Tripodal, cooperative, and allosteric transphosphorylation metallocatalysts

Article abstract of DOI:10.1021/jo061754k

Three artificial amino acids derived from L-serine by replacing the hydroxyl moiety with 1,4,7-triazacyclononane, 1,5,9-triazacyclododecane, and 1,4,7,10-tetraazacyclododecane, respectively, have been connected to the three arms of the tetraamine tris(2-a

Full text of DOI:10.1021/jo061754k

Tripodal, Cooperative, and Allosteric Transphosphorylation
Metallocatalysts
Alessandro Scarso,† Giovanni Zaupa, Florence Bodar Houillon, Leonard J. Prins,
and Paolo Scrimin*
UniVersity of PadoVa, Department of Chemical Sciences and CNR- ITM, PadoVa Section,
Via Marzolo, 1-35131 PadoVa, Italy
paolo.scrimin@unipd.it
ReceiVed August 25, 2006
Three artificial amino acids derived from L-serine by replacing the hydroxyl moiety with 1,4,7-
triazacyclononane, 1,5,9-triazacyclododecane, and 1,4,7,10-tetraazacyclododecane, respectively, have been
connected to the three arms of the tetraamine tris(2-aminoethyl)amine, Tren, to obtain tripodal ligands.
They are able to bind up to four metal ions (like CuII and ZnII), three with the polyazamacrocycles and
one with the Tren platform. Some of the ZnII complexes of these tripodal ligands proved to be good
catalysts for the cleavage of the RNA model substrate 2-hydroxypropyl-p-nitrophenylphosphate (HPNP).
Studies of the catalytic activity in the presence of increasing amounts of ZnII show that the complexes
represent minimalist examples of metallocatalysts with cooperativity between the metal centers and
allosteric control by a metal ion. The Tren binding site constitutes the allosteric regulation unit, while
the three ZnII-azacrown complexes provide the cooperative, catalytic site. The allosteric role of the ZnII
ion located in the Tren binding site was unambiguously demonstrated by studying the catalytic activity
of a derivative unable to complex ZnII in that site. In this case, the cooperativity between the three ZnII
ions bound to the peripheral azacrowns was totally suppressed. The kinetic analysis has shown that
cooperativity is due to neither the occurrence of general-acid/general-base catalysis nor a decreased binding
of the substrate because of the deprotonation of a water molecule bound to the complex but, rather,
stabilization of the complexed substrate in its transformation into the transition state.
Introduction
far from this sluggish reactivity should be the time required for
hydrolytically cleaving the P-O bond of DNA. RNA is more
labile because the nucleophilic attack on phosphorus is per-
formed intramolecularly by the -O(H) in the 2′ position of the
ribose. Thus, the half-life is, in this case, only 104 years. These
processes can be accelerated by several orders of magnitude
by metal ions and a considerable number of laboratories has
embarked in the challenging goal of preparing powerful, metal
ion-based catalysts with, in some cases, interesting results.3
The hydrolytic cleavage of the phosphate bond under physi-
ological conditions is incredibly slow. So slow that, in the case
of phosphate diesters, the determination of reliable rate constants
has been elusive, with numbers that have been updated to a
slower figure almost yearly. The latest values indicate a half-
life of 1010 years for the cleavage of dimethyl phosphate.1,2 Not
* To whom correspondence should be addressed. Tel: +39-049-8275276.
Fax: +39-049-8275239.
(1) William, N. H.; Wyman, P. Chem. Commun. 2001, 1268-1269.
(2) For an update see: Wolfenden, R. Chem. ReV. 2006, 106, 3379-
3396.
† Current address: University of Venice, Calle Larga Santa Marta 2137, 30123
Venezia, Italy.
10.1021/jo061754k CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/20/2006
376
J. Org. Chem. 2007, 72, 376-385

Transphosphorylation Metallocatalysts
Chin4 has estimated that by simply adding up independent
contributions to the catalysis by two cooperating metal ions, a
catalyst could bring up to about 18 orders of magnitude in rate
acceleration. Such a catalyst will, however, remain the Holy
Grail for scientists for many years to come. In the meantime,
addressing fundamental points pertinent to the metal-catalyzed
hydrolytic process may provide key information for its design.
One of these issues is cooperativity: indeed, not only the above-
mentioned estimates made by Chin but most of the enzymes
devoted to the hydrolytic cleavage of the phosphate ester bond
point to a key role played by more than one metal ion in the
catalytic process.5 Following these insights, dinuclear and
trinuclear complexes of CoIII, CuII, and ZnII have been synthe-
sized and shown to be better catalysts for the cleavage of these
substrates.6 Because of the faster reactivity than that of DNA,
RNA and RNA model derivatives are quite popular substrates.
Another important feature present in many enzymes (includ-
ing metalloenzymes) is the presence of allosteric sites devoted
to the control of the structure of the catalyst. They are not
involved directly in the catalytic process but, rather, fine-tune
the activity of the catalyst by stabilizing its active conformation.
An example is provided by E. coli alkaline phosphatase, an
enzyme that catalyzes the cleavage of phosphate monoesters.
In its active site, two ZnII ions are directly involved in the
catalytic process, while 6 Å apart there is a MgII ion that acts
as a strong allosteric activator. Thus, all three ions are necessary
for catalysis but with rather different roles.
FIGURE 1. Conformational change associated to the binding of a
metal ion (MII) to Tren. The diamonds represent hypothetical functional
groups linked to the three arms.
efficiently phosphate anions.13 The tetraamine, upon metal ion
binding, changes its conformation from an open one, where the
three arms stay far apart in order to minimize charge repulsion
between the protonated amines, to a closed one necessary for
metal ion coordination (see Figure 1).14
In this latter conformation, the three arms get closer, and if
properly functionalized with metal complexes, cooperativity
between them in an hydrolytic process may be observed. Thus,
the three arms must bear suitable ligands for metal ion
coordination but should also not get too close to avoid the
formation of µ-complexes that are known to be very little active
in hydrolytic catalysis.15,16 The interesting results obtained with
our original system based on polypeptide TP3 prompted us to
investigate simpler, synthetically more accessible catalysts where
the ligand subunits could be tuned to better enhance their
catalytic properties. Furthermore, an unambiguous proof of the
allosteric role of the Tren-bound metal ion was still lacking.
The tripodal systems we have designed and synthesized are
reported in Chart 1. The metal ion coordination units are
provided by artificial amino acids derived from L-serine where
the -OH has been replaced by a cyclic polyamine. These cyclic
ligands are known to coordinate very strongly transition metal
ions like CuII and ZnII. Furthermore, previous work by Kimura
has shown that the pKa of a water molecule bound to these ZnII-
polyamine complexes varies within the 7.3-9.8 interval.17
Accordingly, it should be possible to tune the activity of the
system also by modulating the acidity (and nucleophilicity) of
this metal-bound water molecule within a physiologically
relevant pH interval. A simple 1,4-disubstituted benzene acts
as spacer to avoid the collapse of the arms which would result
in inactive complexes.
Recently,7 in search of systems where metal ion cooperativity
in a catalytic process could be triggered by appropriate con-
formational changes induced by a metal ion placed in a remote
position, we have reported the catalytic activity of the tripodal
polypeptide TP3 in the transphosphorylation of the RNA model
substrate 2-hydroxypropyl-p-nitrophenylphopsphate (HPNP).
Allosteric synthetic catalysts have also been reported by others.8
Kra¨mer has reported9 a 2 + 1 metal ion synthetic phosphodi-
esterase where a metal ion acts as an allosteric regulator and
the remaining two constitute a functional (catalytic) site. A
similar example was reported by Shinkai and Takeuchi.10
Our control unit was based on tris(2-aminoethyl)amine (Tren),
a tripodal tetraamine that has proven to be a very versatile
platform for the allosteric control of the activity of rather
different synthetic systems ranging from HIV-1 protease inhibi-
tors11 to membrane permeability affectors.12 Recent work by
Anslyn has also shown that similar systems recognize very
(3) Williams, N. H.; Takasaki, B.; Wall, M.; Chin, J. Acc. Chem. Res.
1999, 32, 485-493.
Results and Discussion
(4) Mancin, F.; Scrimin, P.; Tecilla, P.; Tonellato, U. Chem. Commun.
2005, 2540-2548.
Syntheses. Our strategy toward the synthesis of Boc-protected
ligand-amino amides 2 and 3 follows the general procedure
reported in Scheme 1 and already employed by us for the
(5) Stra¨ter, N.; Lipscomb, W. N.; Klabunde, T.; Krebs, B. Angew. Chem.
1996, 108, 2158-2191; Angew. Chem., Int. Ed. Engl. 1996, 35, 2024-
2055. Wilcox, D. E. Chem. ReV. 1996, 96, 2435-2458.
(6) Molenveld, P.; Engbersen, J. F. J.; Reinhoudt, D. N. Chem. Soc. ReV.
2000, 29, 75-86.
(7) Scarso, A.; Scheffer, U.; Go¨bel, M.; Broxterman, Q. B.; Kaptein,
B.; Formaggio, F.; Toniolo, C.; Scrimin, P. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 5144-5149.
(12) (a) Scrimin, P.; Veronese, A.; Tecilla, P.; Tonellato, U.; Monaco,
V.; Formaggio, F.; Crisma, M.; Toniolo, C. J. Am. Chem. Soc. 1996, 118,
2505-2506. (b) Scrimin, P.; Tecilla, P.; Tonellato, U.; Veronese, A.; Crisma,
M.; Formaggio, F.; Toniolo, C. Chem. Eur. J. 2002, 8, 2753-2763.
(13) Tobey, S. L.; Jones, B. D.; Anslyn, E. V. J. Am. Chem. Soc. 2003,
125, 4026-4027.
(14) Scrimin, P.; Tecilla, P.; Tonellato, U.; Valle, G.; Veronese A.
J. Chem. Soc., Chem. Commun. 1995, 1163-1165.
(15) Williams, N. H.; Cheung, W.; Chin, J. J. Am. Chem. Soc. 1998,
120, 8079-8087.
(16) Hegg, L. E., Burstyn, J. N. Coord. Chem. ReV. 1998, 173, 133-
165.
(8) (a) Tee, O. S.; Bozzi, M.; Clement, N.; T. Gadosy, T. A. J. Org.
Chem. 1995, 60, 3509-3517. (b) Iglesias, E. J. Am. Chem. Soc. 1998, 120,
13057-13069. Rebek, J. Jr.; Costello, T.; Wattley, R. J. Am. Chem. Soc.
1985, 107, 7487-7493.
(9) Fritsky, I. O.; Ott, R.; Pritzkow, H.; Kra¨mer, R. Chem. Eur. J. 2001,
7, 1221-1231. Fritsky, I. O.; Ott, R.; Kra¨mer, R. Angew. Chem., Int. Ed.
2000, 39, 3255-3258.
(10) Takebayashi, S.; Ikeda, M.; Takeuchi, M.; Shinkai, S. Chem.
Commun. 2004, 420-421.
(11) Valente, S.; Gobbo, M.; Licini, G.; Scarso, A.; Scrimin, P. Angew.
Chem., Int. Ed. 2001, 40, 3899-3902.
(17) Kimura, E.; Koike, T. Comments Inorg. Chem. 1991, 11, 285-
301.
J. Org. Chem, Vol. 72, No. 2, 2007 377

Scarso et al.
CHART 1
synthesis of 1.18 This requires the Boc-protection of all amino
groups but one, of the corresponding cyclic polyamine and
reaction of the latter with the Z-protected lactone of serine. The
reactions give predominantly the desired amino acid and minor
amounts of the undesired amide (up to 20%). The amino acids
were then converted into the N-methyl amides by treatment with
methylamine and standard coupling reagents. The Z-protecting
group was removed by hydrogenation immediately before
coupling to the Tren platform. The most delicate step of this
procedure is the protection of the cyclic polyamines for which
dichloromethane (contrary to what we had reported previously)18
must be avoided, and a very slow addition of the required
amount of (Boc)2O is mandatory (see the Experimental Section
for details). Additionally, Boc-protected ligand 4, a structurally
simpler analogue of 1, was synthesized in two steps via
alkylation of di-Boc-protected 1,4,9-triazacyclononane with
N-(2-bromoethyl)phthalimide and subsequent deprotection with
hydrazine.
Subsequently, the Boc-protected azacrown ligands 1-4 (after
removal of the Z-group) were coupled to the modified Tren-
platform 10 as reported previously.12b After a final deprotection
step to remove the Boc-groups, the expected tripodal ligands
5-8 were obtained in moderate yield (Scheme 2). The synthesis
of the control compound 9, in which the secondary amines of
the Tren-binding site are acylated, required some additional
steps. Initially, the Boc-groups of compound 1112b were
removed, and the deprotected amines were reacted with acetic
anhydride. Next, the methyl ester was hydrolyzed under basic
conditions to give 12 which was finally reacted with ligand 4
in a similar manner as discussed before. Final deprotection of
the Boc-groups gave compound 9.
Metal Ion Binding. We have studied complex formation of
ligands 5-7 with two metal ions, CuII and ZnII. Tripodal ligands
5-7 have two different types of binding sites: the Tren-
templating unit and the three cyclic polyamines. Accordingly,
each of them can bind up to four metal ions with different
binding strength. We were not particularly interested in the
determination of the binding constants, which, on the basis of
(18) Rossi, P.; Felluga, F. Scrimin, P. Tetrahedron Lett. 1998, 39, 7159-
7162.
378 J. Org. Chem., Vol. 72, No. 2, 2007

Transphosphorylation Metallocatalysts
SCHEME 1. Synthesis of the Z-Protected Amino Acids 1-3 and of the Free Base 4
SCHEME 2. Synthesis of the Tripodal Ligands 5-9
the values reported in the literature19 should be large enough to
ensure the complete formation of the 4:1 complex even under
the most diluted reaction conditions. We were, on the contrary,
extremely interested in knowing exactly the sequence of binding
of the metal ions in the two different sites as the tripodal ligands
are progressively loaded with increasing amounts of CuII or ZnII.
Luckily enough, due to the different maximum of the absorption
bands of the complexes with CuII ions of the cyclic polyamines
(ca. 650 nm) and Tren (ca. 850 nm), this information could be
easily obtained for this metal via spectrophotometric titrations.
Figure 2a-c reports the number of ions bound to each site
during the titration of each tripodal ligand 5-7 with CuII.
Analysis of this curve reveals that in all cases the CuII ions
(19) Smith, R. M.; Martell A. E. In Critical Stability Constants;
Plenum: New York, 1989; Vol. 6.
J. Org. Chem, Vol. 72, No. 2, 2007 379

Scarso et al.
FIGURE 4. Kinetic effect on the observed rate constant for the
cleavage of HPNP exerted by ligands 5 (filled squares), 6 (filled circles),
and 7 (open circles) in the presence of increasing amounts of (a) ZnII
and (b) CuII ions. The open squares in (a) show the behavior of a
mixture of the trismethylamide of deprotected 10 (1 equiv) and fully
deprotected 1 acetylated at the R-amino group (3 equiv).7 (Conditions:
[catalyst] ) 2 × 10-4 M, [HPNP] ) 2 × 10-5 M, 40 °C, pH ) 7). The
solid lines are drawn to guide the eye.
FIGURE 2. Binding of CuII ions to the two different binding sites
present in the tripodal ligands 5-8: (a) 5, (b) 6, (c) 7, (d) 8. In all
cases, the open circles refer to the binding to the Tren site, while the
filled circles refer to the binding to the azacrown subunits. Condi-
tions: [ligand] ) 1.0 × 10-3 M, pH ) 6.3 (0.1 M MES buffer). The
solid lines are drawn to guide the eye.
putative catalytic site (see Scheme 3). In this site, the three
metals form a cluster where they may cooperate in the catalytic
process. The observed site-selectivity of CuII binding for 6
follows the relative strength of CuII complexation to Tren20 and
triazacyclododecane.21 We would have expected a similar
selectivity for 5, too, as the reported binding affinity of
triazacyclononane22 for CuII is more than 3 orders of magnitude
smaller than that for Tren, while in the case of 7 the inverse
should have occurred (as the affinity of cyclen for CuII is higher
than that for Tren23). Clearly, the substituents on the ligands
(particularly Tren or cyclen) decrease their affinity for the metal
ion in a way which is dependent on the final structure of the
tripodal catalyst.
A direct spectroscopic investigation could not be performed
with ZnII because of the lack of distinct absorption bands for
the complexes with this metal ion. However, competition
experiments with the CuII complexes revealed a quite similar
pattern in terms of selectivity (data not shown). Furthermore,
with ZnII, by following the shift of the signals of selected protons
of the ligands in the 1H-NMR spectrum we were able to
unambiguously confirm that the binding first occurs with the
Tren site and only subsequently with the azamacrocycles. Figure
3 reports this behavior for ligand 6. Only in the case of tripodal
ligand 5 does the selectivity appear to be slightly lower than
that observed with CuII. In general, we may conclude that the
FIGURE 3. Shifts of the 1H NMR signals of selected protons of ligand
6 upon addition of ZnII: CHCH2N- signal close to the triazacy-
clododecane (open symbols) and ortho protons of the aromatic spacer
close to the Tren arms (filled symbols).
first bind to the Tren site and only subsequently to the cyclic
amines. However, this selectivity of binding is modest in the
case of triazacyclononane (after the first equivalent of copper
is added to 5 only 50% of the Tren subsite is occupied by the
metal) and excellent in the case of both triazacyclododecane
and cyclen (more than 80% of the first equivalent of CuII binds
to Tren with these polyamines present in ligands 6 and 7,
respectively). Thus, upon progressively adding CuII ions to the
tripodal ligands, the metal first binds to Tren changing the
conformation of the system and, subsequently, the remaining
three metal ions bind to the cyclic polyamines defining the
(20) Bencini, A.; Valtancoli, B.; Golub, G.; Cohen, H.; Paoletti, P.;
Meyerstein, D. Inorg. Chim. Acta, 1997, 255, 111-115.
(21) Kodama, M. Bull. Chem. Soc. Jpn. 1997, 70, 1361-1368.
(22) Yang, R.; Zompa, L. J. Inorg. Chem. 1976, 15, 1499-1502.
(23) Akinori, J.; Akiyoshi, N.; Takashi, K.; Akira, O. Bull. Chem. Soc.
Jpn. 1983, 56, 3062-3064.
380 J. Org. Chem., Vol. 72, No. 2, 2007

Transphosphorylation Metallocatalysts
SCHEME 3. Proposed Scheme for the Sequential Occupation of the Metal-Binding Binding Sites in Tripodal Ligand 6 by CuII
and ZnII
SCHEME 4. Cleavage of HPNP
pattern described in Scheme 3 for CuII and ZnII binding to 6 is
also valid for the other ligands.
Transphosphorylation Catalysis. We have tested our tri-
podal metallocatalysts in the cleavage of HPNP, an activated
phosphate ester frequently used as a model for RNA. The release
of p-nitrophenol (or p-nitrophenolate, depending on pH) is
accompanied by the formation of a cyclic phosphate24 (Scheme
4) and can be easily followed spectrophotometrically. In order
to assess what is the best tripodal ligand and the nuclearity of
the most active complex, we have run kinetics at a fixed ligand
concentration and pH ([ligand] ) 2 × 10-4 M, pH ) 7, 40 °C)
in the presence of increasing amounts of metal ions (ZnII or
CuII). The results are reported in Figure 4. They reveal the
following: (a) the ZnII complexes are far better catalysts than
the CuII complexes; (b) in all cases the best catalyst is the
tetranuclear complex;25 (c) the best ligand appears to be 6, while
the poorest one is 7; (d) the sigmoidal shape of the curves for
the ZnII complexes may be indicative of allosteric control by a
metal ion or cooperativity between several metal centers.
Noteworthy, a mixture of the Tren platform and 3 equiv of Boc-
deproteced 2 showed almost no activity.7 Hence, it is a peculiar
behavior of the entire ligand (and not of the unassembled parts)
that we observe in the plots of Figure 4a. Because of the
structure of the ligands, it is conceivable that both allosteric
control and cooperativity are present with these systems. The
above data, however, do not give a clear-cut answer to this
question. Accordingly, this point will be examined in more detail
in a separate section.
Very surprisingly, the CuII complexes are poor catalysts, but
also the kinetic profiles do not indicate neither cooperativity or
allosteric control (Figure 4b). Our best explanation for the
observed behavior is that the substrate binds to the free apical
position of the metal ion of the Tren platform instead of to those
of the azamacrocycles. This is supported by the fact that the
maximum rate acceleration with this metal is observed when,
according to the plots of Figure 2, there is complete saturation
of the Tren subunit of ligands 5 and 6 (compare Figure 4b with
Figure 2a,b). It has been shown24 that metal complexes of Tren
are rather poor transphosphorylation catalysts in accord with
the modest rate accelerations we observe here. The reason why
HPNP interacts with the Tren-bound CuII rather than with the
metal ions bound to the azamacrocycles could be related to the
formation of inactive dimeric complexes of the latter.16
A
summary of the rate accelerations observed with these catalysts
is reported in Table 1.
The kinetics of Figure 4a were performed at pH 7, and under
those conditions, the best catalyst appears to be the tetranuclear
ZnII complex of ligand 6. In order to determine the kinetic pKa
(24) Bonfa`, L.; Gatos, M.; Mancin, F.; Tecilla, P.; Tonellato, U. Inorg.
Chem. 2003, 42, 3943-3949.
(25) Noteworthy saturation is attained after at least 5 equiv of ZnII ions
are added. This indicates that the apparent binding constant for ZnII at this
pH is not particularly high because of the competition with the protons.
J. Org. Chem, Vol. 72, No. 2, 2007 381

Scarso et al.
TABLE 1. Transesterification of HPNP Catalyzed by the
Tetranuclear Complexes of Ligands 5-7 and TP3 with ZnII
trip dal
kobs
° /
c
unassembled mixture
catalysta
105kobsb (s-1
)
kobs/ko
kobs
5-4ZnII
5-4ZnII
6-4ZnII
6-4ZnII
7-4ZnII
TP3-4ZnII
0.55
1.35e
1.1
140
n.d.
275
n.d.
17
23d
n.d.
30f
n.d.
n.d.
50d
1.1 e
0.07
1.2
300
a [catalyst] ) 2 × 10-4 M. b At pH ) 7.0 and 40 °C. c Estimated ko
unassembled mixture
from initial rate measurements is 4.0 × 10-8 s-1
.
d kobs
is
f
unassembled mixture
2.2 × 10-7 s-1
.
e At pH ) 8.5 and 40 °C. kobs
is 3.7 ×
10-7 s-1
.
FIGURE 6. Kinetic effect on the observed rate constant for the
cleavage of HPNP exerted by ligands 8 (filled circles) and 9 (open
circles) upon addition of ZnII ions. Conditions: [catalyst] ) 2 × 10-4
M, [HPNP] ) 2 × 10-5 M, 40 °C, pH ) 7.
our complexes, likely because of the presence of three metal
ions, the acidity of the effective nucleophile becomes slightly
higher. Morrow and Richard27 have reported a decrease of the
pKa of a ZnII-bound water molecule of 1.4 on moving from a
mononuclear to a dinuclear complex.
Cooperativity and Allosteric Control. In order to unam-
biguously prove the allosteric role of the Tren-bound ZnII metal,
we have synthesized tripodal ligands 8 and 9 and investigated
their catalytic behavior in the presence of ZnII. The only
difference between 8 and 9 is the fact that, in the latter, the
secondary amines of Tren are acetylated, thus preventing binding
of a metal ion to this site. In fact, a spectrophotometric titration
of ligand 8 with CuII shows the appearance of two new
absorption bands at 650 and 850 nm corresponding to the
complexes of CuII with the cyclic polyamines and Tren,
respectively (Figure 2d) in accord with the behavior of ligands
5-7. At variance with the previous ligands, the selectivity for
Tren in binding is almost lost and both binding sites are more
or less simultaneously occupied upon addition of CuII. On the
other hand, the same titration with ligand 9 only gives an
increase of the absorption band at 650 nm, indicating that
acetylation of Tren indeed suppresses CuII binding at this site.
Next, the kinetics were repeated using ligands 8 and 9 in the
presence of increasing amounts of ZnII. The results are given
in Figure 6. For compound 8, a curve was obtained that was
very similar to those obtained for compounds 5 and 6, with the
characteristic jump in activity upon the addition of the fourth
ZnII, indicating a similar behavior in terms of cooperativity.
Interestingly, compound 8 is significantly more reactive with
respect to compound 5 having identical azacrowns. For the
acetylated compound 9, on the other hand, a low activity is
observed, with a smooth increase of rate until the addition of 3
equiv of ZnII after which the curve flattens. This clearly proves
the allosteric role of the ZnII ion in the Tren binding unit.
We can now dissect the different contributions of the metal
complexes in the kinetic profiles of Figure 4a for ligands 5 and
6. Since the first metal binds to the Tren platform, the first event
is a conformational change of the ligand while the putative
catalytic site remains metal free. Accordingly, very little rate
acceleration is observed, likely due to the interaction of the
substrate with the protonated cyclic amines28 or with the ZnII
FIGURE 5. Effect of pH on the observed rate constants measured for
the cleavage of HPNP by catalysts 5-4ZnII (filled squares) and 6-4ZnII
(filled circles). Conditions: [catalyst] ) 2 × 10-4 M; [HPNP] ) 2 ×
10-5 M.
of the catalytically active species, we performed a series of
experiments at different pH for the two most active complexes,
5-4ZnII and 6-4ZnII. The results, reported in Figure 5, reveal
that the kinetic pKa for the 6-4ZnII complex is ca. 1.2 units
lower than that of 5-4ZnII (6.8 and 8.0, respectively, for the
two complexes).
Accordingly, the difference in activity measured at pH 7
simply reflects the different amount of active species present
at that pH, and in fact, at pH 7.8 the two catalysts become
equally active and, above this pH, 5-4ZnII is slightly better so
that the intrinsic reactivity of this latter catalyst appears to be
superior to the one based on the triazacyclododecane macro-
cycle. The apparent second-order rate constants for the fully
deprotonated complexes 5-4ZnII and 6-4ZnII are 0.125 and
0.063 M-1 s-1, respectively. This finding appears to be in full
accord with what has been recently reported in the literature24
where it has been shown that, in the case of HPNP, the
dependence of the reactivity on the acidity of the nucleophile
is rather pronounced (Brønsted ânuc ) 0.75) and the most active
nucleophile is the least acidic. Interestingly, the kinetic pKa’s
we find for the tripodal complexes are ca. 0.5 units lower than
those reported26 for the ZnII complexes of the parent cyclic
amines. This provides an indication that in the catalytic site of
(27) Iranzo, O.; Kovalevsky, A. Y.; Morrow, J. R.; Richard, J. P. J. Am.
Chem. Soc. 2003, 125, 1988-1993.
(28) Dalby, K. N.; Kirby, J. A.; Hollfelder, F. J. Chem. Soc., Perkin
Trans. 2 1993, 1269-1281.
(26) Rossi, P.; Felluga, F.; Tecilla, P.; Formaggio, F.; Crisma, M.;
Toniolo, C.; Scrimin, P. J. Am. Chem. Soc. 1999, 121, 6948-6949.
382 J. Org. Chem., Vol. 72, No. 2, 2007

Transphosphorylation Metallocatalysts
(S)-2-Benzyloxycarbonylamino-3-[1-(5,9-bis(tert-butyloxycar-
bonyl)-1,5,9-triazacyclododecane)] N-Methyl Propanamide, 2.
To a solution of 1,5,9-triazacyclododecane (462 mg, 2.7 mmol)
and Et3N (1.10 mL, 7.86 mmol) in 30 mL of dry CHCl3 was added
di-tert-butyl dicarbonate (886 mg, 4.02 mmol) dissolved in 15 mL
of dry CHCl3 over a period of 4 h using a syringe pump. The
solution was stirred for 24 h, the solvent removed under reduced
pressure, and the crude product purified by column chromatography
(SiO2, CH2Cl2/AcOEt) to yield 498 mg of di-Boc-protected 1,5,9-
triazacyclododecane. This material was dissolved in 8 mL of dry
CH3CN and added to a solution of the â-lactone of Z-(L)-serine
(333 mg, 1.51 mmol) in CH3CN. The solution was kept at 40 °C
for 7 days under stirring under a N2 atmosphere. After evaporation
of the solvent, the crude product was dissolved in 20 mL of CH2-
Cl2, di-tert-butyl dicarbonate (255 mg, 1.31 mmol) was added, and
the solution was stirred for 24 h. This procedure allowed the
conversion of the excess of diprotected azacrown into the tripro-
tected material that could be separated more easily from the product.
Evaporation of the solvent under reduced pressure and purification
of the crude product by flash chromatography (SiO2, AcOEt/CH3-
OH) gave 220 mg (28% yield) of the intermediate free acid of 2:
1H NMR (CDCl3) δ (ppm): 1.46 (s, 18H), 1.7-2.1/2.8-3.6 (br,
ion bound to Tren. The following metal ions start binding to
the triazamacrocycle units but a significant rate acceleration is
observed only when at least two ZnII complexes are formed
with these ligands. The complexation of the last metal ion is
required in order to observe the maximum catalytic efficiency
of the system. Thus, these experiments indicate that the first
metal is a structural (allosteric) ion, while the remaining three
are functional (catalytic) ions, which cooperate in the accelera-
tion of the transesterification process.
Conclusions
While the plots of Figure 4a strongly support the cooperativity
between the metal centers in the catalytic sites, the kinetic
profiles of Figure 5 indicate that the role of the metal ions is
that of stabilizing the complexed substrate toward the transition
state where a further negative charge develops and in facilitating
deprotonation of the nucleophilic species. We do not observe a
bell-shaped profile for these plots indicating that the cooperat-
ivity is not due to the occurrence of general-acid/general-base
catalysis;29 neither there is a decreased binding of the substrate
because of the deprotonation of a water molecule bound to the
complex.30 This might imply that the binding of the substrate
to ZnII does not require the displacement of this metal-bound
hydroxide.
If we compare the efficiency of the peptide-based catalyst
TP3-4ZnII with its simpler sibling 5-4ZnII, based on the same
ligand subunits (see Table 1), we note that the polypeptide-
based system is just 2-fold faster. This is not a particularly
exciting result in view of the synthetic effort put into its
synthesis. This different reactivity does not come from a
difference in pKa of the effective nucleophilic species (they are
the same within the experimental error), but from a better
binding of the substrate due to a slightly more hydrophobic
environment present in the catalytic site of the peptide-based
catalyst. This is, obviously, a reasonable speculation in the
absence of experimental binding data. On the contrary, the effect
of cooperativity is immediately evident if one compares the
activity of the tripodal systems with that observed when three
copies of the N-acetylamino amide 1 (after Boc-deprotection)
and the Tren platform are mixed together: 25-fold with respect
to 5-4ZnII and ca. 50-fold with respect to the peptide-based
catalyst. Allowing for the difference in pKa of the nucleophile
between the monomeric and tripodal, peptide-based catalyst the
cooperative gain is of ca. 1 order of magnitude. Morrow and
Richard have recently reported27 a 12-fold gain in comparing
the activity against HPNP transphosphorylation of a fully
deprotonated mononuclear catalyst with that of a dinuclear
catalyst.
20H), 4.20 (br, 1H), 5,06 (s, 2H), 6.13 (br, 1H), 7.31 (br, 5H); 13
C
NMR (CDCl3) δ (ppm): 171.1, 156.3, 136.4, 128.4, 128.0, 79.4,
67.7,66.3, 60.3, 49.7, 46.8, 43.8, 29.7, 28.4, 26.2, 21.0, 14.2, 13.8;
ESI-MS (CH3OH, m/z) calcd 592.7, found 593 [M + H]+, 615 [M
+ Na]+. The acid (0.48 mmol) was dissolved in a 5 mL CH2Cl2
solution, and at 0 °C under a N2 atmosphere were added EDC‚HCl
(206 mg, 1.05 mmol) and, subsequently, HOAT (133 mg, 0.958
mmol). After 20 min, CH3NH2‚HCl (100 mg, 1.48 mmol) was
added, and the reaction mixture was made basic (pH ca. 10) with
900 µL of diisopropylethylamine (5.14 mmol). The reaction was
then stirred at room temperature for 6 days. Next, the solvent was
evaporated, and the residue was taken up in 25 mL of AcOEt,
washed with KHSO4 10% (2 × 10 mL), NaHCO3 5% (2 × 10
mL), and H2O (2 × 10 mL), and dried over Na2SO4. Evaporation
of the solvent gave a crude that was purified by chromatography
(SiO2, light petroleum/AcOEt) yielding 2 (260 mg, 90% yield): 1H
NMR (250 MHz, CDCl3) δ (ppm) 1.46 (s, 18H), 1.7-2.1/2.8-3.6
(br, 20H), 2.76 (d, 3H), 4.20 (br, 1H), 5.06 (s, 2H), 6.13 (br, 1H),
7.31 (br, 5H); ESI-MS (CH3OH, m/z) calcd 605.8, found 606 [M
+ H]+, 628 [M + Na]+.
(S)-2-Benzyloxycarbonylamino-3-[1-(4,7,10-tris(tert-butyloxy-
carbonyl)-1,4,7,10-tetraazacyclododecane)] N-Methyl Propana-
mide, 3. The procedure followed for the preparation of this material
was essentially the same as for the preparation of 2. The intermedi-
ate acid was obtained in 25% yield: 1H NMR (CDCl3) δ (ppm)
1.35-1.48 (m br, 27 H), 2.67-3.70 (br, 18H), 4.25-4.65 (br, 1H),
5.05 (br, 2H), 7.27 (m, 5H); ESI-MS (CH3OH, m/z) calcd 693.8,
found 694 [M + H]+, 716 [M + Na]+. Coupling with CH3NH2 in
analogous conditions as before gave 3 (119 mg, 35% yield): 1H
NMR (250 MHz, CDCl3) δ (ppm) 1.39-1.45 (s br, 27H), 2.0-3.7
(br, 18H), 2.76 (d, 3H), 4.27 (br, 1H), 5.05 (AB, 2H), 6.32 (br,
1H), 6.69 (br, 1H), 7.32 (s br, 5H); ESI-MS (CH3OH, m/z) calcd
706.9, found 707 [M + H]+, 729 [M + Na]+.
In conclusion, we have shown in this paper that the conjuga-
tion of three arms of a Tren derivative with ligands units for
ZnII ions provides an entry to simple, cooperative catalysts where
the Tren platform acts as a regulation site: only when a metal
ion is bound to it cooperativity between the metal ions in the
three arms in the cleavage of a RNA model substrate is
observed.
2-(1-(4,7-Bis(tert-butyloxycarbonyl)-1,4,7-triazacyclonane)-
ethylamine, 4. To a solution of di-Boc-protected triazacyclononane
(150 mg, 0,45 mmol) in CH3CN (10 mL) were added N-(2-
bromoethyl)phthalimide (115 mg, 0,45 mmol), diisopropylethy-
lamine (250 µL), and a small amount of K2CO3. The solution was
stirred at 60 °C and followed by TLC (SiO2, CHCl3/diethyl ether
9/1, UV/ninhydrin), Rf 0.0, diBoc-TACN; 0.3, product; 0.8, N-(2-
bromoethyl)phthalimide. After completion of the reaction (7 days),
the solvent was evaporated and the residue purified by column
Experimental Section
Synthesis. Synthetic procedures for 1,18 tris[4-(carboxy)phenyl-
methyl-2-(tert-butyloxycarbonylamino)ethyl]amine 10,12a and the
corresponding methyl ester derivative 1312a have already been
reported by us.
(29) Worm, K.; Chu, F.; Matsumoto, K.; Best, M. D.; Lynch, V.; Anslyn,
E. V. Chem. Eur. J. 2003, 9, 741-747.
(30) Molenveld, P.; Kapsabelis, S.; Engbersen, J. F. J.; Reinhoudt,
D. N. J. Am. Chem. Soc. 1997, 119, 2948-2949.
J. Org. Chem, Vol. 72, No. 2, 2007 383

Scarso et al.
chromatography (SiO2, CHCl3). The intermediate (153 mg, 0.3
mmol) was dissolved in ethanol (5 mL), and hydrazine monohydrate
(100 µL, 2 mmol) was added. The solution was stirred at room
temperature, and after 3 h, a white precipitate formed. After 6 h,
the solvent was evaporate under reduced pressure and the residue
treated with CH2Cl2 (15 mL). The precipitate was filtered off and
the solution dried on Na2SO4. After evaporation, compound 4 (67
mg, 60%) was obtained as a colorless oil: 1H NMR (CDCl3) δ
(ppm) 1.47 (s, 18H), 2.63 (br m, 8H), 3.28 (br m, 4H), 3.40-3.48
(m, 4H); ESI-MS (CH3OH, m/z) calcd 372.3, found 373.2 [M +
H+].
3.6 (br, 66H), 2.77 (d, 9H), 4.40 (br, 9H), 7.05-7.85 (m br, 12H).
The above material was dissolved in 3 mL of a 33% solution of
HBr/CH3COOH and stirred at room temperature for 1 h. Addition
of 4 mL of ethyl ether gave a white precipitate of the HBr salt of
7 (8 mg) that was filtered and dried: 1H NMR (300 MHz, D2O) δ
(ppm) 2.67 (d, 9H), 2.75-3.25 (m br, 66H), 4.21 (m br, 6H), 4.55
(tr, 3H), 7.52-7.81 (m br, 12H); 13C NMR (D2O) δ (ppm) 173.1,
148.2, 130.5, 128.3, 50.8, 50.6, 48.4, 44.4, 42.1, 41.5, 26.0; ESI-
MS (CH3OH, m/z) calcd 1311.8, found 656 [M + 2H]2+, 437 [M
+ 3H]3+, 678 [M + 2Na]2+, 459 [M + 3Na]3+
.
Tris{4-[2-(1-(1,4,7-triazacyclononane))ethanamide]-
carboxyphenylmethyl-2- aminoethyl}amine, 8. A procedure
similar to that described for the preparation of 4 was followed but
using 20.0 mg (24 µmol) of 1012a as starting material. The Boc-
protected compound 8 was dissolved in CH2Cl2 (2 mL) and TFA
(1 mL) was added. The mixture was stirred for 4 h after which the
volatiles were removed under reduced pressure. Compound 8 (28.0
mg, 27%) was obtained as TFA salt: 1H NMR (250 MHz, CD3-
OD) δ (ppm) 2.76 (br, 4H), 2.91 (br, 4H), 3.07 (br, 2H), 3.30 (br,
4H), 3.45 (br, 6H), 4.16 (s, 2H), 7.47 (d, 2H), 7.76 (m, 2H); ESI-
MS (CH3OH, m/z) calcd 1010.9; found 503.2 [M + 2H]2+, 337.9
[M + 3H]3+, 253.7 [M + 4H]4+; RP-HPLC (Phenomenex Jupiter
4 µm, 90 Å, 230 nm) 26.5 min.
Tris[4-carboxymethylphenylamino(N-acetyl)ethyl]amine, 12.
Compound 1112a (700 mg, 0.78 mmol) was dissolved in 10 mL of
HBr (33% in CH3COOH) and 7 mL of acetic acid and stirred for
2 h at room temperature. Diethyl ether (40 mL) was added, and
the formed precipitate was filtered off and washed with diethyl
ether. The resulting BOC-deprotected compound 11 was dissolved
in CH2Cl2 (15 mL) and DIPEA (2 mL) and cooled to 0 °C. Acetic
anhydride (15 mL) was added, and the mixture was stirred at room
temperature for 20 h. Next, the mixture was washed three times
with an aqueous solution of NaOH (1 M, 50 mL), dried over Na2-
SO4, and evaporated to dryness. Compound 12 was obtained as a
reddish oil (320 mg, 57%): 1H NMR (250 MHz, CDCl3) δ (ppm)
2.10 (m, 3H), 2.54 (m, 2H), 2.33 (m, 2H), 3.92 (d, 3H), 4.56 (m,
2H), 7.25 (m, 2H), 7.98 (m, 2H); ESI-MS (CH3OH, m/z) calcd
716.3, found 739.2 [M + Na]+.
Tris{4-[(S)-2-yl-3-(1-(1,4,7-triazacyclononane))-N-methylpro-
panamide]carboxyphenylmethyl-2-aminoethyl}amine, 5. Com-
pound 116 was dissolved in 6 mL of CH3OH to which, after purging
with N2, 35 mg of Pd/C 10% was added. H2 was fluxed through
the solution and the reaction flask saturated with H2. After 18 h,
the reaction mixture was filtered through a Celite pad. The Celite
was washed several times with methanol and the solvent removed
to give an oil, which was used as such for the coupling. Compound
1012a (100 mg) was dissolved in 15 mL of CH2Cl2 and the solution
cooled to 0 °C. Subsequently, HOBt (100 mg, 0.74 mmol) followed
by EDC (120 mg, 0.62 mmol) were added, and the reaction mixture
was stirred under a N2 atmosphere. For the coupling, 210 mg of
deprotected 1 was added to the above solution that was stirred at
room temperature for 2 days with occasional addition of N-
methylmorpholine to adjust the pH to ca. 9. The solvent was then
removed under reduced pressure and the residue was taken up with
AcOEt (25 mL) and extracted with 10% aqueous KHSO4 (25 mL),
H2O (25 mL), 5% aqueous NaHCO3 (25 mL), and H2O (25 mL).
After the organic solvent was dried over Na2SO4 and evaporated,
230 mg of crude material was obtained, which was purified by
chromatography (SiO2, CH2Cl2/AcOEt) to yield 65 mg (26%) of
Boc-protected 5: 1H NMR (300 MHz, CDCl3) δ (ppm) 1.35-1.45
(81 H), 2.54-3.72 (m, 54 H), 2.82 (d, 9H), 4.41 (br, 6H), 4.58 (m
br), 7.59 (d br, 3H), 7.20-7.79 (AA“BB”, 12H); 13C NMR (75
MHz, CDCl3) δ (ppm) 171.9, 171.1, 166.9, 156.3, 155.8, 132.8,
127.6, 126.8, 80.2, 80.0, 77.2, 62.0, 60.4, 55.6, 54.9, 52.7, 52.5,
51.4, 51.1, 50.4, 50.0, 49.3, 49.1, 45.6, 28.5, 28.4, 26.3, 21.0, 14.2;
ESI-MS (CH3OH, m/z) calcd 2083, found 1064 [M + 2Na]2+. This
compound was dissolved in 3 mL of a 33% solution of HBr/CH3-
COOH and stirred at room temperature for 1 h. Addition of 4 mL
of ethyl ether precipitated the HBr salt of 5 (60 mg) that was filtered
and dried: 1H NMR (300 MHz, D2O) δ (ppm) 2.69 (s, 9H), 2.85-
3.58 (m, 54H), 4.26 (s, 6H,), 7.54-7.57-7.80-7.83 (AA“BB”,
12H).
Tris{4-[2-(1-(1,4,7-triazacyclononane))ethanamide]-
carboxyphenylmethyl-2-amino(N-acetyl)ethyl}amine, 9. Com-
pound 12 (320 mg, 0.4 mmol) was dissolved in ethanol (5 mL),
and a solution of LiOH‚xH2O (165 mg, 7 mmol) in H2O (1 mL)
was added. The mixture was stirred overnight, after which time
solvent was removed via liophilization. The residue was taken in
H2O (10 mL), and 1 M HCl was added until the formation of a
precipitate was observed. The precipitate was isolated via filtration
and dried under over P2O5. Compound 13 (270 mg) was analyzed
Tris{4-[(S)-2-yl-3-(1-(1,5,9-triazacyclododecane))-N-methyl-
propanamide]carboxyphenylmethyl-2-aminoethyl}amine, 6. A
procedure similar to that described for the preparation of 5 was
followed but using 67.1 mg (79.1 µmol) of 1012a as starting material.
The Boc-protected compound 6 was obtained with a yield of 50%:
1H NMR (250 MHz, CDCl3) δ (ppm) 1.36-1.44 (br, 81H), 1.7-
1.9/2.3-3.5 (br, 81H), 4.40 (br, 9H), 7.16-7.97 (m br, 12H); 13C
NMR (CDCl3) δ (ppm) 171.1, 156.3, 127.1, 80.0, 79.7, 79.6, 79.2,
63.7, 60.4, 46.0, 44.7, 44.0, 28.4, 21.0, 14.1.
1
by H NMR to verify the disappearance of the methyl ester and
used directly for the coupling step: 1H NMR (250 MHz, DMSO)
δ (ppm) 2.07 (s, 3H), 3.37 (m br, 2H), 3.61 (m br, 2H), 3.92 (d,
3H), 4.60 (m, 2H), 7.35 (m, 2H), 7.96 (m, 2H). Compound 13 (17
mg, 2.5 µmol) was dissolved in anhydrous CH2Cl2 (4 mL) and
DIPEA (100 µL) and cooled to 0 °C. Subsequently, HOBt (20 mg,
0.15 mmol) and EDC (27 mg, 0.14 mmol) were added, and the
mixture was stirred for 45 min at 0 °C, after which time a solution
of compound 4 (38 mg, 0.1 mmol) in anhydrous CH2Cl2 (2 mL)
was added. The mixture was stirred at room temperature for 7 days,
after which time the solvent was removed under reduced pressure.
The residue was taken in EtOAc (5 mL), and the product was
extracted in 10% KHSO4 (6 mL). The aqueous phase was basified
to pH 10 using NaOH (1 M) and extracted with CHCl3 (4 × 15
mL). The combined organic phases were dried over Na2SO4 and
evaporated to dryness. The crude product was purified using
preparative TLC (Al2O3, CHCl3/MeOH/aq NH3 ) 92.5/7/0.5), and
17 mg of a yellowish oil was obtained. This compound was
dissolved in CH2Cl2 (2 mL), and TFA (1 mL) was added, after
which the mixture was stirred for 4 h. The volatiles were removed
under reduced pressure to give compound 9 (15 mg) as its TFA
salt: 1H NMR (250 MHz, CD3OD) δ (ppm) 2.20 (s, 3H), 2.90 (br,
The above material was dissolved in 3 mL of a 33% solution of
HBr/CH3COOH and stirred at room temperature for 1 h. Addition
of 4 mL of ethyl ether gave a white precipitate of the HBr salt of
6 (92 mg) that was filtered and dried: 1H NMR (250 MHz, D2O)
δ (ppm) 1.75-2.20/2.55-3.25 (m br, 72H), 2.62 (d, 9H), 4.20 (s,
9H), 7.48-7.79 (m br, 12H); 13C NMR (D2O) δ (ppm) 170.0, 156.3,
127.0, 124.9, 124.4, 47.2, 46.3, 45.3, 40.5, 38.4, 37.8, 37.4, 37.3,
35.3, 14.5; ESI-MS (CH3OH, m/z) calcd 1308.8, found 655 [M +
2H]2+, 437 [M + 3H]3+, 677 [M + 2Na]2+, 459 [M + 3Na]3+
.
Tris{4-[(S)-2-yl-3-(1-(1,4,7,10-tetraazacyclododecane))-N-me-
thylpropanamide]carboxyphenyl methyl-2-aminoethyl}amine,
7. A procedure similar to that described for the preparation of 5
was followed but using 13.1 mg (15.4 µmol) of 1012a as starting
material. The Boc-protected compound 7 was obtained with a 27%
yield: 1H NMR (250 MHz, CDCl3) δ (ppm) 1.43 (s, 108H), 2.4-
384 J. Org. Chem., Vol. 72, No. 2, 2007

Transphosphorylation Metallocatalysts
4H), 3.08 (m br, 4H), 3.30 (br, 4H), 3.50-3.65 (m br, 6H), 3.76
1-piperazineethanesulfonic acid; pH 8-8.5 EPPS, 4-(2-hydroxy-
ethyl)piperazine-4-(3-propansulfonic acid); pH 9÷9.5 CHES, 2-(cy-
clohexylamino)ethanesulfonicacid;pH10CAPS,2-(cyclohexylamino)-
1-propanesulfonic acid), 80 µL of methanol (spectrophotometric
grade), and the proper volume of water to reach a final volume of
880 µL and then thermostated at 40 °C. After a couple of minutes
of equilibration time, HPNP (3.6 µL, 5 mM in water) was injected,
and the increase in UV absorbance at λ ) 400 nm due to the release
of p-nitrophenolate (or 317 nm at low pH) was recorded. All of
the solutions remained clear during the kinetic measurements (at
(br, 2H), 4.83 (s, 2H), 7.44 (d, 2H), 7.94 (m, 2H); ESI-MS (CH3-
OH, m/z) calcd 1136.8, found 569 [M + 2H]2+, 380 [M + 3H]3+
;
RP-HPLC (Phenomenex Jupiter 4 µm, 90 Å, 230 nm) 14.0 min.
Metal Ion Binding Experiments. Via Spectrophotometry. To
a cuvette containing 200 µL of H2O and 1000 µL of buffer solution
MES (4-morpholineethanesulfonic acid) 0.2 M pH 6.3 were added
800 µL of a stock solution of the ligands (1.51 mM). The increase
in absorbance at 845 and 605 nm at 25 °C were followed upon the
stepwise addition of 1 µL of 50 mM Cu(NO3)2 in water up to 7
equiv of metal. The absorbance readings were corrected for the
dilution upon titration.
least 4 half-lives). The pseudo-first-order rate constants kobs (s-1
)
were calculated by fitting of the data by standard methods.
1
Via H NMR. Compound 6·13HBr (44.7 mg, 18,7 µmol)) was
dissolved in D2O (5 mL). From this stock solution 2.5 mL was
transferred into a 5 mL volumetric flask to which 6 equiv of Zn-
(NO3)2 was added. The pD of both solutions was adjusted to 7.0
using a solution of NaOD in D2O, after which both volumes were
adjusted to 5 mL. The resulting stock solutions A and B had a
final concentration of 1.90 mM in compound 6, and stock solution
B had additionally a 11.4 mM concentration of Zn Zn(NO3)2. NMR
samples were prepared by mixing solutions A and B in the following
ratios (6:0; 5:1; 4:2; 3:3; 2:4; 1:5; 0:6) resulting in six NMR tubes
with 0-6 equiv of ZnII, respectively.
Kinetics. In a typical experiment, the ligand (120 µL, 1.51 mM
in water) and Zn(NO3)2 (50 mM) were added to a cuvette containing
500 µL of a 0.2 M buffer solution (adjusted with NaOH to the
desired pH, pH 6-6.5 MES; pH 7-7.5 HEPES, 4-(2-hydroxyethyl)-
Acknowledgment. Support by the European Community’s
Human Potential Programme under contract HPRN-CT-1999-
00008, ENDEVAN (fellowship to F.B.H.), the Ministry of
Education, University, and Research of Italy (MIUR, PRIN
2002) and the University of Padova (Project CPDA054893) is
gratefully acknowledged.
Supporting Information Available: 1H NMR spectra of
compounds 2-9 and 12 and materials and methods used in the
syntheses. This material is available free of charge via the Internet
at http://pubs.acs.org.
JO061754K
J. Org. Chem, Vol. 72, No. 2, 2007 385