Dinuclear Metal Complexes Based on all-cis-2,4,6-Triaminocyclohexane-1,3,5-triol as Catalysts for Cleavage of Phosphate Esters

Article abstract of DOI:10.1002/ejoc.200300381

Two dimetallic ligands 2 and 3 for transition metal ions were obtained by connecting two all-cis-2,4,6-triamino-cyclohexane-1,3,5-triol (TACI, 1) subunits via 1,3- or 1,4-xylyl linkers. Their dimetallic Cu^II and Zn^II complexes were i

Full text of DOI:10.1002/ejoc.200300381

FULL PAPER
Dinuclear Metal Complexes Based on all-cis-2,4,6-Triaminocyclohexane-
1,3,5-triol as Catalysts for Cleavage of Phosphate Esters
Fabrizio Mancin,*[a] Enrico Rampazzo,[a] Paolo Tecilla,[b] and Umberto Tonellato[a]
Keywords: Catalysis / Dimetallic complexes / Enzyme models / Phosphate ester hydrolysis / Tripodal ligands
Two dimetallic ligands 2 and 3 for transition metal ions were
obtained by connecting two all-cis-2,4,6-triamino-cyclohex- plex in promoting the hydrolysis of HPNP at pH values close
dimetallic systems are more efficient than the TACI·ZnII com-
ane-1,3,5-triol (TACI, 1) subunits via 1,3- or 1,4-xylyl linkers.
Their dimetallic CuII and ZnII complexes were investigated
as catalysts for the cleavage of the phosphate diesters 2,4-
to neutrality (7.0−7.8). In this case, the effects of cooperativity
between the two metal centers were highlighted in a detailed
kinetic study; the catalytic efficiency seems to be related to
dinitrophenyl ethyl phosphate (DNPEP) and 2-hydroxypro- the stronger binding of the substrate to the dimetallic ZnII
pyl p-nitrophenyl phosphate (HPNP) and the triester 2,4-di-
complexes. Additionally, in this case, although to a much
nitrophenyl diethyl phosphate (DNPDEP). The results of a lesser extent than in the case of the CuII counterparts, the
comparative kinetic study using the monometallic complexes
of TACI as a reference indicate that the CuII complexes of 2
formation of µ-hydroxo bridges apparently hampers the cata-
lytic efficiency, as indicated also by the observation that the
and 3 are virtually inert; this finding is ascribed to the forma- activity of the dimetallic complexes increases as the distance
tion of intra-complex µ-hydroxo bridges that prevent the re-
quired interactions with the substrate. On the other hand, the
dimetallic ZnII complexes produce remarkable accelerations,
particularly in the case of the HPNP transesterification. The
between the two metal centers increases, which, thus,
thwarts the formation of intermetallic bridges.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2004)
Introduction
toward the cleavage of the phosphate ester bonds of DNA,
the development of synthetic catalysts that may compete
with their natural counterparts is a formidable problem.[3]
As a matter of fact, the reactivities of the synthetic models
that have been reported, as either mono-, di- or tri-nuclear
complexes, are far from the target of approaching those of
enzymes.
Enzymes that catalyze the hydrolytic or nucleophilic
cleavage of phosphate esters and nucleic acids are normally
activated by more than one metal ion.[1,2] The search for
simple, yet effective, synthetic catalysts is being addressed
by the study of systems that somehow mimic natural metal-
loenzymes and may exploit the additivity and, possibly, the
synergistic effects of two or more metal ions bound into the
structure.[3,4,5] The large interest in the realization of such
biomimetic systems is stimulated by their potential appli-
cations, such as the development of artificial restriction en-
zymes for molecular biology and biotechnology and of anti-
viral and antitumor drugs.[6] Extensive mechanistic studies
of the metal ion-promoted hydrolysis of phosphates are
now available[3,7Ϫ10] so that the design of effective binuclear
metal ion complexes has apparently been made easier.[3] As
pointed out by Chin, however, when considering the ef-
ficiency (up to 1017-fold accelerations) of metalloenzymes
In such artificial enzymes, very large catalytic effects
could be achieved, at least in principle, if the direct modes
of activation that metal ions can provide for hydrolyzing
phosphate esters (at least in the case of diesters) are simply
combined even without relying on cooperative effects. Such
‘‘direct’’ activation effects are essentially (i) Lewis acid acti-
vation, (ii) intramolecular nucleophile activation, and (iii)
leaving group activation.[3] Even if identifying and evaluat-
ing these catalytic roles provides encouraging perspectives,
combining the estimated benefits that result from them still
faces quite a number of problems that make the design of
catalysts very difficult and quite challenging. For instance,
a few variables that play crucial roles, and that must be
solved mostly in the absence of established guidelines, are
the nature of the metal ion involved, the distance between
the metal ions, the degree of conformational freedom al-
lowed by the linker of the two ligand subunits, the nature
of the ligand with regard to the coordinating atoms, and
the coordination geometry. Moreover, solubility in aqueous
solutions is another property that adds to the above prob-
[a]
Dipartimento di Chimica Organica
e Istituto CNR di
`
Tecnologia delle Membrane Ϫ Sezione di Padova, Universita
di Padova,
Via Marzolo 1, 35131 Padova, Italy
Fax: (internat.) ϩ 39-049-8275239
E-mail: fabrizio.mancin@unipd.it
[b]
`
Dipartimento di Scienze Chimiche, Universita di Trieste,
Via Giorgieri 1, 34127 Trieste, Italy
Fax: (internat.) ϩ 39-040-5583903
E-mail: tecilla@dsch.univ.trieste.it
Eur. J. Org. Chem. 2004, 281Ϫ288
DOI: 10.1002/ejoc.200300381
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F. Mancin, E. Rampazzo, P. Tecilla, U. Tonellato
FULL PAPER
lems if the systems are intended for biological appli- dinitrophenyl ethyl phosphate (DNPEP) and 2-hydroxypro-
cations.[11]
pyl p-nitrophenyl phosphate (HPNP), and the triester 2,4-
Recently, we reported the results of a study, on the hydro- dinitrophenyl diethyl phosphate (DNPDEP).
lytic reactivity of ZnII complexes of polyamino structures
toward phosphodiesters, that highlighted, among several
interesting mechanistic features, the role played by the li-
Results and Discussion
gand in modulating the catalytic efficiency of the com-
plexes.[10] Among the several polyamino ligands investi-
Design of the Ditopic Ligands 2 and 3
gated, we found that all-cis-2,4,6-triamino-cyclohexane-
Our target catalytic species was a binuclear complex that
1,3,5-triol (TACI, 1), is a suitable structure for preparing
reactive ZnII complexes. As a matter of fact, complexes of
TACI are not only good catalysts towards model phosphate
triesters and diesters, but, more interestingly, toward super-
coiled DNA: TACI·CuII, in particular, ranks among the
most effective of the simple DNA-cleaving agents.[12] Fur-
thermore, a dicopper complex of a hexamethyl derivative of
TACI is quite effective in promoting phosphodiester cleav-
age.[13] These findings stimulated studies now in progress on
the reactivity of derivatives of TACI and related ligands
that are aimed at the realization of binuclear catalysts.
Here, we report the synthesis of two ditopic ligand struc-
tures, DTMX 2 and DTPX 3 (Scheme 1), that bear two
TACI subunits held together at a convenient distance by
1,3- or 1,4-xylyl linkers, and a study of the reactivity of their
CuII and ZnII complexes towards two phosphodiesters, 2,4-
could operate in neutral aqueous solutions as an artificial
phosphatase and could somehow mimic its natural counter-
parts.
As anticipated, the choice of TACI as the ligand subunit,
notwithstanding the synthetic problems concerning modifi-
cations of its basic structure, whose chemistry is rather un-
predictable, was mainly motivated by the known reactivities
of its complexes and by the hydrophilic properties of its
three hydroxy groups that could contribute to the overall
solubility of the species in water. Moreover, the same
hydroxy groups could play a relevant role in increasing the
reactivity of the complexes toward DNA.
The linkers were selected among those that have been
used successfully to realize other binuclear complexes.[14Ϫ17]
We employed preliminary force field calculations (MM2)[18]
to choose the most suitable structures; we examined the
structures of the ternary complexes between dimetallic ZnII
complexes having different spacers and dimethylphosphate
(dmp), which was chosen as a model of the phosphodiesters
investigated. Structures featuring p-xylyl and m-xylyl spa-
cers turned out to have the proper geometries to act as pre-
dicted or, at least, they do not hint at any adverse features,
and are shown in Figure 1.
Figure 1. Computed structures of (A) [Zn2(DTMX)-
(dmp)(H2O)2]2ϩ and (B) [Zn2(DTPX)(dmp)(H2O)2]2ϩ. Hydrogen
atoms omitted for clarity
The intermetallic distances calculated for the two com-
˚
˚
plexes are 5.0 A in the case of DTMX and 5.2 A in DTPX;
such distances are within the range of those reported for
metalloenzymes. The same kind of calculations for TACI-
based dimetallic complexes having different, but apparently
suitable, spacers, such as 1,8- or 2,7-dimethylnaphthalene,
led to strained structures and greater inter-metal distances.
These results directed our choice to the two meta- and para-
xylyl linkers, which was also guided in view of their de-
creased hydrophobic character.
As for the substrates, HPNP, which bears a 2-hydroxy-
propyl residue, is the activated diester usually taken as an
Scheme 1. Structures of ligands and substrates used in this work
282
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Catalysts of Phosphate Esters Cleavage
FULL PAPER
RNA model and it may be compared with the diester
DNPEP, a DNA model, and with the triester DNPDEP.
Synthesis and Characterization of DTMX and DTPX
Handling the TACI molecule to realize its desired N-
monoalkylation is a difficult task, as is predictable in view
of its multifunctionality.[19] Furthermore, the number of ac-
cessible synthetic pathways is greatly reduced because the
solubility of TACI is limited to water and methanol. Early
attempts aimed at achieving the BOC protection of two of
the three amino groups failed to give reasonable yields of
the desired intermediate since the reaction of TACI with
even less than one equivalent of (BOC)2O yielded, in each
case, only the triprotected product. Then, was uncovered a
successful route (Scheme 2) by the synthesis of the di-N-
BOC-protected precursor 4. This compound was obtained
in reasonable yield only by an extremely slow (1 day) ad-
dition of (BOC)2O to a very dilute (1Ϫ2 m) solution of
TACI in methanol at low temperature, followed by column
chromatography. Compound 4, once purified, was treated
with the requisite meta- or para-α,αЈ-dibromoxylene to give
5 and 6 and these compounds were deprotected to give the
final product(s) (as hexahydrochloride salts) in fair yields.
Figure 2. UV/Vis titration of DTMX (squares, 0.18 m) and
DTPX (black circles, 0.19 m) with Cu(NO3)2 at 647 nm. (MES
buffer 0.05 , pH 6.3, 25 °C)
In the case of the ZnII complex, the binding of the metal
ion was investigated by potentiometric titrations, which al-
lowed us to evaluate the values of pKa of the amino groups
(pKn), the formation constant of the ZnII complexes (log
βn), and the values of pKa of the acid dissociation of the
water molecule(s) bound to the metal ion(s) (pKan) (Table 1).
The values of pKa of the ammonium ions are reasonably
similar to those of TACI when taking into account the elec-
trostatic effects at play in polyacidic compounds. These ef-
fects are likely responsible for the lower complex-formation
constants, particularly the second one, for DTMX. The rel-
atively small binding constant of the second ZnII ion in this
dinuclear complex was a matter of concern; under the con-
ditions used for the reactivity experiments (see below), it
can be evaluated that at values of pH larger than 8Ϫ9, ca.
80% of the ligand is in the form of dimetallic complexes.
Kinetic Studies
We carried out our first preliminary kinetic measure-
ments in water at pH 9 and 25 °C, employing DTMX and
DTPX and either CuII or ZnII in a 1:2 molar ratio, for the
cleavage of the three substrates of choice, namely DNPEP,
DNPDEP, and HPNP. Table 2 shows the apparent pseudo-
first-order rate constants (kψ) together with those measured
for TACI under the same conditions.
The main findings are the following. The effects of the
metal ion complexes depend strongly on the structure of
the complex and, in several cases, the efficiency of the di-
Scheme 2. Synthetic pathway to DTMX and DTPX
Both ditopic ligands are soluble in water at up to 2 m metallic ones turns out to be undeniably disappointing
at room temperature. They can complex two CuII or ZnII when the control complexes of TACI are taken as a refer-
metal ions. In the case of the copper complex, this binding ence. In fact, the dinuclear CuII complexes are remarkably
is clearly indicated by changes in the absorbance at 647 nm less effective than the mononuclear TACI analog, particu-
(the region of the dϪd transition of the ion), as shown in larly in the transesterification of HPNP (by two orders of
Figure 2 for DTMX and DTPX following the addition of magnitude) and in that of DNPEP for which a measurable
Cu(NO3)2 at pH 6.3. The titration profiles of both ligands reactivity (after reasonable observation times) was recorded
display a linear increase up to a ratio [CuII]/[ligand] of 2:1, only for CuII·TACI. On the other hand, when using the ZnII
which indicates rather large formation constants for the complexes, the effects of all the complexes used (including
mono- and di-nuclear complexes.
that of TACI) are comparable and larger that those of the
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F. Mancin, E. Rampazzo, P. Tecilla, U. Tonellato
FULL PAPER
Table 1. Ligand deprotonation constants (Kn), stepwise ZnII complex-formation constants (βn), and deprotonation constants of ZnII-
bound H2O [Kan (H2O)] for TACI, DTMX, and DTPX at 25 °C, in 0.1  NaCl (estimated errors in the values of these thermodynamic
constants are Ϯ5%)
Ligand[a][b]
pK1
pK2
pK3
pK4
pK5
pK6
log β1
log β2
pKa1 (H2O)
pKa2 (H2O)
TACI[c]
DTMX
DTPX
8.86
8.98
9.30
7.40
8.39
8.87
6.02
7.33
7.65
8.39
7.75
8.05
8.56
7.66
8.68
6.73
6.95
5.67
5.65
4.99
5.26
4.71
7.21
8.44
Ϫ[d]
[a]
[b]
[c]
[d]
Kn ϭ [HnϪ1L][H3Oϩ]/[HnL]. βn ϭ [ZnnL]/[ZnII][ZnnϪ1L]. Ref.[10]
.
Not accessible.
Table 2. Rates for the transesterification of HPNP and cleavage of
DNPEP and DNPDEP in the presence of ZnII and CuII complexes
of DTMX, DTPX, and TACI at 25 °C and pH 9 (CHES buffer,
0.05 ). The estimated errors in the vales of the kinetic constants
are Ϯ1%
Ligand
Substrate
kψ (CuII), sϪ1
kψ (ZnII), sϪ1
DTMX[a]
DTPX[b]
TACI[c]
HPNP
HPNP
HPNP
DNPDEP
DNPDEP
DNPDEP
DNPEP
DNPEP
DNPEP
5.0 ϫ 10Ϫ6
6.1 ϫ 10Ϫ6
1.7 ϫ 10Ϫ4
4.9 ϫ 10Ϫ5
5.2 ϫ 10Ϫ5
6.0 ϫ 10Ϫ4
Ϫ[d]
4.2 ϫ 10Ϫ5
7.8 ϫ 10Ϫ5
1.8 ϫ 10Ϫ4
8.9 ϫ 10Ϫ5
1.4 ϫ 10Ϫ4
2.1 ϫ 10Ϫ4
Ϫ[d]
Figure 3. Proposed structure of a µ-hydroxo-bridged dimetallic
complex
DTMX[a]
DTPX[b]
TACI[c]
as an RNA model but also because we have recently investi-
gated in detail the mechanism of its transesterification pro-
moted by ZnII·TACI.[10]
First we analyzed the effect of pH on the reaction rate
using the same concentrations and ratios employed in the
preliminary experiments. Figure 4, A, displays the rateϪpH
profiles obtained with the two dimetallic ZnII complexes.
DTMX[a]
DTPX[b]
TACI[c]
Ϫ[d]
Ϫ[d]
4 ϫ 10Ϫ5
Ϫ[d]
[a] [DTMX] ϭ 0.44 m, [Metal] ϭ 0.88 m. [b] [DTPX] ϭ 0.47 m,
[Metal] ϭ 0.94 m.
No reaction observed.
[c]
[d]
[TACI] ϭ 0.98 m, [Metal] ϭ 0.98 m.
CuII analogs, at least in the cases of DNPDEP and HPNP.
The much greater efficiencies of the dinuclear ZnII com-
plexes relative to those of the CuII complexes, which is a
finding that is at variance with those commonly reported
for mononuclear complexes, is not unprecedented. The
main reason for this finding is quite likely to be ascribed to
the easier formation of µ-hydroxo bridges between two
copper ions than between two zinc ions. Such bridged spec-
ies are known to be poorly active also in monometallic sys-
tems,[20,21] as in the case of CuII·TACI complexes, which
have a greater tendency to form dimeric forms than do
complexes of other tripodal ligands.[22,23] In the dimetallic
complex, the two CuII ions may face each other readily so
that the formation of intramolecular bridges (Figure 3) is
further favored and, hence, their disruption, which is a pre-
requisite for the productive binding of a phosphate ester, is
more difficult. The combination of the formation of bridges
and the limited affinity of phosphate esters to the metal ion
leads to the poor reactivity displayed by the CuII complexes
of DTMX and DTPX.
Figure 4. (a) The pH dependence of the rate constant for HPNP
transesterification promoted by the ZnII2·DTMX (squares) and
ZnII2·DTPX complexes (black circles). Conditions: [ligand] ϭ 5.0
ϫ 10Ϫ4 , [ZnII] ϭ 1.0 ϫ 10Ϫ3 , [Buffer] ϭ 0.05  (see Exp. Sect.),
25 °C. (b) The pH dependence of the rate constant for HPNP trans-
esterification promoted by the ZnII·TACI complex. Conditions: [li-
gand] ϭ 1.0 ϫ 10Ϫ3 , [ZnII] ϭ 1.0 ϫ 10Ϫ3 , [Buffer] ϭ 0.05 ,
25 °C
In both cases, we obtained bell-shaped curves having
maxima at ca. pH 8.7Ϫ9. The two complexes exhibit
slightly different behavior and the DTPX system is more
reactive by a factor of ca. 2 than is DTMX in the pH range
8Ϫ9.5. This behavior is diagnostic of the deprotonation of
two acidic functions of the complex: the first deprotonation
event leading to a reactivity increase and the second to the
The ZnII Complexes as Catalysts of HPNP
Transesterification
The preliminary results led us to restrict our further kine- opposite effect. At variance with the rate profile of the di-
tic studies to the dinuclear ZnII complexes as catalysts of metallic complexes, that of ZnII·TACI describes a clean sig-
HPNP transesterification. The attention devoted to this moidal curve (Figure 4, B) that is diagnostic of a single fa-
substrate not only was due to the fact that it can be taken vorable deprotonation. A comparison of the data at differ-
284
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Catalysts of Phosphate Esters Cleavage
FULL PAPER
ent values of pH indicates that the dinuclear complexes are ZnII2·DTMX, and ZnII2·DTPX complexes. Under these
slightly more efficient (1.5-fold) than the mononuclear one conditions ([complex] ϭ 0.05 m; [substrate] ranging from
under neutral conditions (pH 7Ϫ7.8), but the opposite is 0.2 to 3 m; pH 9), complete cleavage of the substrate was
true at higher values of pH. More interestingly, the achieved and we observed no detectable rate effects that are
rateϪpH profiles obtained for the monometallic TACI due to product formation. Thus, the complexes employed
complex and for the dimetallic catalysts have quite different act as real catalysts (i.e., with turnover). The profiles of rate
shapes, which is diagnostic of different mechanistic pat- versus substrate concentration possess a moderate curva-
terns, as is expected for the dimetallic systems assuming co- ture as a result of the modest degree of binding of the sub-
operativity of the two reactive sites.
strate to the catalyst; at any rate, the analysis of data al-
In the concentration range 0.1Ϫ0.6 m, the profiles of lowed us to estimate (albeit with a rather large error) the
reaction rate versus complex concentration at pH 9 were binding constant of the substrate to the catalyst, Kb, and
linear, which indicates that no intramolecular reaction oc- the limiting rate constant, klim, which are reported in
curs. The apparent second-order rate constants determined Table 3.[24]
for these profiles are 0.19, 0.12, and 0.060 Ϫ1 secϪ1 for
ZnII·TACI, (Zn)2·DTPX, and (Zn)2·DTMX, respectively.
To evaluate the possible benefits or disadvantages of such
cooperativity in terms of reactivity, we carried out kinetic
experiments using a fixed concentration of ditopic ligand
and increasing amounts of the metal ion (pH 8.0, 25 °C).
The results are shown in the diagram (Figure 5) of the rate
vs. the [ZnII]/[DTPX] ratio.
Table 3. Kinetic and thermodynamic parameters for the transesteri-
fication of HPNP promoted by the ZnII complexes of TACI,
DTMX, and DTPX. Estimated errors are within 10%
Complex[a]
Kb (Ϫ1
)
klim (sϪ1
)
TACI·ZnII [b]
33
107
67
9.7 ϫ 10Ϫ3
1.5 ϫ 10Ϫ3
2.6 ϫ 10Ϫ3
[c]
DTMX·ZnII
2
DTPX·ZnII
[c]
2
[a]
[b]
Conditions: CHES buffer 0.05 , pH 9.0, 25 °C.
[ligand] ϭ
5.0 ϫ 10Ϫ4 , [ZnII] ϭ 5.0 ϫ 10Ϫ4 . [ligand] ϭ 2.5 ϫ 10Ϫ4 ,
[c]
[ZnII] ϭ 5.0 ϫ 10Ϫ4 .
It appears that, on one hand, the intrinsic reactivity of
ZnII·TACI is larger than that of the dimetallic complexes
and, on the other, that they have a larger affinity (the abso-
lute values are in each case rather small) for the substrate;
on the whole, the balance of these parameters still makes
the TACI system the most effective catalyst.
The cleavage of HPNP promoted by ZnII·TACI occurs
by a transesterification process that results in the formation
of a cyclic phosphate ester and p-nitrophenol. In this case,
the suggested mechanism involves direct coordination to
the metal ion of the substrate alcoholic function and its
subsequent nucleophilic attack on phosphorus leading to
the cyclic phosphate (Scheme 3, A).[10] There are no reasons
Figure 5. Kinetic profile for the transesterification of HPNP as a
function of the ZnII/DTPX ratio, in HEPES buffer (0.05 ) at pH
8. [DTPX] ϭ 5.0 ϫ 10Ϫ4 , 25 °C
The rate profile describes a smooth sigmoidal curve that to assume that the outcome of the reaction catalyzed by the
reaches a plateau for ratios between 2 and 2.5. The reaction dimetallic ZnII complexes investigated here are different.
rate observed after the introduction of the second metal ion The mechanistic details, however, are substantially different
is ca. 2.3-times larger than that observed in the presence of insofar as the reported results point to the fact that the two
one equivalent of ZnII, which indicates that the effect of reacting sites somehow cooperate, as is indicated clearly by
`
the two complexed subunits is more than additive and is the pHϪrate profiles, vis-a-vis that of TACI in Figure 4 and
diagnostic of synergistic effects. A different profile (not by the kinetic titration diagram of Figure 5. Bell-shaped pH
shown) was observed with the ligand DTMX; in this case, profiles are often observed in the cleavage of phosphodies-
the kinetic data describe an almost linear curve up to 2 ters by dimetallic complexes.[15,16] In principle, the cooper-
equiv. of added metal ion that then proceeds smoothly to ative effects of the two ZnII ions and the increased affinity
saturation such that a plateau of reactivity is not reached of the dimetallic complexes for the substrate may be ex-
even after addition of 3 equiv. This behavior can probably plained by two main reasonable, and kinetically indis-
be ascribed to the low formation constant of the dimetallic tinguishable, mechanistic hypotheses (Scheme 3, B؊C):
complex of DMTX (see Table 1), such that the transition case B indicates that the bound substrate is activated by the
from the mono- to dinuclear complex is rather difficult and two metal ions for a nucleophilic attack and that a ZnII-
may mask the synergistic effects at play in the case of coordinated water molecule, in turn, acts as a base to de-
DPTX.
protonate the substrate’s hydroxy group that subsequently
Finally, we performed kinetic experiments using HPNP acts as the nucleophile; case C involves substrate activation
concentrations that largely exceed those of the ZnII·TACI, again by the two metal ions and concomitant coordination
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F. Mancin, E. Rampazzo, P. Tecilla, U. Tonellato
FULL PAPER
to one of them to the substrate’s hydroxy group, which is, culties implicit in the design of the dimetallic hydrolytic
thus, activated as the nucleophile. The bell-shaped profile catalysts cannot be underestimated. As a matter of fact,
can be explained by assuming that each metal ion weakly comparison of the efficacy of the ZnII complexes of TACI
binds a water molecule and the substrate in a competitive and of DTMX or DTPX, and the inefficacy of the CuII
way up to the pH value at which deprotonation of the water complexes, could simply suggest that the synthetic effort ex-
molecule occurs to give a stronger base, but also a stronger pended to obtain the ditopic ligands did not pay off, at least
ligand. Thus, there are two opposite effects at play in the it has not so far. Much work is needed to understand all the
bell-like region: (i) acid dissociation of a metal-bound water factors at play to design and realize a really potent catalyst.
molecule (or of the substrate hydroxy group) to give a better Placing two metal ions at a short distance in not a sufficient
base or nucleophile that still does not impair substrate bind- requirement for obtaining great rate accelerations. Factors
ing; (ii) acid dissociation of the second water molecule to such as the formation of intracomplex µ-hydroxo bridges
generate a bound hydroxide ion that weakens the associ- must be taken in the account as they can lead to significant
ation of the substrate. In the region of low pH of the ‘‘bell,’’ reactivity losses. Unexpected, modest accelerations ob-
the balance of these effects is favorable to an enhanced reac- tained by other dimetallic catalyst, at least in aqueous solu-
tivity while the opposite effect occurs in more alkaline solu- tions, are likely to meet similar problems.[26,28]
tions. The mechanistic hypotheses above offer a rationale
for the bell-shaped behavior, but not for the rather modest
reactivity, at least when compared to that of ZnII·TACI.
This complex is slightly less reactive than the dinuclear
Experimental Section
complexes only below pH 7.5, but is more reactive by a
factor of ca. 2 at pH 8.7, at the top of the bell-like profile, commercially available reagents and substrates were used as re-
General Remarks: Solvents were purified by standard methods. All
ceived. TLC analyses were performed using Merck 60 F254 pre-
coated silica gel glass plates. Column chromatography was carried
out on MachereyϪNagel silica gel 60 (70Ϫ230 mesh). Melting
points were determined with a Buchi 510 capillary melting point
apparatus and are uncorrected. NMR spectra were recorded using
a Bruker AC250F (250 MHz). Chemical shifts are reported relative
to internal Me4Si. Multiplicity is given as usual. Elemental analyses
were performed by the Laboratorio di Microanalisi of the Inor-
ganic and Analytic Chemistry Department of the University of Pa-
dova. UV/Vis absorption measurements were performed with a
and is remarkably more reactive at higher values of pH.
Clearly, other factors at play defy a simple explanation. One
of them, supported by the behavior of the CuII complexes,
could imply the formation, also in the case of ZnII, of µ-
hydroxo bridges that have a severe adverse effect on the
reactivity. A similar effect has been suggested for a related
di-ZnII complex that features a m-xylyl spacer.[25] In ad-
dition, the greater reactivity of the DTPX complex, relative
to that of DTMX, supports this hypothesis; in fact, inspec-
tion of molecular models indicates that the increased inter- PerkinϪElmer Lambda 16 spectrophotometer equipped with a
thermostatted cell holder. Potentiometric titrations were performed
using a Metrohom 716 DMS Titrino dynamic titrator. Cu(NO3)2
and Zn(NO3)2 were analytical grade products. Metal ion stock
solutions were titrated against EDTA following standard pro-
cedures. The buffer components were used as supplied by the
manufacturers: acetic acid (Aldrich), 2-morpholinoethanesulfonic
acid (MES, Fluka), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES, Sigma), 4-(2-hydroxyethyl)-1-piperazinepropanesul-
fonic acid (EPPES, Sigma), 2-[cyclohexylamino]ethanesulfonic acid
(CHES, Sigma), and 3-(cyclohexylamino)-1-propanesulfonic acid
(CAPS, Sigma). The 2,4-dinitrophenyl ethyl phosphate (DNPEP)
lithium salt,[29] the 2,4-dinitrophenyl diethyl phosphate
(DNPDEP),[29] and the 2-hydroxypropyl p-nitrophenyl phosphate
barium salt (HPNP)[30] were prepared and purified following litera-
ture methods. all-cis-2,4,6-Triamino-cyclohexane-1,3,5-triol (TACI,
1) was prepared as reported.[31] DTMX and DTPX were prepared
starting from TACI according to Scheme 2.
metallic distance in the ‘‘para’’ complex makes the forma-
tion the µ-hydroxo bridges more difficult.
Scheme 3. Proposed mechanisms for the transesterification of
HPNP promoted by the ZnII complexes
Conclusion
The results of this present investigation, on the use of all-cis-2-Amino-4,6-bis(tert-butoxycarbonylamino)cyclohexane-
1,3,5-triol (4): A solution of (BOC)2O (1.16 g, 5.32 mmol) in dry
CH2Cl2 (950 mL) was added dropwise (ca. 60 mL/min) to a solu-
tion of TACI (0.42 g, 2.4 mmol) in dry methanol (950 mL) that was
cooled in a ice/brine bath. At the end of the addition, the reaction
mixture was stirred at room temperature for 12 h with monitoring
by TLC [CH2Cl2/MeOH/NH3(aq), 10:2:0.2; Rf (4) ϭ 0.36]. The sol-
vent was evaporated under reduced pressure and purification of
the crude product was performed by flash column chromatography
[SiO2; CH2Cl2 /MeOH/NH3(aq), 10:1:0.1] to yield 4 as a white solid
(0.36 g, 41%). 1H NMR (250 MHz, CD3OD, 25 °C, TMS): δ ϭ
dimetallic complexes based on TACI ligand subunits as
catalysts for the cleavage of phosphate esters in water, offer
a mixed picture. The ZnII complexes rank among the good
artificial ZnII-based catalysts for the transesterification of
HPNP;[5,11,15,26Ϫ28] the kinetic enhancements over the rate
of spontaneous hydrolysis recorded at pH 9 are in the range
of two order of magnitude and compare well to those re-
ported for other binuclear catalytic complexes. Moreover,
they indicate cooperativity of the two reactive sites. On the
other hand, the results obtained also highlight that the diffi- 1.46 (s, 18 H, tBu), 2.78 (s, 1 H, ϪCHϪNH2), 3.60 (s, 2 H,
286  2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.eurjoc.org
Eur. J. Org. Chem. 2004, 281Ϫ288

Catalysts of Phosphate Esters Cleavage
FULL PAPER
ϪCHϪNHϪBOC), 3.84Ϫ3.97 (m, 3 H, ϪCHϪOH) ppm. 13C produce was eluted through an ion-exchange column (DOWEX
NMR (63 MHz, CD3OD, 25 °C, TMS): δ ϭ 27.7, 52.6, 52.9, 71.8, 50WX2, ClϪ form) and 3 (white solid) was obtained as its hexahy-
1
73.6, 79.6, 156.3 ppm.
drochloride salt (0.062 g, 89%). H NMR (250 MHz, D2O, 25 °C,
TMS): δ ϭ 3.55 (t, J ϭ 3 Hz, 2 H, ϪCHϪNHϪ), 3.61 (t, J ϭ
3 Hz, 4 H, ϪCHϪNH2), 4.35 (m, 6 H, ϪCHϪOH), 4.46 (s, 4 H,
ArϪCH2ϪNHϪ), 7.51 (s, 4 H, HAr) ppm. 13C NMR (63 MHz,
D2O, 25 °C, TMS): δ ϭ 50.0, 52.4, 58.4, 66.4, 67.7, 132.9, 133.5
ppm. C20H42Cl6N6O6·6H2O (783.4): calcd. C 30.67, H 6.89, N
10.73; found C 30.71, H 6.35, N 10.23.
all-cis-4,4Ј,6,6Ј-Tetrakis(tert-butoxycarbonylamino)-6,6Ј-m-
xylylidenebis(cyclohexane)-1,3,5-triol (5): α,αЈ-Dibromo-p-xylene
(0.103 g, 0.390 mmol) was added to a solution of 4 (0.303 g,
0.804 mmol) in dry DMF (4 mL) in the presence of diisopropyle-
thylamine (0.50 mL, 2.9 mmol). The reaction mixture was heated at
70 °C for 48 h with monitoring by TLC [CHCl3/MeOH/NH3(aq),
10:2:0.2; Rf (5) ϭ 0.51]. After this time, CH2Cl2 (150 mL) was ad-
ded and the organic layer was extracted with 10% Na2CO3 solution
(3 ϫ 50 mL) and brine (3 ϫ 50 mL). The organic phase was dried
(Na2SO4) and the solvent was evaporated. Purification of the crude
product was performed by flash column chromatography [SiO2;
CHCl3/MeOH/NH3(aq), 10:1.5:0.15] to obtain 5 as a pale-yellow
Potentiometric Titrations: Protonation constants and ZnII complex-
formation constants for ligands 1Ϫ3 were determined by pH
potentiometric titrations (25 °C, 0.10  NaCl). Solutions contain-
ing ca. 1 ϫ 10Ϫ3  of the hydrochloride salts of the ligands and,
when necessary, Zn(NO3)2 were titrated using a 0.1  sodium hy-
droxide solution. The electrode system was calibrated by titrating
a 0.01  solution of HCl so that the value of pKw was 13.78. The
pH and the volume of added NaOH data were fitted with the com-
puter program BEST[32] to obtain the desired protonation and
complex-formation constants.
1
solid (0.128 g, 39%). H NMR (250 MHz, CD3OD/CDCl3 4:1, 25
°C, TMS): δ ϭ 1.47 (s, 36 H, tBu), 2.77 (s, 2 H, Ar-CH2-NH-CH),
3.59 (s, 4 H, ϪCHϪNHϪBOC), 3.93 (m, 6 H, ϪCHϪOH), 4.13
(s, 4 H, arom.ϪCH2ϪNHϪ), 7.34Ϫ7.45 (m, 3 H, HAr), 7.69 (s, 1
H, HAr) ppm. 13C NMR (63 MHz, CD3OD/CDCl3, 4:1, 25 °C,
TMS): δ ϭ 28.5, 52.0, 58.0, 69.1, 72.3, 80.0, 129.4, 155.7 ppm.
Kinetic Measurements: The kinetic traces were recorded on a
PerkinϪElmer Lambda 16 spectrophotometer equipped with a
thermostatted cell holder. The reaction temperature was main-
tained at 25 Ϯ 0.1 °C. The reactions were started by adding a solu-
tion of substrate (1Ϫ2 ϫ 10Ϫ3 ; 20 µL) to a solution of metal
complex in the appropriate buffer (2 mL) and monitored by follow-
ing the absorption of p-nitrophenoxide at 400 nm. The initial con-
centration of substrate was 1Ϫ2 ϫ 10Ϫ5  and the kinetics were in
each case first-order up to 90% of the reaction. The pseudo-first-
order rate constants were obtained by non-linear regression analy-
sis of the data from a plot of absorbance vs. time and the fit error
on the rate constant was always less than 1%.
all-cis-4,4Ј,6,6Ј-Tetrakis(tert-butoxycarbonylamino)-6,6Ј-m-
xylylidenebis(cyclohexane)-1,3,5-triol (DTMX, 2) Hexahydrochlo-
ride Salt: 5 (0.128 g, 0.149 mmol) was treated with CH2Cl2/TFA
(2:1, 9 mL) for 4 h with monitoring by TLC [CHCl3/MeOH/
NH3(aq), 10:2:0.2]. The solvent was evaporated under reduced
pressure and then water (a few mL) was added to the resulting
solid. The crude product was eluted through an ion-exchange col-
umn (DOWEX 50WX2, ClϪ form) and 2 (white solid) were ob-
tained as its hexahydrochloride salt (0.082 g, 70%). 1H NMR
(250 MHz, D2O, 25 °C, TMS): δ ϭ 3.62Ϫ3.74 (m, 6 H, ϪCHϪNH
and ϪCHϪNH2), 4.45 (m, 6 H, ϪCHϪOH), 4.59 (s, 4 H,
ArϪCH2ϪNHϪ), 7.44Ϫ7.67 (m, 4 H, HAr) ppm. 13C NMR
(63 MHz, D2O, 25 °C, TMS): δ ϭ 50.1, 52.3, 58.4, 66.3, 67.6, 132.1,
132.7, 133.4, 133.7 ppm. C20H42Cl6N6O6·6H2O (783.4): calcd. C
30.67, H 6.89, N 10.73; found C 30.76, H 6.11, N 10.04.
In the case of the experiments performed in the presence of an
excess of HPNP, the reactions were started by adding a solution of
the substrate (2.57 ϫ 10Ϫ2 ; 30 µL) to a solution of the metal
complex in CHES buffer (2 mL) at pH 9.0 and were monitored
by following the absorption of p-nitrophenoxide. Reactions were
followed up to ca. 1% of substrate hydrolysis (ca. 10 h). The
pseudo-first-order rate constants were obtained from the slope of
the plot of absorbance versus time (the fit error was always less
than 1%) divided by the absorbance of p-nitrophenoxide and the
concentration of the substrate.
all-cis-4,4Ј,6,6Ј-Tetrakis(tert-butoxycarbonylamino)-6,6Ј-p-
xylylidenebis(cyclohexane)-1,3,5-triol (6): α,αЈ-Dibromo-p-xylene
(0.061 g, 0.23 mmol) was added to a solution of 4 (0.18 g,
0.48 mmol) in dry DMF (2 mL) in the presence of diisopropylethyl-
amine (0.5 mL, 2.9 mmol). The reaction mixture was heated at 70
°C for 48 h with monitoring by TLC [CHCl3/MeOH/NH3(aq),
10:2:0.2; Rf(6) ϭ 0.56]. After this time, CH2Cl2 (150 mL) was added
and the organic layer was extracted with 10% Na2CO3 solution (3
ϫ 50 mL) and brine (3 ϫ 50 mL). The organic phase was dried
(Na2SO4) and the solvent evaporated. Purification of the crude
product was performed by flash column chromatography [SiO2;
CHCl3/MeOH/NH3(aq), 10:1.5:0.15] to obtain 6 as a pale-yellow
Acknowledgments
Financial support for this research has been partly provided by the
Ministry of Instruction, University and Research (MIUR contracts
2001038212 and 2002031238) and by University of Padova (Young
Researchers Grant CPDG022585).
1
solid (0.088 g, 45%). H NMR (250 MHz, CD3OD/CDCl3 4:1, 25
°C, TMS):
ArϪCH2ϪNHϪCHϪ), 3.55 (s,
δ
ϭ
1.46 (s, 36 H, tBu), 2.67 (s,
2 H,
4
H, ϪCHϪNHϪBOC),
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Eur. J. Org. Chem. 2004, 281Ϫ288