefore
cannot efficiently bind the substrate. In accordance with this explanation,
the phosphoesterolytic activity of Eu(III) complex with octadentate 14
was restored by removal of one of a coordinated amide groups by using
the septadentate ligand 20 [137]. Lanthanide complexes with another
ligand similar to 20 also were proposed for RNA model substrate hydro-
lysis [138].
A combinatorial approach to the screening of lanthanide complexes

was applied to a system involving the four ligand structures 21a-d and six
lanthanide(III) cations: Eu, Gd, Tb, Dy, Ho, and Er [139]. Chromogenic
substrates such as BNPP and 4a were used for the screening on a 96-well
plate reader and the most active complex Gd(21d)3+ was found to be
active also in the hydrolysis of a double-stranded plasmid DNA with kcat
= 7.5×10–5 s–1 and KM = 7.4 M.
Phosphate esters of the RNA type have since the beginning also been
used as substrates for lanthanide-induced hydrolysis [7b,13,14]. The reac-
tivity increases here as expected in the order Tm(III), Yb(III), Lu(III),
which are the most efficient lanthanide(III) ions [7a]. As with DNA more
highly charged ions such as Ce(IV) also promote cleavage of RNA-type
substrates or of cyclic monophosphates particularly well [140]. It has,
however, been found that this reaction is not homogenous [141]. Cleavage
of mononucleotides such as AMP is also efficient with Ce(IV), leading
to mixtures of nucleosides and nucleobases [142]. In the presence of air
cleavage with Ce(III) salts is fast due to formation of Ce(IV) [143]. With
Yb(III) and Pr(III) complexes cyclic oxy and deoxy AMP are hydrolyzed
yielding predominantly 3’-monophosphates [144]. Association constants
of the Ce(IV)-DNA complexes were found to decrease from single-
stranded to double-stranded DNA, with much smaller affinity towards
dinucleotides. In contrast, cleavage rates for single-stranded and double-
stranded DNA were comparable, but much smaller than with dinucleotide
[145]. Recent investigations have almost invariably adressed the design
of suitable lanthanide ligands aiming at activity, transport and selectivity
properties of such artificial ribonucleases [4e,21e,146].
4.4. Complexes with Metal-Bound Hydroxy Anions and Rate

Equations Different from Saturation Kinetics
Besides the Michaelis-Menten type, other rate equations were reported,

which involve kinetics of first-, second-, and third-order in metal. Of
course, all these kinetic types do not exclude the formation of intermediate
catalyst-substrate complexes of too low stability to be observed by the
“saturation” in a given concentration range of metal or substrate. In fact,
the intermediate coordination of a phosphate ester to the metal ion is
always considered as a necessary step in catalysis.
First-order catalyst kinetics were reported for hydrolyses of both
BNPP and para-nitrophenyl phosphate (NPP) by Ce(III) and Eu(III)
cryptates in the low concentration range 0.4-4.0 mM [98]. In contrast,
different rate laws for these two substrates were found with lanthanide
complexes of BTP (Scheme 7): first-order kinetics for BNPP in the range
1-7 mM metal, but “saturation” kinetics with KM about 1 mM for NPP
[89]. Such a difference agrees with the much better ligating properties of
dianionic and more basic NPP capable of metal chelation as compared to
the monoanionic much less basic BNPP [4b,147].
Kinetics of second-order in metal were reported for several systems
in which the active species are dinuclear and the observed second order
reflects their reversible formation. These data involve RNA cleavage in
the presence of LaCl3 in basic solutions with La2(OH)5+ as the active form
[85] and BNPP hydrolysis by Pr(III) in the presence of macrocycle 22,
which forms with the metal cation a dinuclear complex [148] (see Section
4.5 for more details). Second-order kinetics are typical also for lantha-
nide-hydrogen peroxide systems (see Section 5).
However, higher than first-order kinetics can be observed already
with dinuclear complexes. Thus, the cleavage of BNPP by dinuclear
Y(III) complexes of BTP is second-order in total metal concentration
under conditions when all metal is incorporated into dimeric species [88].
It was shown that in the case of BTP complexes the reaction rate is pro-
portional to the product of concentrations of two different species: rate
= k2[Y2(BTP)(OH)42+][Y2(BTP)(OH)5+]. Supposedly, a higher hydroxide
complex Y2(BTP)(OH)5+ acts as a nucleophile providing the coordinated
hydroxide, and a more electrophilic lower complex Y2(BTP)(OH)42+ pro-
vides electrophilic assistance. Even third-order kinetics were reported for

the cleavage of BNPP by a binuclear complex of La(III) with the ligand
23 in 75% EtOH [149]. The active species is a dihydroxo dinuclear com-
plex and the proposed mechanism involves coordination of two such
complexes to both phosphate oxygens of BNPP with subsequent attack
of the electrophilically activated substrate by the third complex from the
opposite side (see also Section 6.2).
The active species were identified for several systems from studies
of pH dependencies of the reaction rates together with speciation studies,
mostly by potentiometric titrations. Such results permit to correlate the
pH-dependence of the reaction rate with species distribution curves and
to see which species contribute to the catalysis. As an example, Fig. 14
shows such a correlation for BNPP hydrolysis in the presence of Pr(III)
and BTP.
Obviously, no single species is responsible for the hydrolytic activity.

Since the reaction rate was found to be first-order in total metal, one can
describe the observed rate constant as a linear combination of contributions
from different species by Eq. (8):
kobs = k1[PrOH2+] + k2[Pr2(BTP)2(OH)24+] + k3[Pr2(BTP)2(OH)42+] (8)
Equation (8) can then be used for a linear multiparameter regression

to calculate the respective second-order rate constants k1-k3. Such
analysis performed for six lanthanide(III) cations (La, Pr, Nd, Eu, Gd,
Dy) showed that for a given hydroxo complex, e.g. M2(BTP)2(OH)24+,
the reactivity decreases with increasing atomic number and for a given
metal the reactivity increases with increasing n for a series of species
M2(BTP)2(OH)n6–n. Such a trend indicates the importance of the basicity

of the coordinated hydroxide.
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].
Species distribution diagrams were also obtained for La(III) com-

plexes with ligand 23 [149]. For the dinuclear complex a clear correla-
tion of the reaction rate with the fraction of the dihydroxo species was
observed; for less reactive mononuclear complexes, however, different
species contributed comparably to the observed reaction rate. The pH-
rate profile for RNA cleavage in the presence of LaCl3 in basic solutions
together with the potentiometric titration of LaCl3 showed La2(OH)5+ to
be the active species [85]. A similar approach was applied already in an
earlier study on the catalysis of phosphotriester hydrolysis by the La(III)
macrocyclic complex La(24)3+ the active form of which was found to be
the hydroxo complex La(24)OH2+ [150]. Qualitative studies of pH-depen-
dencies for lanthanide(III) cryptands [98] and for Ce(IV) micellar systems
[67] agree with hydroxo complexes as reactive species.
25 26
The pH-rate profiles for phosphodiester cleavage by lanthanide(III)

complexes with macrocyclic ligands bearing alcohol pendent groups
(15, 16) have sigmoid shape indicating that the reactive species are the
respective alkoxide or hydroxide complexes (see Section 5) [114,151].
Identification of active species for Ce(IV) is difficult. The structure
proposed for active species formed in aqueous neutral buffered
solutions of Ce(NH4)2(NO3)6 involves a dinuclear fragment Ce2(OH)44+
capable of the double Lewis acid activation of the phosphate group as
shown schematically in 25 [5b], although some other structures for the
phosphate complex were proposed [152]. Potentiometric titrations of the
Ce(IV) complex with the ligand 19 indicated formation of mononuclear
[Ce(19)(OH)]2+ species and dinuclear [Ce2(19)(OH)4]2+ species; the
structure of the latter is shown schematically as 26 [105].
5. LANTHANIDE ALKOXIDE AND HYDROPEROXIDE

COMPLEXES
The serine hydroxy group often participates as a nucleophile in active sites

of hydrolytic enzymes, in particular the metal-bound alcohol nucleophile
operates in the active site of alkaline phosphatase (Section 2.3). It has
been known for a long time that alkoxo anions possess higher nucleo-
philic reactivity than hydroxide anion towards activated esters [153]. With
model Zn(II) complexes (see Section 7) it was shown that coordinated
alkoxo anions are also better nucleophiles than coordinated hydroxide for
both carboxylic acid ester and phosphodiester cleavage [4a,154]. Similar
behavior is expected for lanthanide complexes, although it is not clear
to which degree alkoxide complexes indeed have advantage with these
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
(values of pH = –log[CH3OH2+] in methanol, calculated by adding 2.24 to

the observed pH-meter reading), and catalyze efficiently the methanolysis
of phosphodiesters [157]. It is worth noting that in contrast to aqueous
solutions where formation of hydroxide species at relatively high concen-
trations is impossible without stabilizing ligands because of the very low
solubilities of lanthanide(III) hydroxides [84], lanthanide methoxides are
perfectly soluble in methanol and can be employed without any additional
ligands. Potentiometric titrations in methanol solutions indicate formation
of two dimeric complexes La2(OMe)24+ and La2(OMe)5+ reminiscent of
dimeric hydroxide species in water (vide supra), which act as the active
forms. The reaction kinetics is complicated, but the observed catalytic
effects are large, reaching 1010-fold accelerations for the phosphodiester
(BNPP, diphenyl phosphate, methyl p-nitrophenyl phosphate) methanoly-
sis in the presence of 0.5 mM La(III).
SCHEME 8
Reaction mechanism for phosphate ester cleavage by alkoxide complexes illus-
trated by Ln(15)3+ species [114,155].
27
Another type of a nucleophile used in combination with lanthanides for

the phosphodiester cleavage is hydrogen peroxide. The anion of hydrogen
peroxide possesses an enhanced reactivity (α-effect) in acyl and phosphoryl
transfer reactions [158]. Uncatalyzed cleavage of BNPP by HO2– (second-
order rate constant 7.9×10–4 M–1s–1 at 25°C) is much faster than the alka-
line hydrolysis (see data in Table 2). Unexpectedly one mole of hydrogen
peroxide substitutes both nitrophenyl groups of the substrate [159]. Addi-
tion of La(III) dramatically increases the reaction rate, e.g. in the presence
of 2 mM La(III) and 20 mM H2O2 the observed first-order rate constant for
BNPP cleavage equals 4.8×10–3 s–1 at 25°C and pH 7.0, which represents
perhaps the highest reported reactivity under such conditions (half-life just
2.4 min) [20]. However, low stability of the metal-hydrogen peroxide mix-
tures, which lose the catalytic activity after ca. 30 min is a serious drawback
of this system. Similar reactivity was observed in a more sophisticated
system involving La(III) bound to a cyclodextrin ligand [160]. Differ-
ent lanthanides from La(III) to Eu(III) show similar activities [161]. On
the basis of potentiometric titration and kinetic data the cationic dimeric
complex La2(O2)22+ with the hypothetical structure 28 was proposed as
the active species which interacts with the phosphodiester as shown in
29 [20,161]. The reaction rate as a function of the dimer 28 concentration
shows a saturation with 1/KM = 1.3×103 M–1 for BNPP, indicating a fairly
strong complexation of the substrate to the dimeric complex.
The Ln(III)/H2O2 system also shows high reactivity in RNA and

diribonucleotide cleavage [162]. However, potentiometric titration of the
La(III)/H2O2 system reveals [162] formation of different dimeric neutral
species La2(O2)3, and the reaction kinetics was third-order in total metal
under conditions where all metal is incorporated in the dimeric complex

[162]. It was proposed that the active species is a hexameric complex
formed by reversible association of three neutral dimers. The discrepancy
in the composition of peroxo complexes was attributed to different
concentrations of components used for titrations (2 mM and 5 mM La(III)
in [20] and [162], respectively), although the difference does not seem to
be significant. In addition, according to [162] the Ln(III)/H2O2 system
does not lose activity even after 100 min pre-incubation.
In a more recent study of the Y(III)/H2O2 system potentiometric
titration showed formation of two dimeric complexes: Y2(O2)2(OH)2,
which is the predominant form at pH above 6.5, and Y2(O2)22+ as a
minor component [163]. The kinetic data agreed with Y2(O2)2(OH)2
as the active species. The concentration-rate profile with BNPP as the
substrate shows that the reaction is second-order in Y2(O2)2(OH)2 at metal
concentrations below 1 mM, but a saturation-type profile is observed at
higher concentrations. A probable reaction mechanism involves reversible
formation of a tetranuclear complex [Y2(O2)2(OH)2]2 which binds BNPP
tightly and cleaves it intramolecularly.
6. CATALYSIS WITH DI- AND POLYNUCLEAR COMPLEXES
6.1. Di- and Polynuclear Models with Metal Ions Other than Ln(III)
As discussed in Section 2.3 most metallohydrolases contain two or more

metal ions, separated by a distance between 2.9 and 3.9 Å [2]. As shown in
Scheme 6, the metal ions can promote hydrolysis by Lewis acid catalysis,
by stabilizing the leaving group and by activating the solvent water, gener-
ating reactive OH– anions at neutral pH. Following the footsteps of nature
a variety of multinuclear artificial hydrolases have been prepared, with
often significantly higher activity as compared to the mononuclear ana-
logues. Dinuclear catalysts with metals other than lanthanides have been
discussed in several reviews [4]; here only few and more recent of these
intriguing models will be mentioned. Most of the earlier studies were per-
formed using Zn(II) ions, e.g. on BNPP [164] or on plasmid DNA hydro-
lysis [165]. The reactive species were partially characterized with respect
to their structures and the underlying mechanisms by analyzing the effect
of pH variations. Thus, the ligand (a) in Scheme 9 forms at pH above 7.0

a dinuclear hydroxo-bridged complex, which catalyses the hydrolysis of
p-nitrophenyl acetate [166]. The dizinc complex of the propanol-bridged
octaazacryptand (b) in Scheme 9 has in the crystal structure a distance
of 3.4 Å between the metal ions; it exhibits a considerably accelerated
second-order reaction with mono-nitrophenyl phosphate; formation of a
phosphoramide, however, by attack from the nitrogen atom prevents turn-
over [167,168]. Efficient rate acceleration in ester cleavage occurs also
with dinuclear strontium(II) complexes [169]. Considerably higher hydro-
lytic activity is usually observed with di-copper complexes (see e.g. [170-
173]). The hydrolysis of BNPP with the dicopper complex (c,c’) shown in
Scheme 9 was reported to be about 107 times faster than the uncatalyzed
reaction; saturation kinetics with excess substrate indicated turnover, and
a KM value of 0.026 M with kcat = 5.5×10–3 s–1. The distance between
two Cu ions bridged by OH anions in these complexes varies between 2.9
and 3.8 Å [171]. In other studies dinuclear Ni(II) complexes were found
to be significantly better catalysts than analogous dicopper complexes,
which was attributed to the hexacoordinate Ni(II) in comparison to the
only pentacoordinate Cu(II) systems [174]. In analogy to several enzymes
heteronuclear complexes with asymmetric ligands hold particular promise
[175]. Heterodinuclear [ZnFe(III)]-Fe(II) complexes were found to be
10-fold more reactive than corresponding homonuclear [FeFe(III)]-Fe(II)
systems [176]. Mixed trinuclear complexes were studied of the general
formula (L-H)M-s(M-f), where L is a polyaza ligand having a tetraden-
tate and two bidentate metal binding sites; M-s are different “structural”
metal ions (e.g. Cu, Ni, Pd), and M-f are functional (catalytic) metal ions.
Kinetic analysis with the RNA-analog substrate 4a yielded 1/KM values
(in M–1) of 170 (with M-s = Cu(II)), 340 (with M-s = Ni(II)), and 2600
(with M-s = Pd(II)); the kcat values varied also significantly , with k (in
s–1) = 17×10–3 (M-s = Cu(II)), 3.1×10–3 (M-s = Ni(II)), 0.22×10–3 (M-s
= Pd(II)). In line with crystal structural analysis the results were inter-
preted as allosteric influence of the structural ion M-s on the complex
conformations [177]. Recently also calixarene-based trinuclear metal
complexes were used for the cleavage of RNA-type dinucleotides [178].
SCHEME 9
Some ligands for binuclear Cu or Zn catalysts.
The distance dependence of two metal centers was studied with

di-zinc complexes using the dinucleotide UpU and BNPP as substrates
[179]. Optimal rates were observed with conformations essentially in
agreement with the geometry found in dinuclear metallohydrolases (see
Section 2.3). The observed enhancement factors f = kdinucl/kmononucl rela-
tive to the corresponding mononuclear complexes (with kmononucl) were,
however, only moderate for UpU (f = 2); with BNPP f = 12 was found.
Besides dinuclear Zn(II) complexes for, e.g., dinucleotide cleavage [179-
181] also dinuclear peroxo complexes [161] were investigated, see Sec-
tion 5. Dinuclear complexes containing imidazole ligands showed with
BNPP only an enhancement factor of f = 2, and no cleavage with DNA
[182]. A biomimetic way to fix two metal ions in a desired distance has
been materialized with a heptapeptide carrying 1,4,7-triazacyclononane
binding units for Cu(II), Ni(II), or Zn(II) [183,184]. The peptide forms a
stable helical conformation even at 50°C and shows for the RNA-model
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.
6.2. Dinuclear Lanthanide Complexes
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’-
OH ends), similar to DNA hydrolyzing enzymes [105]. Another series of

dinuclear lanthanide complexes used for a plasmid DNA hydrolysis were
reported using ligand 30 [186].
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
In several instances cooperation between different lanthanide cations

as well as between a lanthanide and a non-lanthanide cation was reported.
One may expect such cooperation to be due to formation of di- or poly-
nuclear mixed metal complexes, although the exact active form was not
identified for the systems discussed below. Thus, the rate of TpT hydro-
lysis by Ce(IV) at pH 7 and 50°C increases ca. ten-fold in the presence
of 0.5 equivalent of Pr(III) or Nd(III) although the trivalent lanthanides
by themselves do not have any hydrolytic activity in this reaction [187].

Cations of Y(III) and Sc(III) do not affect the reactivity of Ce(IV) and
divalent cations of Mg, Mn, Ni, Co, and Fe inhibit the catalytic activity
of Ce(IV). On the other hand, under similar conditions a positive coop-
erativity between La(III) and cations of Fe(III), Mn(III), Sn(IV), In(III),
and Ga(III) in the hydrolysis of BNPP was reported [188]. Cooperation
between Ce(IV) and lanthanides was studied in more detail in homoge-
neous dextran solutions [189]. The activities of ternary Ce(IV)/Ln(III)/
dextran systems in the hydrolysis of TpT at pH 7 and 50°C depend on the
type of trivalent lanthanide added following the order Pr>Nd>Eu>La>Lu.
With Pr(III) the activity of the ternary system is ca. 300 times higher than
that of Ce(IV)/dextran alone and with less active Lu(III) it is still 9 times
higher. Dextran by itself decreases significantly the activity of Ce(IV),
but in the presence of Pr(III) the ternary system is only slightly less active
than Ce(NH4)2(NO3)6 alone under similar conditions. It was proposed that
the increased reactivity of dimetallic systems results from formation of
mixed hydroxides in which both metals participate in Lewis acid substrate
activation. Homogeneous Ce(IV) solutions, prepared by using solubiliz-
ing additives (see Section 3.3), promptly hydrolyzed plasmid DNA. The
inactivity of Ce(IV)-hydroxide gel for this reaction, which is in contrast
with its remarkable efficiency in the hydrolysis of linear DNA, has been
ascribed to the steric hindrance in the reaction between the supercoiled
DNA and the polymeric catalyst [190].
Addition of an equimolar concentration of Zn(II) to Eu(15)3+ increases
15-fold the catalytic activity of the latter in the cleavage of the RNA 5’-cap
structure model compound GpppG [191a]. Less electrophilic Mg(II) does
not affect the reaction rate. The mechanism of the Zn(II)-assisted cleavage
is shown in Scheme 11. Such bifunctional action of two metal complexes,
one of which activates a nucleophile and another one acts as a Lewis acid
catalyst was mentioned above for Y(III) hydroxide complexes stabilized
by BTP (Section 5). Obviously it requires entropically unfavorable
trimolecular interaction and one of the advantages of dinuclear complexes
is a possibility that this bifunctional catalysis occurs inside one species, as
illustrated in Scheme 6b. Unusual 2-carboxyethylgermanium complexes of
Yb(III) and Pr(III) of the general structure 31 were used for the hydrolysis
of cyclic 3’,5’ ribonucleoside and deoxyribonucleoside phosphates [144].
31
SCHEME 11
Cooperation of a lanthanide macrocyclic complex with Zn(II) in the cleavage of
the 5’-cap structure [191a].
7. ANALOGS OF FUNCTIONAL AMINO ACIDS IN

CATALYTIC CENTERS
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
functional elements are found in many natural nucleases such as alkaline

phosphatase (AP). The role of these groups has been the subject of many
recent investigations, often clarified by mutants lacking functions of the
wildtype enzyme. Thus, replacement of Ser102 in AP by, e.g., Ala leads
to an efficiency decrease by about 103 [192]. In synthetic analogs of
metalloenyzmes, such functions can be implemented as part of the ligand,
or they can be added as reactive compound which may have only loose
binding to the metal, to the substrate or to products. Scheme 12 illustrates
how incorporation of hydroxyalkyl groups in, e.g., Zn(II) complexes
can as serine mimics promote hydrolysis of esters. The experimental
analyses of the underlying mechanisms has been discussed in detail in
Section 5. Water bound to, e.g., a [12]aneN3-Zn(II) complex (Scheme
12) shows a pKa value of 7.3, explaining the fast hydrolysis of esters
at neutral condition. If a hydroxyalkyl pendant group is attached this is
also deprotonated with practically the same pKa of 7.4, thus generating a
very powerful alkoxide nucleophile [4a]. The second-order rate constant
is about 10 times larger than that for a corresponding complex lacking
the hydroxyalkyl side group. With Zn(II)-cyclen formation of a mixed
complex including OH– anions and the CH2OH group were observed.
Noticeably, the hydrolysis of the intermediate formed by transfer of the
acetyl group to the CH2OH group was even faster than the first step,
thus allowing fast turnover in this covalent catalysis. Similarly, BNPP
hydrolysis is accelerated by these Zn(II)-hydroxyalkyl complexes at close
to neutral conditions with second-order rate constants up to 10 times
SCHEME 12
Hydrolytic Zn(II) complexes with pendent hydroxylalkyl groups.
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.
SCHEME 13
Polyols as supporting functional groups.
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
of a metal binding unit [165]. The imidazole may be largely blocked by

association with the metal, but some of it could still serve for proton relay,
for acid-base catalysis or even as a nucleophile. Preliminary results shown
in Scheme 15 indicate moderate effects on BNPP or plasmid DNA with a
crown ether bearing two imidazole groups; the acceleration would be larger
if the complexation with Eu(III) would be stronger [203]. However a com-
bination of imidazole and a carboxylic function within one single cofactor
as shown in Scheme 15 was reported to produce the largest rate of DNA
cleavage with Eu(III) salts known until now.
SCHEME 14
Carboxylic groups as cofactor in BNPP hydrolysis [198].
Natural amino acids and peptides offer combinations of functional

side chains which like in enzymes may contribute efficiently to ester
hydrolysis if they are assembled in a productive way. Metal complexes
with macrocyclic peptides were claimed to provide considerable accelera-
tion of DNA cleavage [204-206]; the reports were, however, later refuted
[207-209 ]. More promising was a screening of a combinatorial library of
625 different undeca-peptides containing Arg, His and Trp [210]. With a
special assay based on developing an indigo dye from an indole phosphate
ester (Scheme 16) all combinations were screened with Zn(II), Eu(III),
Ce(IV) and Zr(IV) as metal ions. The best results were obtained with some
SCHEME 15
Imidazole-containing cofactors [203].
peptides after isolation in the presence of Zr(IV) salts, which accelerated

the cleavage of p-nitrophenyl phosphate also in solution, however only by
a factor of five. The problem with such peptides can be that orientation of
all “productive” functions around the metal-substrate complex may require
thermodynamically unfavorable peptide conformations. This is not so with
libraries of single amino-acids, or e.g. dipeptides which have been tested
as possible cofactors in Eu(III)-catalyzed cleavage experiments [203].
The disadvantage of this approach is of course that the ternary, if not qua-
ternary, complexes between substrate, metal, and one or more cosubstrates
are quite weak, for which reason the observed rate effects were rather low.
SCHEME 16
Assay used for screening a combinatorial library [210].
Nevertheless, several combinations of Ser, His and Asp were found to be

more effective, leading with BNPP to rate enhancements of only up to
2.5, whereas others showed mostly decreased efficiency of Eu(III). With
plasmid DNA even smaller accelerations, and these only with Z-protected
amino acids, were observed. Combinatorial libraries have also been used
to select catalysts on the basis of polyallylamines substituted with eight
different carboxylic acids and Mg(II), Zn(II), and Fe(III) salts [211]; the
largest hydrolysis acceleration observed with BNPP was about 10-fold
smaller than that with Eu(III) alone.
Other ligands attached to lanthanide complexes aim at better binding
to nucleic acids; they can thus lead to better KM values. An example is a
complex with the DNA groove binder bisnetropsin linked to hydroxamic
acids [212]. Attachment of a hydrolytically active metal complex to an
intercalator ligand may lead to a significant increase in DNA cleavage
activity. A series of intercalator-linked hydroxamic acids 32 were prepared
and used for a plasmid DNA cleavage with both transition metals (Fe2+
and VO2+) and trivalent lanthanides [213].
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
major aim here is the strategy to achieve selectivity in DNA cleavage by

attaching oligonucleotides, which is the topic of Chapter 12 in this volume
[214].
8. OTHER LANTHANIDE-BASED SYSTEMS
Phosphodiester cleavage by lanthanides was studied in micellar solutions

with two aspects: using micellar media to stabilize the highly charged
Ce(IV) catalyst and using simple lanthanide aqua ions to hydrolyze
phosphate-functionalized surfactants. Another aspect is the possibility
to cleave biocidic hydrophobic esters including warfare agents in an
efficient and economical way. In the presence of micelles of 2 mM non-
ionic surfactant Brij-35 (C12H25(OCH2CH2)23OH), cerium(IV) 1-2 mM
solutions are stable up to pH 5 and the cleavage of BNPP occurs with
a large first-order rate constant ca. 0.01 s–1 at 37°C [67]. Additions of
hydrophobic ligands 33a-c to the micellar solution in 1:1 or 2:1 metal:
ligand ratios allow to stabilize Ce(IV) up to pH 11 with conservation of
phosphodiesterolytic activity. A more detailed study which demonstrated
the Michaelis-Menten-type kinetics was performed for the Ce(IV)-
palmitate (33b) micellar system in phosphonate hydrolysis [215].
33
Phosphodiesters like 34 form micelles at concentrations above

0.045 mM and their cleavage in the presence of lanthanides(III) at pH
7 and 37°C proceeds ca. 10 times faster than that of ethyl p-nitrophenyl
phosphate taken as a nonmicellar analog [216]. The increased reactivity
was attributed to enhanced binding of cations to anionic micelles that is
manifested in lower KM values observed with micellar substrates. Like
in the case of BNPP hydrolysis the reactivity in the lanthanide series
increased on going from La(III) to Tm(III) and then decreased again for
Yb(III) (cf. Table 9).
34 35 36
Phosphate-functionalized lipids 35 and 36 were prepared and the

hydrolysis of their phosphodiester bonds by lanthanide(III) aqua ions was
used to provide important information regarding the mobility of vesicles
formed of these lipids [217]. Phosphate bilayer membranes were used as a
reaction medium for DNA cleavage by Ce(III) [218].
Lanthanide complexes with the Schiff-base macrocylic ligands 24 and
37 were employed for catalytic RNA hydrolysis with turnover [21b] and
showed high site selectivity as compared to hydrolysis by free cations
[124,219]. Schiff-base chelates of lanthanide ion were reported to cleave
nucleotides selectively at the 5’-end, whereas the free metal ions cleave
also at the 3’ position. Europium(III) texaphyrin 38 conjugated to a
synthetic oligonucleotide complementary in sequence to a synthetic RNA
30-mer used as a substrate was employed for sequence-selective RNA
cleavage [23]. Such complexes were also conjugated with antisense DNA
fragments to achieve a ribozyme mimic [220]. Oligonucleotide conjugates
37 38
of Eu(III) complexes of two bifunctional tetraaza macrocyclic ligands

with pendent hydroxyethyl groups were prepared and used to promote the
hydrolytic cleavage of an oligoribonucleotide containing complementary
(antisense) sequences [221]. Cleavage is not observed in the presence of
Eu(III) conjugates containing scrambled sequences, nor by the unmodified
complex.
A lead-dependent catalytic DNA (DNAzyme) was found to be
catalytically active towards ribonucleotides in the presence of trivalent
lanthanide cations [222]. The activity increases slowly on going from
La(III) to Tb(III) and than rapidly becomes one order of magnitude higher
for Tm(III) and Lu(III). The observed first-order rate constant is a linear
function of Lu(III) concentration in the range 0.02-0.1 mM indicating
rather weak binding of the metal cation to DNA. The lanthanide DNAzyme
cannot hydrolyze polyribonucleotides like native RNA, but cleaves the
phosphodiester bond in an oligodeoxyribonucleotide containing one
ribonucleotide with a first-order rate constant ca. 5×10–4 s–1 at pH 7.4 and
0.1 mM Lu(III) concentration. Such activity is similar to those reported
for lanthanide complexes with synthetic ligands.
A new approach to lanthanide-based chemical nucleases is based
on specially designed chimeric peptides, which combine DNA-binding
and metal-binding fragments [223]. The chimeric 33- and 34-residue
peptides incorporate helices from the DNA-binding protein engrailed and
the Ca-binding protein calmodulin. The dissociation constants of peptide
complexes with Eu(III) are 10 and 3 μM. They hydrolyze BNPP with
second-order rate constants of 0.1 and 0.3 M–1s–1 at 37°C and pH 7, which
are considerably higher than the rate constants for the Eu(III) aqua ion
under similar conditions. It was also demonstrated that Eu(III) complexes
of chimeric peptides hydrolyze supercoiled plasmid DNA.
9. A FINAL PERFORMANCE COMPARISON OF

LANTHANIDES WITH OTHER METAL IONS
Table 11 collects [15,51,64,66,67,88,89,98,148,151,171,224-228]

selected kinetic data for BNPP cleavage by lanthanide and some other
metal complexes, which allows to compare efficiencies of different
systems. Since different systems follow different rate laws and the exact
TABLE 11
Selected Kinetic Parameters for BNPP Cleavage by Lanthanide and Some Other
Metal Complex Catalysts
a) Tris(3-aminopropyl)amine, see Scheme 5

b) See Scheme 9c
c) See Scheme 12
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
Zn(II) and Pr(III), which again clearly show the advantage of lanthanide
cations.
39 40
10. CONCLUSIONS AND OUTLOOK
There are few enzyme-analog catalysts which have already achieved

such enormous rate accelerations as lanthanide-based synthetic systems
for phosphate ester hydrolysis. Rate enhancements of up to 108 under
physiological conditions have been reported [230]; antisense or antigene
strategies are clearly at hand to introduce selectivity in cleaving nucleic
acids. Nevertheless, enzymatic cleavage is still superior in many respects,
and much needs to be done before we come closer to design artificial
analogs as effective as the natural systems, and to fully understand how
they are achieving this.
Future aims will be to develop complexes with preferably several
metal ions of high charge density placed in the correct distances; in addi-
tion they should be kinetically very stable without hampering at the same
time their catalytic activity. Dissociation of metal ions out of the complex
can lead to damage at other parts and to loss of acitvity before the target is
reached; furthermore, an exchange in the cell is faster by other metal ions
abundant in proteins [231]. Obviously, the carboxylic groups which are so
effectively complexing lanthanides in NMR relaxation agents or fluores-
cent labels (see other chapters of this volume) are counterproductive in the
build-up of negative charges in the transition state and/or in intermediates
associated with phosphate ester hydrolysis. One can expect that the future
belongs to a large degree to macrocyclic ligands, which serve the purpose
to fix lanthanide ions sufficiently strongly, and in desired distances. Such
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].
ACKNOWLEDGMENTS
A.K.Y. thanks for financial support of CONACyT (project 25183-E)

and DGAPA-UNAM (project IN214998). H.-J. S. appreciates support
by the Deutsche Forschungsgemeinschaft, Bonn, by the A.-v. Humboldt
Foundation, Bonn, and by the Fonds der Chemischen Industrie,
Frankfurt.
ABBREVIATIONS
[18]aneN6 hexacyclen (1,4,7,10,13,16-

hexaazacyclooctadecane)
[2.2.1]crypt 4,7,13,16,21-pentaoxa-1,10-diazabicyclo[8.8.5]tri-
cosane
ACES [N-(2-acetamido)-2-aminoethanesulfonic acid

AcO acetate anion or residue (CH3COO–)
AMP adenosine 5’-monophosphate
ATP adenosine 5’-triphosphate
AP alkaline phosphatase
bipy 2,2’-bipyridine
Bis-Tris 2,2-bis(hydroxymethyl)-2,2’,2”-nitrilotriethanol
BNPP bis(p-nitrophenyl) phosphate
BTP bis-tris propane (1,3-bis[tris(hydroxymethyl)-
methylamino]propane)
CMP cytidine 5’-monophosphate
cyclen 1,4,7,10-tetraazacyclododecane
DMSO dimethylsulfoxide
DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid
e.u. entropy unit (J/K mol))
EDTA ethylenediamine-N,N,N’,N’-tetraacetate
EPPS 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic
acid
Gly glycine
GMP guanosine 5’-monophosphate
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HPLC high-pressure liquid chromatography
MES 4-morpholine ethanesulfonic acid
MOPSO 3-(N-morpholinol)-2-hydroxypropanesulfonic acid
NPP p-nitrophenyl phosphate
Nu nucleophile
tdci 1,3,5-trideoxy-1,3,5-tris(dimethylamino)-cis-
inositol
Tris tris(hydroxymethyl) aminomethane
trpn tris(3-aminopropyl) amine
Z-protected N-carbobenzyloxy protected amino acid
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Ln-CATALYSED HYDROLYSIS OF PHOSPHATE ESTERS 369

Lanthanide-Catalysed Hydrolysis of Phosphate

Hans-Jörg Schneider1 and Anatoly K. Yatsimirsky2

1FR Organische Chemie, Universität des Saarlandes,

1. INTRODUCTION AND SCOPE 370

4. KINETICS OF CATALYTIC PHOSPHATE

1. INTRODUCTION AND SCOPE

Phosphate ester bonds belong to the kinetically most stable binding

the glycerol phosphate model (Scheme 1). Their scission is controlled by

Important phosphate diesters in nature (B1, B2: nucleobases), with estimated

The development of artificial restriction enzymes is of particular

antigene strategies. Of medicinal interest is that selective nucleases can in

1.1. Historical Perspectives

2. MECHANISMS OF PHOSPHATE ESTER HYDROLYSIS

2.1. Non-catalytic Hydrolysis

2.1.1. Mechanisms of Nucleophilic Substitution in Phosphate Esters

Several reviews are available on mechanisms of nucleophilic substitution

but non-catalytic hydrolysis may occur by considerable or even dominant

However, a purely dissociative mechanism is rather improbable [30],

of the racemization expected for the SN1 mechanism speaks in favor of

Hydrolysis of monoanions of phosphate monoesters ROPO3H–

Some special mechanism for the monoanion hydrolysis is required in

H2O + ROPO3H– OH– + ROPO3H2 → ROH + H2PO4– (3)

H2O + ROPO32– OH– + ROPO3H– → ROH + HPO42– (4)

Generally, triesters of phosphoric acid are hydrolyzed in basic solutions

2.1.2. Hydrolysis Rates and Half Life Times under “Physiological”

the observed first-order rate constant 3×10–19 M–1s–1 in neutral solution.

a Observed rate constant at pH 8.0.

Rate constants and (when available) activation parameters for the

The kinetics of hydrolysis of dinucleoside phosphates were studied in

In neutral solution the dominant route is the alkaline hydrolysis [56].

a Calculated from data at 333.2 and 363.2 K.

2.2. Role of Metal Ions

2.2.1. General Considerations

General aspects of metal catalysis in phosphate ester hydrolysis are

Common types of mechanisms of metal ion catalysis in phosphate

leaving group (mechanism “b”) is equivalent to a decrease in the leav-

Co(III) in complexes shown in Scheme 5 is lowered by about 10 units

Cleavage of BNPP by some Co(III) di-aqua complexes (0.01 M complex at

2.2.2. Studies with Model Compounds

Kinetic studies with substrates bearing coordinating groups provide useful

Hydrolysis in the presence of La3+ can be first-, second- and third-

hydrolysis of 8 at pH 8. The features of La3+ which explain its ability to

Although other lanthanides were not studied with these substrates,

2.3. A Look at Natural Enzymes

Many enzymes which catalyze the hydrolysis of phosphate esters are

Mg(II) [2b,4f]. In most hydrolytic metalloenzymes the metal ions are

group [2d]. An additional electrophilic activation is provided by Arg166.

Kinetic Parameters for BNPP Hydrolysis by Some Biological Catalysts

a) Estimated from reported Vmax = 24 μmol min–1 mg–1, assuming the

Staphylococcal nuclease is an example of a single-metal phospho-

FIG. 2. Mechanism of the hydrolysis of a phosphomonoester by alkaline phos-

which however needs not to be part of a polynucleotide chain; simple

FIG. 3. Mechanism of staphylococcal nuclease. Adapted with permission of the

KM = 3.5 μg/mL [80a,b]. An enzyme in which Ca2+ is substituted by Sr2+

3. FORMATION OF LANTHANIDE COMPLEXES IN WATER

The development of thermodynamically and even kinetically stable ligands

it was proposed that it may be related to a complex pattern of dehydration

Although lanthanides do not form complexes with acyclic polyamines

cryptands bind lanthanides in water with stability constants around 106

Considerable progress has been reached in molecular modeling of

4. KINETICS OF CATALYTIC PHOSPHATE ESTER