Catalysis of diribonucleoside monophosphate cleavage by water soluble copper(II) complexes of calix[4]arene based nitrogen ligands

Article abstract of DOI:10.1021/ja0632106

Calix[4]arenes functionalized at the 1,2-, 1,3-, and 1,2,3-positions of the upper rim with [12]ane-N₃ ligating units were synthesized, and their bi- and trimetallic zinc(II) and copper(II) complexes were investigated as catalysts in the cleavage of phosphodiesters as RNA models. The results of comparative kinetic studies using monometallic controls indicate that the subunits of all of the zinc(II) complexes and of the 1,3-distal bimetallic copper(II) complex 7-Cu₂ act as essentially independent monometallic catalysts. The lack of cooperation between metal ions in the above complexes is in marked contrast with the behavior of the 1,2-vicinal bimetallic copper(II) complex 6-Cu₂, which exhibits high catalytic efficiency and high levels of cooperation between metal ions in the cleavage of HPNP and of diribonucleoside monophosphates NpN′. A third ligated metal ion at the upper rim does not enhance the catalytic efficiency, which excludes the simultaneous cooperation in the catalysis of the three metal ions in 8-Cu ₃. Rate accelerations relative to the background brought about by 6-Cu₂ and 8-Cu₃ (1.0 mM catalyst, water solution, pH 7.0, 50 °C) are on the order of 10⁴-fold, largely independent of the nucleobase structure, with the exception of the cleavage of diribonucleoside monophosphates in which the nucleobase N is uracil, namely UpU and UpG, for which rate enhancements rise to 10⁵-fold. The rationale for the observed selectivity is discussed in terms of deprotonation of the uracil moiety under the reaction conditions and complexation of the resulting anion with one of the copper(II) centers.

Full text of DOI:10.1021/ja0632106

Published on Web 08/18/2006
Catalysis of Diribonucleoside Monophosphate Cleavage by
Water Soluble Copper(II) Complexes of Calix[4]arene Based
Nitrogen Ligands
Roberta Cacciapaglia,† Alessandro Casnati,‡ Luigi Mandolini,*,†
David N. Reinhoudt,*,§ Riccardo Salvio,† Andrea Sartori,§ and Rocco Ungaro*,‡
Contribution from the Dipartimento di Chimica and IMC - CNR Sezione Meccanismi di
Reazione, UniVersita` La Sapienza, Box 34 - Roma 62, 00185 Roma, Italy, Dipartimento di
Chimica Organica e Industriale, UniVersita` degli Studi di Parma, V.le G. P. Usberti 17/A,
43100 Parma, Italy, and Laboratory of Supramolecular Chemistry and Technology, MESA+
-
Research Institute, UniVersity of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands
Received May 8, 2006; E-mail: d.n.reinhoudt@tnw.utwente.nl; luigi.mandolini@uniroma1.it; rocco.ungaro@unipr.it
Abstract: Calix[4]arenes functionalized at the 1,2-, 1,3-, and 1,2,3-positions of the upper rim with [12]ane-
N3 ligating units were synthesized, and their bi- and trimetallic zinc(II) and copper(II) complexes were
investigated as catalysts in the cleavage of phosphodiesters as RNA models. The results of comparative
kinetic studies using monometallic controls indicate that the subunits of all of the zinc(II) complexes and of
the 1,3-distal bimetallic copper(II) complex 7-Cu2 act as essentially independent monometallic catalysts.
The lack of cooperation between metal ions in the above complexes is in marked contrast with the behavior
of the 1,2-vicinal bimetallic copper(II) complex 6-Cu2, which exhibits high catalytic efficiency and high
levels of cooperation between metal ions in the cleavage of HPNP and of diribonucleoside monophosphates
NpN′. A third ligated metal ion at the upper rim does not enhance the catalytic efficiency, which excludes
the simultaneous cooperation in the catalysis of the three metal ions in 8-Cu3. Rate accelerations relative
to the background brought about by 6-Cu2 and 8-Cu3 (1.0 mM catalyst, water solution, pH 7.0, 50 °C)
are on the order of 104-fold, largely independent of the nucleobase structure, with the exception of the
cleavage of diribonucleoside monophosphates in which the nucleobase N is uracil, namely UpU and UpG,
for which rate enhancements rise to 105-fold. The rationale for the observed selectivity is discussed in
terms of deprotonation of the uracil moiety under the reaction conditions and complexation of the resulting
anion with one of the copper(II) centers.
Many enzymes that catalyze the hydrolytic cleavage of
phosphodiesters incorporate two or three metal ions in their
active site.1 In the aim at mimicking the extraordinary catalytic
activity of such enzymes, di- and trinuclear complexes of di-
and trivalent metal ions have been synthesized and shown to
be better catalysts than analogous mononuclear complexes in
the cleavage of di- and oligoribonucleotides, as well as of RNA
model compounds.2
Our previous studies in the field have shown that the calix-
[4]arene scaffold, blocked in the cone conformation by proper
alkylation of the lower rim hydroxyls, is suitable for the design
of di- and trimetallic phosphodiesterase models.2g,3 Efficient
catalysis by synergic action of the metal centers was observed,
for instance, for the zinc(II) complexes of 2,6-bis[(dimethy-
lamino)methyl]pyridine (BAMP) functionalized calix[4]arenes
1-3. Analogous copper(II) complexes were investigated, but
much poorer results were obtained.4 A major drawback of the
BAMP functionalized catalysts was the limited solubility in
water which required the use of mixed aqueous-organic
solvents. Another limitation, a more serious one, was the
moderate affinity of BAMP for zinc(II). The binding constant
K ≈ 1 × 105 M-1 in CH3CN/H2O 1:1, pH 7.0,5 which is large
enough for studies carried out at 1 mM catalyst concentration,
drops to 1.1 × 103 M-1 in water at the same pH.6
(2) For review articles, see: (a) Niittyma¨ki, T.; Lo¨nnberg, H. Org. Biomol.
Chem. 2006, 4, 15-25. (b) Mancin, F.; Scrimin, P.; Tecilla, P.; Tonellato,
U. Chem. Commun. 2005, 2540-2548. (c) Morrow, J. R.; Iranzo, O. Curr.
Opin. Chem. Biol. 2004, 8, 192-200. (d) Liu, C.; Wang, M.; Zhang, T.;
Sun, H. Coord. Chem. ReV. 2004, 248, 147-168. (e) Cowan, J. A. Curr.
Opin. Chem. Biol. 2001, 5, 634-642. (f) Kimura, E. Curr. Opin. Chem.
Biol. 2000, 4, 207-213. (g) Molenveld, P.; Engbersen, J. F. J.; Reinhoudt,
D. N. Chem. Soc. ReV. 2000, 29, 75-86. (h) Bashkin, K. K. Curr. Opin.
Chem. Biol. 1999, 3, 752-758. (i) Williams, N. H.; Takasaki, B.; Wall,
M.; Chin, J. Acc. Chem. Res. 1999, 32, 485-493. (j) Hegg, E. L.; Burstyn,
J. N. Coord. Chem. ReV. 1998, 173, 133-165. (k) Franklin, S. J. Curr.
Opin. Chem. Biol. 2001, 5, 201-208. (l) Komiyama, M.; Sumaoka, J. Curr.
Opin. Chem. Biol. 1998, 2, 751-757.
† Universita` di Roma.
(3) Cacciapaglia R.; Casnati, A.; Mandolini, L.; Reinhoudt, D. N.; Salvio, R.;
Sartori, A.; Ungaro, R. J. Org. Chem. 2005, 70, 624-630.
‡ Universita` di Parma.
§ University of Twente.
(4) Molenveld, P.; Engbersen, J. F. J.; Kooijman, H.; Spek, A. L.; Reinhoudt,
D. N. J. Am. Chem. Soc. 1998, 120, 6726-6737.
(1) (a) Weston, J. Chem. ReV. 2005, 105, 2151-2174. (b) Jedrzejas, M. J.;
Setlow, P. Chem. ReV. 2001, 101, 607-618. (b) Cowan, J. A. Chem. ReV.
1998, 98, 1067-1087. (c) Wilcox, D. E. Chem. ReV. 1996, 96, 2435-
2458. (d) Strater, N.; Lipscomb, W. N.; Klabunde, T.; Krebs, B. Angew.
Chem., Int. Ed. Engl. 1996, 35, 2024-2055.
(5) Molenveld, P.; Kapsabelis, S.; Engbersen, J. F. J.; Reinhoudt, D. N. J.
Am. Chem. Soc. 1997, 119, 2948-2949.
(6) From spectrophotometric titration at 266 nm of 0.20 mM BAMP with Zn-
(ClO4)2 in H2O, 20 mM HEPES, pH 7.0, 25 °C (this work).
9
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J. AM. CHEM. SOC. 2006, 128, 12322-12330
10.1021/ja0632106 CCC: $33.50 © 2006 American Chemical Society

Water-Soluble Synthetic CuII-Nucleases
A R T I C L E S
Chart 1
To investigate further the potential of calix[4]arene-based
artificial phosphodiesterases, and with the specific aim at
developing metal catalysts soluble enough in water to avoid
the use of organic cosolvents, we turned our attention to 1,5,9-
triazacyclododecane ([12]aneN3) as a ligating unit for the
construction of di- and tritopic ligands. Zinc(II) and copper(II)
complexes of [12]aneN3 and other polynitrogen macrocycles
have well documented catalytic properties in phosphodiester
cleavage.2a-j,7 In water solvent [12]aneN3 has a very high affinity
for zinc(II) (log K ) 8.41 at 25 °C),8 and even higher is the
affinity for copper(II) under the same conditions (log K )
12.63).9 This ensures extensive binding of the metal ion to the
ligand, on condition that the pH is not too low.10
2-hydroxypropyl p-nitrophenyl phosphate (HPNP). The copper-
(II) complexes were further investigated as catalysts of the
cleavage of a number of diribonucleoside 3′,5′-monophosphates
that more closely mimic the structure of RNA.
(7) (a) O’Donoghue, A.; Pyun, S. Y.; Yang, M.-Y.; Morrow, J. R.; Richard, J.
P. J. Am. Chem. Soc. 2006, 128, 1615-1621. (b) Iranzo, O.; Elmer, T.;
Richard, J. P.; Morrow, J. R. Inorg. Chem. 2003, 42, 7737-7746. (c) Iranzo,
O.; Kovalevsky, A. Y.; Morrow, J. R.; Richard, J. P. J. Am. Chem. Soc.
2003, 125, 1988-1993. (d) Bonfa`, L.; Gatos, M.; Mancin, F.; Tecilla, P.;
Tonellato, U. Inorg. Chem. 2003, 42, 3943-3949. (e) Bencini, A.; Berni,
E.; Bianchi, A.; Giorni, C.; Valtancoli, B.; Chand, D. K.; Schneider H.-J.
Dalton Trans. 2003, 793-800. (f) Deck, K. M.; Tseng, T. A.; Burstyn, J.
N. Inorg. Chem. 2002, 41, 669-677. (g) Kaukinen, U.; Bielecki, L.;
Mikkola, S.; Adamiak, R. W.; Lo¨nnberg, H. J. Chem. Soc., Perkin Trans.
2 2001, 1024-1031. (h) Sissi, C.; Rossi, P.; Felluga, F.; Formaggio, F.;
Palumbo, M.; Tecilla, P.; Toniolo, C.; Scrimin, P. J. Am. Chem. Soc. 2001,
123, 3169-3170. (i) Itoh, T.; Hisada, H.; Usui, Y.; Fujii, Y. Inorg. Chim.
Acta 1998, 283, 51-60. (j) Kimura, E.; Kodama, Y.; Koike, T.; Shiro, M.
J. Am. Chem. Soc. 1995, 117, 8304-8311. (k) Burstyn, J. N.; Deal, K. A.
Inorg. Chem. 1993, 32, 3585-3586.
Herein, we describe the synthesis of calix[4]arenes function-
alized with [12]aneN3 ligating units and the results of a kinetic
investigation of the catalytic activity of their zinc(II) and copper-
(II) complexes in the cleavage of RNA model compound
(8) Kimura, E.; Shiota, T.; Koike, T.; Shiro, M.; Kodama, M. J. Am. Chem.
Soc. 1990, 112, 5805-5811.
(9) Zompa, L. J. Inorg. Chem. 1978, 17, 2531-2536.
9
J. AM. CHEM. SOC. VOL. 128, NO. 37, 2006 12323

A R T I C L E S
Scheme 1a
Cacciapaglia et al.
a Reagents and conditions: (i) bis-Boc-protected [12]aneN3 9, K2CO3, CH3CN; (ii) (a) CF3COOH, CH2Cl2, rt, 2 h; (b) Li2CO3.
Table 1. HPNP Transesterification Catalyzed by Mono-, Di-, and
Trinuclear Zinc(II) Complexes of [12]aneN3-Based Systems, in
50% CH3CN/H2O, 20 mM HEPES, pH 7.0, 25.0 °Ca
Results and Discussion
Ligand Syntheses. To avoid complications arising from
undesired polysubstitution of [12]aneN3, we developed a
synthetic procedure for the preparation of calix[4]arenes 5-8
(Scheme 1) which involved alkylation of bis-Boc-protected [12]-
aneN3 9.11 This strategy offered the additional advantage of an
1
c
catalyst
kobsb (s-
)
kobs/kbg
[12]aneN3-Zn
4-Zn
8.6 × 10-6
3.7 × 10-6
6.3 × 10-6
6.0 × 10-6
2.1 × 10-5
450
190
330
320
6-Zn2
7-Zn2
8-Zn3
1100
a UV-vis monitoring of reaction progress in runs carried out on 0.15
mM substrate solutions, in the presence of 0.20 mM catalyst. b Initial rate
c
method, error limit (10%. kbg) 1.9 × 10-8 s-1, from ref 3.
HPNP Cleavage. Contrary to our hopes, the zinc(II) com-
plexes of ligands 5-8 were not soluble enough in water for
catalytic studies of phosphodiester cleavage. Nevertheless,
complexes 6-Zn2, 7-Zn2, and 8-Zn3 were tested as catalysts of
HPNP transesterification (eq 1) in 50% aqueous CH3CN at pH
7.0 for comparison with analogous BAMP-based complexes.2g,3
easier purification of the protected calix[4]arene intermediates.
Introduction of [12]aneN3 units at the upper rim was carried
out by reaction of the chloromethylated calix[4]arenes 10-13
with 9 in dry acetonitrile, in the presence of K2CO3. The reaction
rate of the latter process was quite low, reasonably due to steric
hindrance in the nucleophile. The monofunctionalized compound
14 was obtained in 70% yield after two days of reaction, whereas
5 days were necessary to obtain good yields of the di- and
trifunctionalized compounds 15-17. Pure products 5-8 were
obtained in quantitative yield upon removal of Boc protecting
groups by reaction with trifluoroacetic acid in dichloromethane.
The trifunctionalized derivative 8 resulted in being water soluble
up to 0.5 mM showing a well resolved 1H NMR spectrum (see
Supporting Information).
(10) Combination of the log K values for binding of zinc(II) and copper(II) to
[12]aneN3 with a value of 12.6 for the pKa of the ligand (from ref 8) shows
that, at pH 7.0 and 1 mM ligand and metal ion, the amount of ligated
metal ion is >99% in the case of copper(II) but drops to less than 50% for
zinc(II). However, because of the insolubility of zinc(II) complexes in water
(see further on), catalytic experiments were carried out in 50% aqueous
CH3CN. If the binding affinity of [12]aneN3 for zinc(II) on going from
pure water to 50% aqueous CH3CN experiences the same enhancement as
that of BAMP, namely, 2 orders of magnitude, then the amount of zinc(II)
bound to [12]aneN3 rises to about 90%.
Since 5-Zn turned out to be insoluble also in 50% aqueous
CH3CN, it was replaced by the N-benzylated model 4-Zn.
Kinetic data (Table 1) are expressed as pseudo-first-order rate
constants for the spectrophotometrically monitored liberation
of p-nitrophenol under a standard set of experimental conditions.
The results of the catalytic experiments reveal a disappointing
lack of synergism between metal ions, which is in marked
contrast with the results previously obtained in the transesteri-
(11) Brandes, S.; Gros, C.; Denat, F.; Pullumbi, P.; Guilard, R. Bull. Soc. Chim.
Fr. 1996, 133, 65-73.
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Water-Soluble Synthetic CuII-Nucleases
A R T I C L E S
Table 2. HPNP Transesterification Catalyzed by Cu2+ Complexes
of [12]aneN3-Based Systems in H2O, 20 mM HEPES, pH 7.0, 25.0
°Ca
1
c
catalyst
kobsb (s-
)
kobs/kbg
[12]aneN3-Cu
4-Cu
3.3 × 10-6
5.8 × 10-6
2.4 × 10-4
8.7 × 10-6
1.3 × 10-4
15
26
1090
40
6-Cu2
7-Cu2
8-Cu3
590
a UV-vis monitoring of reaction progress in runs carried out on 0.1
mM substrate solutions, in the presence of 0.20 mM catalyst. b Initial rate
c
method, error limit (10%. kbg)2.2 × 10-7 s-1, from transesterification
of a 2.0 mM HPNP solution.
Complexes 6-Cu2, 7-Cu2, and 8-Cu3 and mononuclear
controls [12]aneN3-Cu and 4-Cu were analyzed for catalysis
of the transesterification of HPNP in water solution, pH 7.0.
The results are collected in Table 2. Comparison with the data
in Table 1 shows that mononuclear copper(II) complexes are
significantly less efficient than the corresponding zinc(II)
complexes. The different catalytic activity is most likely related
to the acidity of the water coordinated to the [12]aneN3-ligated
metal ion, which is much higher for the zinc(II) complex (pKa
) 7.3)8 than for the copper(II) complex (pKa ) 8.4).7i However
in this case the different substitution pattern of metal ion binding
sites in 6-Cu2 and 7-Cu2 has a marked influence on catalytic
activity, the 1,2-vicinal bimetallic catalyst 6-Cu2 being much
more effective than its 1,3-distal regioisomer 7-Cu2. The
catalytic rate enhancement brought about by the latter is about
twice as great as those exhibited by mononuclear complexes,
showing that there is no rate acceleration per metal center. In
contrast, cooperativity between metal ions is observed in the
reaction catalyzed by 6-Cu2. Here the rate enhancement relative
to a monometallic analogue is either 70-fold or 40-fold,
depending on whether [12]aneN3-Cu or 4-Cu is taken as
reference.
Figure 1. Effect of added 4-Cu on the hydrolysis of 0.1 mM p-nitro-
phenyl acetate (water, pH 7.0, 20 mM HEPES, 25.0 °C; time-course
experiments).
fication of HPNP catalyzed by the zinc(II) complexes of the
BAMP functionalized calix[4]arene ligands.2g,3 In that case a
high degree of synergism between metal centers was observed
for both dinuclear complexes 1-Zn2 and 2-Zn2, with 61- and
380-fold rate-enhancements of dinuclear over mononuclear
catalyst, respectively. It is worth noting that [12]aneN3-Zn is
about 13 times more efficient a catalyst than BAMP-Zn;12 yet
metal complexes 6-Zn2, 7-Zn2, and 8-Zn3 exhibit no significant
synergism between metal centers. These findings clearly indicate
that promising arrangements of catalytically effective ligated
metal ions on a molecular platform by no means guarantee
efficient catalysis. Such a situation is disappointing but not
unprecedented. A number of examples are available in the
literature in which the efficiency of bimetallic complexes is
similar to, or even lower than, that of analogous monometallic
complexes.7b,13
We had better luck with the copper(II) complexes. With the
sole exception of 5-Cu, they were soluble enough in water for
kinetic measurements, the highest solubility being experienced
by 8-Cu3 (ca. 5 mM).
No evidence of trimetallic catalysis is seen in the reaction
catalyzed by the trinuclear complex 8-Cu3, whose catalytic
efficiency turns out to be slightly lower than that of 6-Cu2.
Therefore it appears that 8-Cu3 uses only two vicinal metal
centers in the catalytic mechanism, thus behaving as a 1,2-vicinal
bimetallic catalyst, whose efficiency is somewhat lowered by
the third ligated metal ion, presumably because of steric
hindrance.
In a study of phosphodiester hydrolysis catalyzed by the
copper(II) complex of 1,4,7-triazacyclononane (water solution,
pH 7.24), Burstyn and Deal7k reported that the catalytically
active monomeric form is in equilibrium with an inactive dimer,
1
as indicated by the clean /2-order dependence on the total
copper(II) complex concentration. Furthermore, the catalytic
inertness of copper(II) complexes of ditopic polynitrogen ligands
was ascribed by Mancin et al.13b to the formation of intracom-
plex µ-hydroxo-bridged dinuclear species. Therefore, the [12]-
aneN3-copper(II) system was tested for the possible formation
of unreactive dimeric species. As a convenient test reaction,
we have investigated the rate of p-nitrophenyl acetate hydrolysis
in the presence of increasing amounts of 4-Cu at pH 7.0. The
linear dependence of the pseudo-first-order rate constant on the
total concentration of 4-Cu (Figure 1) excludes any significant
involvement of dimeric species in the investigated concentration
range.
In line with expectations, a plot of initial rate vs substrate
concentration for the 8-Cu3 catalyzed transesterification of
HPNP showed the downward curvature typical of saturation
kinetics (Figure 2). Fitting of the data to the Michaelis-Menten
equation gave a value of 2.0 mM for KM and one of 7.6 × 10-4
s-1 for kcat. Thus, the kinetics are consistent with a two-step
catalytic mechanism in which a reversibly formed, moderately
strong catalyst-substrate complex (K ) 1/KM ) 500 M-1
)
decomposes into products with a first-order rate constant equal
to kcat. Any significant formation of unproductive 1:2 catalyst-
substrate complex must be ruled out, since a bell-shaped
concentration-rate profile should have been observed in such
a case.14 A likely mode of binding of HPNP to the catalyst
involves bridging4 of the phosphate moiety to the ligated copper-
(II) ions in 1,2-vicinal position. Accordingly, in line with
previous suggestions,4 the high rate accelerations observed for
(12) The value kobs/kbg ) 180 reported in ref 3 for the transesterification of 0.2
mM HPNP promoted by 1.0 mM BAMP-Zn has been corrected taking
into account the different catalyst concentration and assuming subsaturating
conditions.
(13) (a) Ozturk, G.; Akkaya, E. U. Org. Lett. 2004, 6, 241-243. (b) Mancin,
F.; Rampazzo, E.; Tecilla, P.; Tonellato, U. Eur. J. Org. Chem. 2004, 281-
288. (c) Worm, K.; Chu, F.; Matsumoto, K.; Best, M. D.; Lynch, V.; Anslyn,
E. V. Chem. Eur. J. 2003, 9, 741-747.
(14) Cacciapaglia, R.; Di Stefano, S.; Kelderman, E.; Mandolini, L. Angew.
Chem., Int. Ed. 1999, 38, 348-351.
9
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A R T I C L E S
Cacciapaglia et al.
Table 3. Cleavage of Diribonucleoside 3′,5′-Monophosphates
a
NpN′ in the Presence of Trinuclear Catalyst 8-Cu3
1
c
NpN
′
kobsb (s-
)
krel
kobs/kbg
CpA
GpA
CpG
CpC
ApG
GpG
GpU
UpG
UpU
<2 × 10-7d
1.4 × 10-6
2.1 × 10-6
2.5 × 10-6
2.7 × 10-6
2.8 × 10-6
3.7 × 10-6
4.2 × 10-5
5.5 × 10-5
<0.14
1.0
1.5
1.8
1.9
2.0
2.6
30
<3 × 102
0.6 × 104
0.6 × 104
1.3 × 104
1.3 × 104
39
1.4 × 105
a Reaction conditions: [8-Cu3] ) 1.0 mM, [NpN′] ) 0.10 mM, water,
pH 7.0 (HEPES 20 mM), 50.0 °C. b Error limits (10%. c 1010 kbg (pH 7.0,
50.0 °C) (s-1): CpA, 6.1; CpC, 4.3; UpU, 3.9; CpG, 3.5; GpG, 2.2; ApG,
2.1. From data reported in ref 17 after extrapolation (see ref 18) to pH 7.0.
d No reaction after 24h.
Figure 2. Initial rate of HPNP transesterification vs substrate concentration
for the reaction carried out in the presence of 0.20 mM 8-Cu3, in water,
pH 7.0 (20 mM HEPES), 25.0 °C. The points are experimental, and the
curve is a plot of the Michaelis-Menten equation with best fit values KM
ligand is most responsible for the lack of cooperativity between
metal ions in 7-Cu2. In contrast, a CPK model of the HPNP
complex with 6-Cu2 reveals a much more open structure, in
which the average planes of the ligating units are far from
parallel, and no steric hindrance to the motion of the 2-hydrox-
ypropyl chain is apparent.
) 2.0 mM and kcat ) 7.6 × 10-4 s-1
.
the transesterification catalyzed by 6-Cu2 and 8-Cu3 are due
to double Lewis acid activation, with the putative involvement
of the metal hydroxide as an intramolecular general base (I).
Diribonucleoside Monophosphate Cleavage. It is well-
known that investigations of phosphodiesters with good leaving
groups may not give conclusions which apply to phosphodiesters
with bad leaving groups, because species that cleave effectively
p-nitrophenyl phosphates are not necessarily good catalysts for
the cleavage of unactivated phosphate esters, and vice versa.13c,15
It was therefore of interest to investigate the catalytic activity
of our copper(II) complexes in the cleavage of diribonucleoside
3′,5′-monophosphates NpN′. The reaction involves intramolecu-
lar nucleophilic attack of the 2′-OH group and liberation of a
nucleoside and of a nucleoside 2′,3′-cyclic monophosphate (eq
2).15,16 The lot of investigated substrates listed in Table 3 is an
Inspection of a CPK molecular model of the complex of
HPNP with the 1,3-distal regioisomer 7-Cu2 shows that
phosphate bridging to the copper(II) ions requires a pinched-
cone conformation in which the two [12]aneN3 units are brought
in a nearly parallel position, thereby defining a narrow corridor
in which the HPNP substrate is hosted (II). It is difficult to
judge whether such a catalyst-substrate complex has a definite
stability, since the existence of, say, 2-3 kcal/mol of strain
energy arising from eclipsing and/or nonbonded interactions can
hardly be estimated on the basis of a CPK model. But what is
clearly suggested by inspection of the model is that one of the
[12]aneN3 units prevents the 2-hydroxypropyl chain from
assuming the pseudocyclic conformation required by OH
addition to the phosphorus. This is tantamount to saying that
7-Cu2 is not a good host for the transition state of the HPNP
transesterification because of steric repulsion between the
2-hydroxypropyl chain and one of the ligating units. We
speculate therefore that the sterically demanding [12]aneN3
arbitrary choice of 9 out of the 16 possible diribonucleoside
3′,5′-monophosphates but sufficient to the purpose of disclosing
nucleobase selective cleavage, if any. A first set of catalytic
experiments was carried out in water solution, pH 7.0 at 50.0
°C, in the presence of 8-Cu3, which is the complex with the
highest solubility in water. The kinetics were monitored by
HPLC. Typical chromatograms are shown in Figure 3. Initial
rates of nucleoside formation were translated into the pseudo-
(15) Oivanen, M.; Kuusela, S.; Lo¨nnberg, H. Chem. ReV. 1998, 98, 961-990.
(16) (a) Cassano, A. G.; Anderson, V. E.; Harris, M. E. Biopolymers 2004, 73,
110-129. (b) Perreault, D. M.; Anslyn, E. V. Angew. Chem., Int. Ed. Engl.
1997, 36, 432-450.
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Water-Soluble Synthetic CuII-Nucleases
A R T I C L E S
about 3 times more rapidly than UpU (Table 4). The lack of
activity of 8-Cu3 toward CpA must be due to the formation of
a presumably strong, nonproductive substrate-catalyst complex,
where an improper substrate orientation is imposed by the third
ligated metal ion.
There is ample evidence that the nucleobase structure has
only a modest influence on the background cleavage of the
phosphodiester bond of diribonucleoside monophosphates, under
both acid and basic conditions.15 Most relevant to the present
study is a set of specific rates (106 kbg, s-1) reported by
Komiyama17 for the cleavage at pH 11.08 and 50.0 °C: CpA,
7.3; CpC, 5.2; UpU, 4.7; CpG, 4.2; GpG, 2.7; ApG, 2.5.
Extrapolation18 of such data to pH 7.0 gave the values reported
in footnote c of Table 3, from which catalytic rate enhancements
Figure 3. HPLC traces of the cleavage of 0.10 mM UpU promoted by 1.0
mM 8-Cu3 (water, 20 mM HEPES, pH 7.0, 50.0 °C). Chromatograms of
the reaction mixture quenched at different time intervals showing chro-
matographic peaks of uridine (a), UpU (b), and internal standard (c). The
axis refers to the bottom trace. Other traces are offset with respect to the
next one by 1.0 min.
k
obs/kbg were calculated (Tables 3 and 4). Complexes 6-Cu2
and 8-Cu3 that provide ca. 1 × 104 rate enhancements (at 1.0
mM catalyst) for cleaving a large number of diribonucleoside
monophosphates (Tables 3 and 4) give ca. 103 rate enhancements
(at 0.2 mM catalyst) for cleaving HPNP (Table 2), which
indicates that replacement of a good leaving group with a bad
leaving group has no adverse effect on catalytic efficiency. In
fact, the monometallic [12]aneN3-Cu complex is much more
effective in the cleavage of diribonucleoside monophosphates
than in the cleavage of HPNP, which implies leaving group
activation through coordination of the poor leaving group
oxygen to the metal.2i The good catalytic efficiency of 6-Cu2
and 8-Cu3 in the cleavage of RNA dinucleoside monophos-
phates is at variance with a recent report by Anslyn et al.13c
that a bis-zinc(II) complex cleaves effectively HPNP with a
remarkable cooperativity between the two metals but shows no
cooperativity in the cleavage of UpU.
Table 4. CpA and UpU Cleavage in the Presence of Cu2+
Complexes of [12]aneN3 and 6-8a
CpA
UpU
1
c
1
c
catalyst
kobsb (s-
)
krel
k
obs/kbg
k
obs (s-
)
krel
kobs/kbg
[12]aneN3-Cu 6.6 × 10-7
1.0 1.1 × 103 2.7 × 10-7
2.0 2.1 × 103 3.8 × 10-7
1.0 6.9 × 102
6-Cu2
7-Cu2
8-Cu3
3.7 × 10-6
1.3 × 10-6
5.6 6.1 × 103 4.2 × 10-5 160
1.1 × 105
1.4 9.7 × 102
d
<2 × 10-7 <0.3 <3 × 102 5.5 × 10-5 200
1.4 × 105
a Reaction conditions: [sub.] ) 0.10 mM, [cat.] ) 1.0 mM (water, pH
7.0, HEPES 20 mM, 50.0 °C). b Error limits (10%. c See footnote c to
Table 3. d Data from Table 3.
first-order rate constants summarized in Table 3. With the sole
exception of CpA, for which no reaction was observed after 24
h, 8-Cu3 effectively cleaves all of the investigated diribo-
nucleoside monophosphates, with a remarkable preference for
UpU and UpG, that are cleaved 39 and 30 times, respectively,
more rapidly than GpA. Rates of transesterification of CpA and
UpU, the least and the most reactive diribonucleoside mono-
phosphate, respectively, in the 8-Cu3 catalyzed reaction, were
also investigated in the presence of [12]aneN3-Cu, 6-Cu2, and
7-Cu2. The results (Table 4) indicate that there are considerable
analogies with the order of catalytic efficiency observed in the
cleavage of HPNP (Table 2). First, in both CpA and UpU
cleavage reactions catalyzed by the 1,3-distal bimetallic catalyst
7-Cu2, there is no rate acceleration per metal center, as shown
by the behavior of the same substrates in the presence of the
mononuclear catalyst [12]aneN3-Cu. Second, cooperativity is
found between metal centers in the 1,2-vicinal bimetallic catalyst
6-Cu2. It is remarkably large in the cleavage of UpU and much
smaller, but still appreciable, in the cleavage of CpA. Finally,
the dinuclear complex 6-Cu2 catalyzes the cleavage of UpU
with an efficiency quite similar to that of 8-Cu3, thus
reinforcing the view that the trinuclear catalyst uses only two
vicinal metal centers, whereas the third metal ion acts as a more
or less innocent spectator.
A final comment is devoted to the most reactive diribo-
nucleoside monophosphates UpU and UpG. The 105-fold rate
accelerations provided by 6-Cu2 and 8-Cu3 are comparable
to those reported for the best synthetic copper(II)- and zinc-
(II)-based di- and trimetallic phosphodiesterases.2i,19 Further-
more, the high selectivity for UpU and UpG reported in this
work is quite remarkable, in view of the findings that in the
cleavage of 3′,5′-NpN’substrates a number of copper(II) com-
plexes of pyridine-based ligands20 are highly selective for
adenine bases vs uracil bases. As shown by Kimura et al.,21 the
acidity of uracil (pKa ) 9.9 for 3-methyluracil) increases to pKa
) 7.14 when it is linked to a doubly protonated [12]aneN4
moiety, as a result of the +2 electrostatic effect on the uracil
NH. Even higher is the acidity enhancement resulting from zinc-
(II) complexation to the [12]aneN4 moiety (pKa < 5). In line
with the above findings, we suggest that the uracil moiety at
the 5′-hydroxyl terminus of UpU and UpG is deprotonated and
(17) Komiyama, M. Carbohydr. Res. 1989, 192, 97-102.
(18) Analysis of pH-rate profiles related to the cleavage of diribonucleoside
monophosphates (Ja¨rvinen, P.; Oivanen, M.; Lo¨nnberg, H. J. Org. Chem.
1991, 70, 5396-5401) has shown that at pH > 7, the dominant reaction
path involves a monoanionic diester having additionally the 2′-hydroxyl
deprotonated. Accordingly, extrapolation of the background rate constants
from pH 11.08 to pH 7.0 was carried out as follows: kbg (pH 7.0) ) 0.83
× 10-4 kbg (pH 11.08).
The background rate of cleavage of CpA is very similar to
that of other diribonucleoside monophosphates (see below).
Hence, the surprising inertness of 8-Cu3 in CpA cleavage is
neither due to an inherently low reactivity of the substrate nor
to its insensitivity to copper(II) catalysis, as clearly shown by
the finding that CpA is cleaved by [12]aneN3-Cu and by 7-Cu2
(19) Molenveld, P.; Engbersen, J. F. J.; Reinhoudt, D. N. Angew. Chem., Int.
Ed. 1999, 38, 3189-3192.
(20) (a) Liu, S. H.; Hamilton, A. D. Chem. Commun. 1999, 587-588. (b)
Komiyama, M.; Kina, S.; Matsumura, K.; Sumaoka, J.; Tobey, S.; Lynch,
V. M.; Anslyn, E. J. Am. Chem. Soc. 2002, 124, 13731-13736.
(21) Kimura, E.; Kitamura, H.; Koike, T.; Shiro, M. J. Am. Chem. Soc. 1997,
119, 10909-10919.
9
J. AM. CHEM. SOC. VOL. 128, NO. 37, 2006 12327

A R T I C L E S
Cacciapaglia et al.
Materials. HPNP was available from previous investigations.3 [12]-
aneN3 was commercially available and used without further purification.
N-Benzyl-1,5,9-triazacyclododecane 4 was synthesized according to a
literature procedure.24 Diribonucleoside 3′,5′-monophosphates UpU,
GpG, and ApG were purchased from Sigma-Aldrich, while UpG, CpC,
GpA, GpU, CpA, and CpG were from Dharmacon Research (Lafayette,
CO). Pure samples of NpN′ and their aqueous solutions were stored at
-4 °C.
the resulting uracil anion forms a stable copper(II) complex.22
In this way one of the copper(II) centers in 6-Cu2 and 8-Cu3
acts as an anchoring site for a nonreacting part of the substrate
(III), whereas a second metal center in the 1,2-vicinal position
enables the entire catalytic jobsnucleophile activation and
delivery to a metal activated phosphoryl group and, possibly,
leaving group activation.23
5-[5,9-Bis(tert-butoxycarbonyl)-1,5,9-triazacyclododec-1-ylmethyl]-
25,26,27,28-tetrakis(2-ethoxyethoxy)calix[4]arene (14). K2CO3 (23
mg, 0.17 mmol) and bis-Boc-protected [12]aneN3 9 (85 mg, 0.229
mmol) were added to a solution of chloromethyl calix[4]arene 10 (115
mg, 0.152 mmol) in dry acetonitrile (4 mL), and the reaction mixture
was stirred under nitrogen for 2 days. The solvent was evaporated,
and the residue was dissolved in CH2Cl2 (40 mL) and washed with a
saturated NaHCO3 aqueous solution (40 mL). The aqueous phase was
extracted again with dichloromethane (40 mL), and the combined
organic layers were evaporated under vacuum. The crude product was
purified by column chromatography (SiO2; Et2O/Hex, 1/1), giving calix-
1
[4]arene 14 (118 mg) as a colorless oil. Yield 70%. H NMR (300
MHz; CDCl3): δ 6.73-6.70 (m, ArH, 4H), 6.62 (d, ArH, J ) 7.4 Hz,
2H), 6.58 (d, ArH, J ) 7.0 Hz, 2H), 6.48 (t, ArH, J ) 7.0 Hz, 1H),
6.43 (s, ArH, 2H), 4.50 (d, ArCH2Ar ax, J ) 13.2 Hz, 2H), 4.49 (d,
ArCH2Ar ax, J ) 13.2 Hz, 2H), 4.19 (t, ArOCH2, J ) 6.0 Hz, 4H),
4.07 (t, ArOCH2, J ) 5.6 Hz, 4H), 3.88 (t, ArOCH2CH2, J ) 6.0 Hz,
4H), 3.83 (t, ArOCH2CH2, J ) 5.6 Hz, 4H), 3.54 (q, OCH2CH3, J )
7.0 Hz, 4H), 3.53 (q, OCH2CH3, J ) 7.0 Hz, 4H), 3.36 (t, BocNCH2-
CH2CH2NBoc, J ) 6.8 Hz, 4H), 3.21 (t, NHCH2CH2CH2NBoc, J )
5.9 Hz, 4H), 3.16 (s, ArCH2N, 2H), 3.14 (d, ArCH2Ar eq, J ) 13.2
Hz, 2H), 3.11 (d, ArCH2Ar eq, J ) 13.2 Hz, 2H), 2.09 (t, ArCH2-
NHCH2, J ) 5.7 Hz, 4H), 1.90 (quin, BocNCH2CH2CH2NBoc, J )
6.8 Hz, 2H), 1.67 (quin, NHCH2CH2CH2NBoc, J ) 5.9 Hz, 4H), 1.47
(s, C(CH3)3, 18H), 1.21 (t, OCH2CH3, J ) 7.0 Hz, 6H), 1.20 (t,
OCH2CH3, J ) 7.0 Hz, 6H). 13C NMR (75 MHz; CDCl3): δ 156.5,
156.2, 155.9, 154.8, 135.3, 134.6, 134.1, 131.5, 128.6, 128.2, 128.1,
128.0, 122.2, 122.0, 79.1, 73.3, 72.9, 69.6, 66.3, 66.2, 56.4, 49.8, 45.3,
43.4, 30.9, 30.8, 28.5, 26.9, 26.5, 15.2. MS (ES+) m/z (%): 1096.3
In conclusion, we have reported an additional example of
the successful use of the calix[4]arene scaffold in the design
and synthesis of efficient dinuclear metallo-phosphodiesterases.
Combination of the present data with analogous data from
previous investigations shows that the catalytic efficiency of
calix[4]arene-based bimetallic complexes is the result of a subtle
interplay between the identity of the metal ion and the structure
of the ligating unit and critically depends on whether the
substitution pattern of ligated metal ions is 1,2-vicinal or 1,3-
distal. It also shows that the role of a third metal ion ranges
from strongly rate enhancing (GpG in the presence of 3-Zn3)19
to strongly rate retarding (CpA in the presence of 8-Cu3, Table
3), thus offering a varied phenomenological picture. In most
cases the synergic action of two metal centers provides ca. 104-
fold rate accelerations, which rise to 105-fold in the cleavage
of UpU and UpG, showing an unprecedented selectivity in
comparison with other base-selective artificial nucleases.
The good catalytic efficiency and selectivity of the copper-
(II) complexes described in this work, coupled with their water
solubility, encouraged further studies in the cleavage of RNA
oligonucleotides, which are currently carried out in our labo-
ratories.
(30) [M + H]+, 1118.0 (100) [M + Na]+, 570.6 (80) [M + 2Na]2+
.
Anal. Calcd for C64H93N3O12 (1096.46): C, 70.18; H, 8.55; N, 3.83.
Found: C, 70.04; H, 8.68; N, 3.77.
5,11-Bis[5,9-bis(tert-butoxycarbonyl)-1,5,9-triazacyclododec-1-yl-
methyl]-25,26,27,28-tetrakis(2-ethoxyethoxy)calix[4]arene (15). Com-
pound 15 was synthesized according to the preparation of 14, starting
from 1,2-dichloromethyl calix[4]arene 11 and using a 1.2 equiv excess
of K2CO3 and 1.35 equiv excess of bis-Boc-protected [12]aneN3 9 per
chloromethyl group. Reaction time: 3 days. The crude was purified
by column chromatography (Hex/EtOAc/Et2O, 6/4/1), giving calix[4]-
1
arene 15 as a colorless oil. Yield 45%. H NMR (300 MHz; CDCl3):
δ 6.67 (dd, ArH, J3 ) 7.6 Hz, J4 ) 1.3 Hz, 2H), 6.63 (dd, ArH, J3 )
7.6 Hz, J4 ) 1.3 Hz, 2H), 6.55 (d, ArH, J ) 1.0 Hz, 2H), 6.53 (t, ArH,
J ) 7.6 Hz, 2H), 6.46 (d, ArH, J ) 1.0 Hz, 2H), 4.50 (d, ArCH2Ar ax,
J ) 13.0 Hz, 1H), 4.48 (d, ArCH2Ar ax, J ) 13.2 Hz, 2H), 4.45 (d,
ArCH2Ar ax, J ) 13.0 Hz, 1H), 4.11 (t, ArOCH2, J ) 5.6 Hz, 8H),
3.85 (t, ArOCH2CH2, J ) 5.6 Hz, 8H), 3.54 (q, OCH2CH3, J ) 7.1
Hz, 4H), 3.53 (q, OCH2CH3, J ) 7.1 Hz, 4H), 3.35 (bs, BocNCH2-
CH2CH2NBoc, 8H), 3.24 (t, NCH2CH2CH2NBoc, J ) 6.7 Hz, 8H),
3.20 (s, ArCH2N, 4H), 3.15 (d, ArCH2Ar eq, J ) 13.0 Hz, 1H), 3.11
(d, ArCH2Ar eq, J ) 13.2 Hz, 2H), 3.07 (d, ArCH2Ar eq, J ) 13.0
Hz, 1H), 2.15 (bs, ArCH2NCH2, 8H), 1.90 (quin, BocNCH2CH2CH2-
NBoc, J ) 6.7 Hz, 4H), 1.69 (bs, ArCH2NCH2CH2, 8H), 1.45 (s,
C(CH3)3, 36H), 1.20 (t, OCH2CH3, J ) 7.1 Hz, 6H), 1.19 (t, OCH2CH3,
J ) 7.1 Hz, 6H). 13C NMR (75 MHz; CDCl3): δ 156.2, 155.2, 135.0,
134.6, 134.4, 131.7, 128.7, 128.6, 128.5, 128.1, 128.0, 127.7, 122.1,
79.1, 73.1, 69.7, 66.3, 49.8, 45.3, 43.5, 30.9, 29.7, 28.5, 26.5, 15.3.
Experimental Section
Instruments and General Methods. NMR spectra were recorded
on a 300 MHz spectrometer. Chemical shifts are reported as δ values
in ppm from tetramethylsilane added as the internal standard. Mass
spectra obtained by electrospray ionization (ESI) and chemical ioniza-
tion (CI) methods were recorded on a Micromass ZMD and on a
Finnigan Mat SSQ710 spectrometer, respectively.
(22) Anchoring of the azacrown-ligated zinc(II) ion of a bimetallic zinc(II)-
nickel(II) cleaving agent to deprotonated N(3) of the uracil moiety of the
3′-hydroxyl terminus of 3′,5′-UpU has been previously suggested. See:
Wang, Q.; Mikkola, S.; Lo¨nnberg, H. Chem. BiodiV. 2004, 1, 1316-1322.
(23) Unfortunately, it was impossible to analyze catalytic data in terms of the
Michaelis-Menten parameters kcat and KM, because of complications in
the kinetic experiments arising from aggregation/precipitation phenomena
in the region of high catalyst concentrations.
(24) Hubsch-Weber, P.; Youinou, M.-T. Tetrahedron Lett. 1997, 38, 1911-
1914.
9
12328 J. AM. CHEM. SOC. VOL. 128, NO. 37, 2006

Water-Soluble Synthetic CuII-Nucleases
A R T I C L E S
MS (ES+) m/z (%): 1479.9 (10) [M + H]+, 1502.4 (35) [M + Na]+,
762.4 (100) [M + 2Na]2+. Anal. Calcd for C84H130N6O16 (1479.99):
C, 68.17; H, 8.85; N, 5.68. Found: C, 68.05; H, 8.99; N, 5.51.
5,17-Bis[5,9-bis(tert-butoxycarbonyl)-1,5,9-triazacyclododec-1-yl-
methyl]-25,26,27,28-tetrakis(2-ethoxyethoxy)calix[4]arene (16). Com-
pound 16 was synthesized according to the preparation of 14, starting
from 1,3-dichloromethyl calix[4]arene 12 and using a 1.2 equiv excess
of K2CO3 and 1.35 equiv excess of bis-Boc-protected [12]aneN3 9 per
chloromethyl group. Reaction time: 4 days. The crude was purified
by column chromatography (Et2O/Hex, 4/1), giving calix[4]arene 16
as a colorless oil. Yield 54%. 1H NMR (300 MHz; CDCl3): δ 6.85 (s,
ArH, 4H), 6.34-6.22 (m, ArH, 6H), 4.48 (d, ArCH2Ar ax, J ) 13.2
Hz, 4H), 4.22 (t, ArOCH2, J ) 5.4 Hz, 4H), 3.98 (t, ArOCH2, J ) 5.7
Hz, 4H), 3.87 (t, ArOCH2CH2, J ) 5.4 Hz, 4H), 3.80 (t, ArOCH2CH2,
J ) 5.7 Hz, 4H), 3.57 (q, OCH2CH3, J ) 6.9 Hz, 4H), 3.50 (q, OCH2-
CH3, J ) 7.2 Hz, 4H), 3.39 (s, 4H, ArCH2N), 3.38-3.29 (m, BocNCH2-
CH2CH2NBoc + NHCH2CH2CH2NBoc, 16H), 3.10 (d, ArCH2Ar eq,
J ) 13.2 Hz, 4H), 2.41 (t, ArCH2NHCH2, J ) 6.0 Hz, 8H), 1.93 (quint,
BocNCH2CH2CH2NBoc, J ) 6.0 Hz, 4H), 1.80 (quint, NHCH2CH2-
CH2NBoc, J ) 6.0 Hz, 8H), 1.45 (s, C(CH3)3, 36H), 1.23 (t, OCH2CH3,
J ) 6.9 Hz, 6H), 1.17 (t, OCH2CH3, J ) 7.2 Hz, 6H). 13C NMR (75
MHz; CDCl3): δ 156.5, 156.2, 155.1, 135.8, 133.8, 132.2, 129.4, 127.6,
122.2, 79.2, 73.6, 72.6, 69.7, 69.6, 66.4, 66.2, 57.9, 50.2, 45.1, 43.6,
30.9, 28.5, 27.1, 26.8, 15.3, 15.2. MS (MALDI-TOF) m/z (%): 1480.5
(100) [M + H]+. Anal. Calcd for C84H130N6O16 (1479.99): C, 68.17;
H, 8.85; N, 5.68. Found: C, 68.29; H, 9.01; N, 5.50.
7.5 Hz, 1H), 6.41 (d, ArH, J ) 7.5 Hz, 2H), 6.25 (s, ArH, 2H), 4.56
(d, ArCH2Ar ax, J ) 13.1 Hz, 2H), 4.49 (d, ArCH2Ar ax, J ) 12.8
Hz, 2H), 4.40 (dt, ArOCHAHB, J2 ) 11.0 Hz, J3 ) 6.3 Hz, 2H), 4.31
(dt, ArOCHAHB, J2 ) 11.0 Hz, J3 ) 6.3 Hz, 2H), 3.99-3.90 (m,
ArOCH2 + ArOCH2CH2, 12H), 3.80-3.73 (m, ArOCH2CH2, 4H), 3.70
(s, ArCH2N, 2H), 3.57 (q, OCH2CH3, J ) 7.0 Hz, 2H), 3.55 (q, OCH2-
CH3, J ) 6.9 Hz, 2H), 3.52 (q, OCH2CH3, J ) 6.9 Hz, 4H), 3.15 (d,
ArCH2Ar eq, J ) 12.8 Hz, 2H), 3.14 (d, ArCH2Ar eq, J ) 13.1 Hz,
2H), 3.08 (bs, HNCH2CH2CH2NH, 4H), 2.95 (bs, NCH2CH2CH2NH,
4H), 2.12 (bs, ArCH2NCH2, 2H), 1.90 (bs, ArCH2NCH2, 2H), 1.66
(bs, NCH2CH2CH2NH, 4H), 1.51 (bs, NHCH2CH2CH2NH, 2H), 1.24
(t, OCH2CH3, J ) 7.0 Hz, 3H), 1.23 (t, OCH2CH3, J ) 6.9 Hz, 3H),
1.18 (t, OCH2CH3, J ) 6.9 Hz, 6H). 13C NMR (75 MHz; CDCl3): δ
156.9, 155.7, 154.7, 136.7, 136.4, 134.5, 133.6, 131.5, 128.8, 128.2,
127.4, 122.9, 122.4, 119.9, 74.4, 74.3, 72.3, 69.6, 69.4, 66.4, 66.3, 66.1,
52.1, 48.0, 47.4, 44.0, 30.7, 23.2, 21.9, 15.3,15.1. MS (ES+) m/z (%):
896.7 (30) [M + H]+, 918. 5 (100) [M + Na]+. Anal. Calcd for
C54H77N3O8 (896.23): C, 72.37; H, 8.66; N, 4.69. Found: C, 72.21;
H, 8.76; N, 4.78.
5,11-Bis(1,5,9-triazacyclododec-1-ylmethyl)-25,26,27,28-tetrakis-
(2-ethoxyethoxy)calix[4]arene (6). Mp (Et2O) ) 61-62 °C. 1H NMR
(300 MHz; CDCl3): δ 6.66-6.63 (m, ArH, 4H), 6.56-6.52 (m, ArH,
4H), 6.46 (s, ArH, 2H), 4.51 (d, ArCH2Ar ax, J ) 13.1 Hz, 2H), 4.49
(d, ArCH2Ar ax, J ) 13.1 Hz, 2H), 4.15 (t, ArOCH2, J ) 5.5 Hz, 4H),
4.13 (t, ArOCH2, J ) 5.5 Hz, 4H), 3.86 (t, ArOCH2CH2, J ) 5.5 Hz,
4H), 3.85 (t, ArOCH2CH2, J ) 5.5 Hz, 4H), 3.54 (q, OCH2CH3, J )
6.9 Hz, 4H), 3.53 (q, OCH2CH3, J ) 6.9 Hz, 4H), 3.30 (d, ArCHAHBN,
J ) 14.0 Hz, 2H), 3.24 (d, ArCHAHBN, J ) 14.0 Hz, 2H), 3.15 (d,
ArCH2Ar eq, J ) 13.1 Hz, 1H), 3.12 (d, ArCH2Ar eq, J ) 13.1 Hz,
2H), 3.08 (d, ArCH2Ar eq, J ) 13.1 Hz, 1H), 2.97 (t, HNCH2CH2CH2-
NH, J ) 4.8 Hz, 8H), 2.72 (t, NCH2CH2CH2NH, J ) 4.8 Hz, 8H),
2.18 (bs, ArCH2NCH2, 8H), 1.88 (bs, HNCH2CH2CH2NH, 4H), 1.67
(bs, NCH2CH2CH2NH, 8H), 1.22 (t, OCH2CH3, J ) 6.9 Hz, 6H), 1.20
(t, OCH2CH3, J ) 6.9 Hz, 6H). 13C NMR (75 MHz; CDCl3): δ 156.2,
155.2, 135.1, 134.7, 134.5, 130.9, 129.2, 128.9, 128.1, 122.2, 73.2,
69.7, 69.6, 66.3, 55.2, 51.8, 49.9, 49.5, 47.1, 31.0, 30.8, 29.7, 27.7,
25.2, 15.3. MS (ES+) m/z (%): 540.2 (100) [M + 2H]2+. Anal. Calcd
for C64H98N6O8 (1079.52): C, 71.21; H, 9.15; N, 7.78. Found: C, 71.38;
H, 9.29; N, 7.59.
5,11,17-Tris[5,9-bis(tert-butoxycarbonyl)-1,5,9-triazacyclododec-
1-ylmethyl]-25,26,27,28-tetrakis(2-ethoxyethoxy)calix[4]arene (17).
Compound 17 was synthesized according to the preparation of 14,
starting from trichloromethyl calix[4]arene 13 and using a 1.15 equiv
excess of K2CO3 and bis-Boc-protected [12]aneN3 9 per chloromethyl
group. Reaction time: 5 days. The crude was purified by column
chromatography using Et2O as eluent, giving calix[4]arene 17 as a
colorless oil. Yield 60%. 1H NMR (300 MHz; CDCl3): δ 6.82 (d, ArH,
J ) 1.0 Hz, 2H), 6.74 (d, ArH, J ) 1.0 Hz, 2H), 6.25 (s, ArH, 3H),
6.09 (s, ArH, 2H), 4.45 (d, ArCH2Ar ax, J ) 13.2 Hz, 2H), 4.42 (d,
ArCH2Ar ax, J ) 13.2 Hz, 2H), 4.21 (t, ArOCH2, J ) 6.2 Hz, 4H),
3.92 (t, ArOCH2, J ) 5.2 Hz, 4H), 3.84 (t, ArOCH2CH2, J ) 6.2 Hz,
4H), 3.75 (t, ArOCH2CH2, J ) 5.2 Hz, 4H), 3.52 (q, OCH2CH3, J )
7.0 Hz, 4H), 3.44 (q, OCH2CH3, J ) 7.0 Hz, 4H), 3.35 (s, ArCH2N,
4H), 3.33-3.26 (m, CH2NBocCH2, 20H), 3.08 (bs, NCH2CH2CH2-
NBoc, 4H), 3.06 (d, ArCH2Ar eq, J ) 13.2 Hz, 2H), 3.02 (d, ArCH2-
Ar eq, J ) 13.2 Hz, 2H), 2.94 (s, ArCH2N, 2H), 2.35 (bs, ArCH2NCH2,
8H), 1.94-1.78 (bs, ArCH2NCH2 + BocNCH2CH2CH2NBoc, 10H),
1.75 (m, ArCH2NCH2CH2, 8H), 1.52 (bs, ArCH2NCH2CH2, 4H), 1.40
(s, C(CH3)3, 54H), 1.27 (t, OCH2CH3, J ) 7.0 Hz, 6H), 1.19 (t, OCH2-
CH3, J ) 7.0 Hz, 6H). 13C NMR (75 MHz; CDCl3): δ 156.4, 156.2,
156.1, 155.1, 154.0, 135.8, 135.7, 133.7, 133.2, 132.1, 131.4, 129.3,
128.1, 127.5, 125.4, 122.0, 79.2, 79.1, 73.7, 72.4, 69.7, 69.6, 66.4, 66.1,
57.9, 49.9, 49.5, 45.4, 45.1, 43.5, 43.4, 30.9, 30.3, 29.6, 28.5, 27.2,
27.1, 26.7, 26.4, 15.3, 15.2. MS (FAB+) m/z (%): 1863.3 (100) [M +
H]+. Anal. Calcd for C104H167N9O20 (1863.53): C, 67.03; H, 9.03; N,
6.76. Found: C, 67.20; H, 9.19; N, 6.67.
5,17-Bis(1,5,9-triazacyclododec-1-ylmethyl)-25,26,27,28-tetrakis-
1
(2-ethoxyethoxy)calix[4]arene (7). H NMR (300 MHz; CDCl3): δ
6.75 (s, ArH, 4H), 6.32-6.22 (m, ArH, 6H), 4.45 (d, ArCH2Ar ax, J
) 13.5 Hz, 4H), 4.15 (t, ArOCH2, J ) 6.0 Hz, 4H), 3.95 (t, ArOCH2,
J ) 5.1 Hz, 4H), 3.80 (t, ArOCH2CH2, J ) 6.0 Hz, 4H), 3.76 (t,
ArOCH2CH2, J ) 5.1 Hz, 4H), 3.50 (q, OCH2CH3, J ) 6.9 Hz, 4H),
3.45 (q, OCH2CH3, J ) 7.2 Hz, 4H), 3.33 (s, ArCH2N, 4H), 3.04 (d,
ArCH2Ar eq, J ) 13.5 Hz, 4H), 2.97 (bs, HNCH2CH2CH2NH, 8H),
2.73 (bs, CH2NHCH2CH2CH2NHCH2, 8H), 2.42 (bs, ArCH2NCH2, 8H),
1.85 (bs, NHCH2CH2CH2NH, 4H), 1.75 (bs, ArCH2NCH2CH2, 8H),
1.16 (t, OCH2CH3, J ) 6.9 Hz, 6H), 1.10 (t, OCH2CH3, J ) 7.2 Hz,
6H). 13C NMR (75 MHz; CDCl3): δ 156.7, 155.3, 135.9, 133.9, 130.6,
129.4, 127.6, 122.3, 73.6, 72.9, 69.8, 69.6, 66.4, 66.2, 55.2, 51.9, 50.0,
46.8, 31.0, 23.9, 15.3, 15.2. MS (FAB+) m/z (%): 1079.6 (100) [M +
H]+. Anal. Calcd for C64H98N6O8 (1079.52): C, 71.21; H, 9.15; N,
7.78. Found: C, 71.39; H, 9.27; N, 7.68.
General Procedure for the Boc Deprotection. Boc protecting
groups were removed from compounds 14-17 by the following
procedure. The Boc-protected calix[4]arene was dissolved in CH2Cl2
(2 mL), and trifluoroacetic acid (1 mL) was added. The solution was
stirred for 2 h and then evaporated under reduced pressure. The residue
was taken up with dichloromethane (20 mL) and a Li2CO3 aqueous
saturated solution (20 mL). The organic phase was separated, the
aqueous one was extracted again with CH2Cl2 (2 × 20 mL), and the
organic fractions were combined and evaporated under vacuum, to give
the corresponding deprotected products 5-8 in quantitative yield.
5-(1,5,9-Triazacyclododec-1-ylmethyl)-25,26,27,28-tetrakis-(2-
ethoxyethoxy)calix[4]arene (5). 1H NMR (300 MHz; CDCl3): δ 7.05-
7.01 (m, ArH, 4H), 6.82 (t, ArH, J ) 7.5 Hz, 2H), 6.56 (t, ArH, J )
5,11,17-Tris(1,5,9-triazacyclododec-1-ylmethyl)-25,26,27,28-tet-
rakis(2-ethoxyethoxy)calix[4]arene (8). Mp (Et2O) ) 59-60 °C. 1H
NMR (300 MHz; CDCl3): δ 6.86 (d, 2H, ArH, J ) 1.1 Hz), 6.82 (d,
ArH, J ) 1.1 Hz, 2H), 6.29 (s, ArH, 3H), 6.14 (s, ArH, 2H), 4.43 (d,
ArCH2Ar ax, J ) 12.8 Hz, 2H), 4.41 (d, ArCH2Ar ax, J ) 13.2 Hz,
2H), 4.20 (t, ArOCH2, J ) 6.2 Hz, 4H), 3.92 (t, ArOCH2, J ) 5.1 Hz,
4H), 3.84 (t, ArOCH2CH2, J ) 6.2 Hz, 4H), 3.74 (t, ArOCH2CH2, J )
5.1 Hz, 4H), 3.50 (q, OCH2CH3, J ) 6.9 Hz, 4H), 3.40 (q, OCH2CH3,
J ) 6.9 Hz, 4H), 3.34 (d, ArCHAHBN, J ) 13.2 Hz, 2H), 3.26 (d,
ArCHAHBN, J ) 13.2 Hz, 2H), 3.07 (d, ArCH2Ar eq, J ) 12.8 Hz,
2H), 3.05 (d, ArCH2Ar eq, J ) 13.2 Hz, 2H), 3.00 (s, ArCH2N, 2H),
9
J. AM. CHEM. SOC. VOL. 128, NO. 37, 2006 12329

A R T I C L E S
Cacciapaglia et al.
2.76 (t, HNCH2CH2CH2NH, J ) 4.8 Hz, 8H), 2.69 (t, HNCH2CH2CH2-
NH, J ) 4.8 Hz, 4H), 2.63 (t, NCH2CH2CH2NH, J ) 5.1 Hz, 8H),
2.48 (t, NCH2CH2CH2NH, J ) 5.2 Hz, 4H), 2.41 (bs, ArCH2NCH2,
8H), 1.92 (bs, ArCH2NCH2, 4H), 1.61 (bs, NCH2CH2CH2NHCH2CH2,
14H), 1.37 (bs, NCH2CH2CH2NH, 4H), 1.17 (t, OCH2CH3, J ) 7.0
Hz, 3H), 1.14 (t, OCH2CH3, J ) 7.0 Hz, 3H), 1.10 (t, OCH2CH3, J )
7.0 Hz, 6H). 13C NMR (75 MHz; CDCl3): δ 156.4, 155.1, 154.2, 135.7,
133.9, 133.3, 131.6, 129.9, 129.4, 129.3, 127.6, 122.3, 73.7, 72.6, 69.7,
69.6, 66.4, 66.2, 55.9, 52.3, 51.2, 49.9, 49.5, 47.1, 47.0, 31.0, 30.3,
29.7, 25.6, 25.2, 25.1, 24.9, 15.3, 15.2. MS (ES+) m/z (%): 1262.8
(30) [M + H]+, 632.0 (100) [M + 2H]2+. Anal. Calcd for C74H119N9O8
(1262.82): C, 70.38; H, 9.50; N, 9.98. Found: C, 70.25; H, 9.41; N,
9.83.
Kinetic Measurements. Spectrophotometric measurements were
carried out on either a double beam or on a diode array spectropho-
tometer. HPLC analyses were performed on a liquid chromatograph
fitted with a UV-vis detector operating at 254 nm. The samples were
analyzed on a Supelcosil LC-18 DB column (25 cm × 4.6 mm I.D.,
particle size 5 µm).
HPNP Transesterification. Kinetic measurements of transesterifi-
cation of HPNP were monitored by UV-vis spectrophotometry at 400
nm. Rate constants were obtained by an initial rate method, error limit
on the order of (10%.
Diribonucleoside 3′,5′-Monophosphate Cleavage. Cleavage of
diribonucleoside 3′,5′-monophosphates NpN′ was monitored by HPLC
analyses of aliquots of the reaction mixture withdrawn at appropriate
time intervals. Reactions were carried out at 50.0 °C on 0.1 mM NpN′
and 1 mM catalyst solutions in water, 20 mM HEPES, pH 7.0. In a
typical experiment the ligand 8 (10 µL, 50 mM in ethanol) and CuCl2
(50 µL, 30 mM in water) were added to 430 µL of the buffered water
solution (20 mM HEPES, pH 7.0) thermostated at 50.0 °C. After 2 h
NpN′ (10 µL, 5 mM in water) was added. At proper time intervals
aliquots (80 µL) of the reaction mixture were withdrawn and quenched
with 80 µL of a saturated water solution of EDTA. After addition of
the internal standard (50 µL of 0.16 mM p-hydroxybenzoic acid), the
solution was filtered and subjected to HPLC analysis by elution with
H2O (0.1% trifluoroacetic acid)/MeCN, linear gradient from 100:0 to
85:15 in 20 min, flow 0.9 mL/min.
The aqueous solutions were prepared from deionized (Millipore)
distilled water.
Acknowledgment. Financial contribution from MIUR COFIN
2003 Progetto Dispositivi Supramolecolari is acknowledged for
the work carried out in Parma and Roma. The Centro Interfacolta`
di Misure (CIM) of the University of Parma is also acknowl-
edged for the use of NMR and MS facilities.
Metal complexes were formed in situ by addition of the calculated
stoichiometric amount of the aqueous solution of the metal salt (Zn-
(ClO4)2 or CuCl2) to the buffered reaction mixture. Due to the slow
formation of the complexes,25 the solutions were incubated for 2 h
before the start of the kinetic run by fast addition of a small volume of
the substrate solution.
Nonlinear least-squares calculations of kinetic data were carried out
using the program SigmaPlot 2002 for Windows, version 8.0 (SPSS,
Inc.).
Supporting Information Available: 1H NMR spectrum of
compounds 5-8 in CDCl3 and of compound 8 in D2O (PDF).
This material is available free of charge via the Internet at
http://pubs.ac.org.
(25) Riedo, T. J.; Kaden T. A. HelV. Chim. Acta 1979, 62, 1089-1096.
JA0632106
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12330 J. AM. CHEM. SOC. VOL. 128, NO. 37, 2006