Ribonuclease activity of an artificial catalyst that combines a ligated CuII ion and a guanidinium group at the upper rim of a cone -Calix[4]arene platform

Article abstract of DOI:10.1021/acs.joc.5b00965

A cone-calix[4]arene derivative, featuring a guanidinium group and a Cu^II ion ligated to a 1,4,7-triazacyclononane (TACN) ligand at the 1,3-distal positions of the upper rim, effectively catalyzes the cleavage of 2-hydroxypropyl p-nitrophenyl phosphate (HPNP) and a number of diribonucleoside 3′,5′-monophosphates (NpN′). Kinetic and potentiometric measurements support the operation of a general-base/general-acid mechanism and demonstrate that the hydroxo form of the ligated Cu^II ion is the sole catalytically active species. Rate enhancements relative to the background hydrolysis reaction at 1 mM catalyst concentration are 6 × 10⁵-fold for HPNP and cluster around 10⁷-fold with the most favorable catalyst-NpN′ combinations.

Full text of DOI:10.1021/acs.joc.5b00965

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Article
Ribonuclease activity of an artificial catalyst that combines a ligated CuII ion
and a guanidinium group at the upper rim of a cone calix[4]arene platform
Riccardo Salvio, Stefano Volpi, Roberta Cacciapaglia,
Alessandro Casnati, Luigi Mandolini, and Francesco Sansone
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.5b00965 • Publication Date (Web): 11 May 2015
Downloaded from http://pubs.acs.org on May 17, 2015
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Ribonuclease activity of an artificial catalyst that combines a ligated CuII
ion and a guanidinium group at the upper rim of a cone calix[4]arene
platform.
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Riccardo Salvio,*a Stefano Volpi,b Roberta Cacciapaglia,a Alessandro Casnati,b Luigi Mandolini,a
Francesco Sansoneb
aDipartimento di Chimica and IMC - CNR Sezione Meccanismi di Reazione, Università La Sapienza,
00185 Roma, Italy
bDipartimento di Chimica, Università degli Studi di Parma, Viale delle Scienze 17/A, 43124 Parma,
Italy
ABSTRACT: A cone-calix[4]arene derivative, featuring a guanidinium group and a CuII ion ligated
to a 1,4,7-triazacyclononane (TACN) ligand at the 1,3-distal positions of the upper rim, effectively
catalyzes the cleavage of 2-hydroxypropyl p-nitrophenyl phosphate (HPNP) and a number of
diribonucleoside 3’,5’-monophosphates (NpN’). Kinetic and potentiometric measurements support
the operation of a general-base/general-acid mechanism and demonstrate that the hydroxo form
of the ligated CuII ion is the sole catalytically active species. Rate enhancements relative to the
background hydrolysis reaction at 1 mM catalyst concentration are 6×105-fold for HPNP and
cluster around 107-fold with the most favorable catalyst-NpN’ combinations.
INTRODUCTION
In view of the biological importance of the phosphodiester bond, notably in DNA and RNA, a great
effort has been devoted by many workers to the design and synthesis of supramolecular catalysts
endowed with phosphodiesterase activity.1,2,3 In its simplest expression, a supramolecular catalyst
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Figure 1. Suggested mechanism of HPNP cleavage catalyzed by the metal complexes of 1H+
(MII = CuII, ZnII).
consists of a recognition unit and a catalytic unit connected by a suitable linker. In a large number
of phosphodiesterases reported so far, the recognition unit consists of one or two guanidinium
units,3 whereas the catalytic unit is either a general base or a nucleophile, depending on whether
the catalyst is expected to display RNAase or DNAase activity, respectively.
In recent works, we4 and others5 have used a m-xylylene spacer for connecting a guanidinium
group to a 1,4,7-triazacyclononane (TACN) ligand, and the role of general base was played by the
hydroxo form of a ligated metal ion (ZnII, CuII), Figure 1.
These bifunctional catalysts showed from modest (ZnII) to fairly high (CuII) degrees of
cooperation between catalytic units in the transesterification of HPNP (eq 1) in 4:1 (v/v)
DMSO-water (heretofore referred to as 80% DMSO), and rate enhancements relative to
background hydrolysis of 1×104-fold and 4×104-fold, respectively, at 5 mM catalyst
concentration.
4
(1)
Catalytic efficiency requires that in the transition state, substrate and catalyst form a well-
matched pair in terms of size and geometrical features. Consequently, the catalytic performance
of a bifunctional catalyst critically depends on the nature of the scaffold on which the catalytic
units are implanted.
The use of the upper rim of cone-calix[4]arenes as a convenient platform for the introduction
of catalytic units is well documented.6 The high catalytic efficiency achieved in a number of cases
has been ascribed, inter alia, to a high level of adaptability resulting from the conformational
mobility of the calix[4]arene scaffold itself.6e It seemed therefore worthwhile to synthesize
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compound 2, and to compare the phosphodiesterase activity of its ZnII and CuII complexes to that
of the corresponding complexes of 1.
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Calix[4]arenes functionalized with two or more different units at the upper rim are quite rare7
and, to the best of our knowledge, there are no important examples where calixarene catalysts or
receptors can benefit from the cooperation of these different functional groups. The exploitation
of such an unexplored arrangement of functions is, moreover, also hampered by the paucity of
synthetic methodologies able to differentiate between the groups present at the upper rim of a
calixarene scaffold.
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In this paper we report on the synthesis of the bifunctional compound 2 and a kinetic
investigation of the cleavage of HPNP in the presence of the CuII complex of 2H+. The finding that
the catalyst turned out to cleave HPNP much more effectively than the CuII complex of 1H+
encouraged an extension of the catalytic study to the cleavage of a number of diribonucleoside
monophosphates (eq 2).
(2)
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RESULTS AND DISCUSSION
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Synthesis of the ligand. Compound 2 was synthesized as described in Scheme 1, starting
from 5,17-bis(chloromethyl)-25,26,27,28-tetrakis(2-ethoxyethoxy)calix[4]arene (3).8
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Scheme 1. Synthesis of 2 a
a
a: Potassium phthalimide; toluene, 110 °C. b: N,N’-Bis-(tert-butoxycarbonyl)-1,4,7-
triazacyclononane, K2CO3; CH3CN, rt. c: N2H4∙H2O; MeOH, 60 °C. d: N,N’-Bis-(tert-butoxycarbonyl)-
N’’-triflylguanidine, NEt3; CH2Cl2, rt. e: (1) CF3COOH (TFA), Et3SiH (TES); CH2Cl2, rt; and then (2) 1M
aqueous HCl; EtOH, rt.
The first step resulted to be the crucial one in the desymmetrization of the two functional
groups present at the upper rim of the calixarene scaffold. By using a stoichiometric amount of
potassium phthalimide and dichloromethyl calixarene 3 a nearly statistical distribution of isolated
products could be found. Besides the monochloromethyl-monophthalimidomethyl-derivative 4 as
main-product (38%), a mixture of the diphthlimidomethyl-calixarene (16%) and of unreacted 3
(23%) was obtained. The following synthetic steps proceeded smoothly. The chloromethyl group in
4, was reacted with N,N’-bis-(Boc)-1,4,7-triazacyclononane to obtain 5 (70% yield), which was
quantitatively deprotected from phthalimide to 6. Guanidinylation of the CH2NH2 group with
[N,N’–bis-(Boc)]triflylguanidine9 afforded 7 (68% yield) and final Boc removal with trifluoroacetic
acid, followed by trifluoroacetate anion exchange with chloride, gave compound 2.4HCl (98%
yield). All the newly synthesized compounds 4-7 and 2 show NMR and mass spectra consistent
1
13
with their structures. H- and C- NMR spectra of compounds 5, 6 and 7 show patterns of signals
compatible with the presence of different rotamers as a consequence of the presence of the
bis-Boc protected triazacyclononane on their upper rim and in agreement with what found with
other bis-Boc-TACN functionalized derivatives10 . These rotamers and their sets of signals
completely disappear upon removal of the Boc groups, and the NMR spectra of 2 become
perfectly compatible with the presence of a single chemical species.
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Potentiometric titrations. Preliminary to the kinetics, potentiometric titrations of 2∙4HCl
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in the absence and presence of ZnCl2 or CuCl2 were carried out in 80% DMSO at 25 °C. In this
medium the autoprotolysis of water is strongly suppressed, pKw = 18.4 at 25 °C,11 and this implies
that pH 9.2 corresponds to a neutral solution. The results of the potentiometric titrations (Figures
2 and 3) are summarized in Table 1. Titration of 2∙4HCl with Me4NOH (Figure 2a) showed, as
expected, four titratable protons. The least acidic proton (pKa = 13.8) belongs to the guanidinium
unit, whereas the three most acidic protons are those of the fully protonated nitrogen ligand.
Addition of 1 mol equiv of ZnCl2 strongly modified the shape of the titration curve (Figure 2b) and
caused an increase to five of the number of titratable protons. The three most acidic protons were
again assigned to the fully protonated azamacrocycle, whose increase in apparent acidity was
ascribed to the strong binding of the ligand to the metal ion (log KZn = 7.6). The pKa value of 9.9
was assigned to a ZnII-coordinated water molecule.
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Titration of 2-4HCl in the presence of 1 mol equiv of CuCl2 gave similar results (Figure 3). The
affinity of CuII for the triazacyclononane ligand (log KCu = 9.2) is larger than that of ZnII, and the
metal-coordinated water molecule is significantly more acidic (pKa = 8.8).
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pH
pK = 13.8
a
14
pK = 13.7
a
(a)
(b)
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pK = 9.3
pK = 9.9
a
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II
Zn
pK = 4.2
a
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pK = 2.4
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0
0
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Me NOH (molar equiv)
4
Figure 2. Potentiometric titration of 1.0 mM 2(H+)4 with Me4NOH in 80% DMSO in the absence (a)
and presence (b) of 1 molar equiv of ZnII. Data points are experimental and the lines are
calculated.
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pH
pK = 13.8
a
pK = 13.9
a
(a)
(b)
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pK = 9.3
a
pK = 8.8
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Cu
pK = 4.2
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pK = 2.4
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Me NOH (molar equiv)
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Figure 3. Potentiometric titration of 1.0 mM 2(H+)4 with Me4NOH in 80% DMSO in the absence (a)
and presence (b) of 1 molar equiv of CuII. Data points are experimental and the lines are
calculated.
Table 1. Acidity Constants (pKa) of 2·4HCl, in the absence and
presence of 1 molar equiv of ZnII or CuII, (80% DMSO, 25 °C)a
entry additive
pKa1
2.4
< 2
pKa2
4.2
< 2
pKa3
9.3
6.9
5.9
pKa4
13.8 b
9.9
pKa5
1
2
3
none
ZnII c
CuII d
13.7
13.9
< 2
< 2
8.8
apKa data from potentiometric titration with Me4NOH of 1.0 mM
substrate solutions in the presence of 10 mM Me4NClO4.
b
Experimental uncertainty = ± 0.1 pK units or less. Under the
c
same conditions, the pKa of guanidine∙HCl is 13.7 (see ref 4).
From the potentiometric titration: log KZn = 7.6 ± 0.4, for the
binding of ZnII to the triazacyclononane moiety in 2H+. d From the
potentiometric titration: log KCu = 9.2 ± 0.4, for the binding of CuII
to the triazacyclononane moiety in 2H+.
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2. Cleavage of HPNP. In a first set of catalytic runs the pH was equal to the pKa of the
metal-coordinated water molecule, namely, pH 9.9 for the ZnII complex and pH 8.8 for the CuII
complex. At the given pH values the guanidine unit is fully protonated, the triazacyclononane
ligand is fully bound to the metal ion, and the latter is 50% in the hydroxo form. The results of the
kinetic experiments are listed in Table 2 as pseudo-first-order specific rates kobs = vo/[HPNP],
where vo is the spectrophotometrically determined initial rate of p-nitrophenol release.
The metal complexes of the bifunctional compound 2H+ are catalytically more effective than
their monofunctional controls (compare entries 1 and 3 with entries 2 and 4, respectively),12 which
indicates the existence of cooperation of the hydroxo complexes of the ligated metal ions with the
neighboring guanidinium. However, the ZnII complex of 2H+ is only 5 times more effective than the
corresponding complex of TACN. This disappointingly moderate degree of synergism between
catalytic units is very similar to that observed for the ZnII complex of 1H+, showing that as long as
the ZnII complexes are involved, replacement of a m-phenylene spacer with an upper rim 1,3-distal
calix[4]arene does not improve catalytic efficiency.
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Excellent results, on the other hand, were obtained with the CuII complex. The catalytic
efficiency of 2H+-Cu is 103 times higher than that of the monofunctional control TACN-Cu, and 25
times higher than that of the corresponding complex of 1H+-Cu,
4 showing that the remarkably
high catalytic performance arises from a very large degree of cooperation between catalytic units.
Notably, the catalytic rate enhancement relative to background hydrolysis (kobs/ kbg) is as high as
3.2×105.
The strictly linear dependence of initial rate on substrate concentration (Figure 4) shows that
association of the catalyst to HPNP, if any, is too weak to affect the kinetics in the investigated
concentration range, as previously found to be the case with catalyst 1H+-Cu.
4 In other words,
there is no significant binding in the reactant state that may be ascribed to two-point hydrogen
bonding interaction of the guanidinium with the anionic phosphate group. All of the available
binding energy arising from the above interaction is utilized in transition state stabilization and,
consequently, is fully translated into catalysis.
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Table 2. Pseudo-first order rate constants and acceleration
factors relative to background for the HPNP cleavage in the
presence of additives, (80% DMSO, 25 °C)a
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c
kobs (s−1) b
2.0 × 10−5
1.1 × 10−4
entry conditions
additives
kobs / kbg
400
1
2
pH 9.9
pH 8.8
TACN + ZnCl2
2H+ + ZnCl2
2200
1.4 × 10−6
1.3 × 10−3
3
4
350
TACN + CuCl2
2H+ + CuCl2
320000
a0.20 mM HPNP; 0.50 mM additive; 0.10 M N,N’-diisopropyl-N-
ethanolamine buffer. bkobs calculated as vo/[HPNP]; error limits on
the order of ±10%. From ref 4: kbg = 5.0 × 10−8 s−1 at pH 9.9, and
c
kbg = 4.0 × 10−9 s−1 at pH 8.8.
2.0
1.5
1.0
0.5
0.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
[HPNP] (mM)
Figure 4. Plot of initial rates for the cleavage of HPNP in 80% DMSO catalyzed by 0.50 mM 2H+-Cu
vs substrate concentration, pH 8.8, 25 °C. From the slope of the straight line
kobs = (1.36 ±0.06) × 10−3 s−1.
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Investigation of the catalytic efficiency of 2H+-Cu as a function of pH gave a sigmoid-shaped
pH-rate profile (Figure 5). The catalyst is inactive at pH < 7, but becomes increasingly more active
at higher pH values until saturation is reached at pH > 11. Data points were fitted to eq 3, where
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k
ꢄ
ꢅꢆꢇ
ꢀꢁꢂꢃ
=
(3)
ꢋ
ꢊ
ꢌ
ꢈꢉ H /ꢍ
ꢆ
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k is the second-order rate constant for reaction of the fully deprotonated form of a species whose
acidity constant is Ka and stoichiometric concentration is Ccat. A least-squares fitting procedure
gave pKa = 8.7 ± 0.1 and k = 5.3 ± 0.2 M−1 s−1. The nice fit of data points to eq 3 and the good
agreement of the kinetically determined pKa value with the potentiometric value of 8.8 ± 0.1 are
clearly consistent with the idea that the hydroxo form of the ligated CuII ion is the sole catalytically
active species. The limiting value of (2.65 ± 0.10) × 10−3 s−1 approached by the quantity kobs = k Ccat
in the high pH domain (i.e., when [H+]/Ka <<1) is twice as large as the kobs value measured at pH 8.8
(Table 2, entry 4 and caption to Figure 4), where only half of the catalyst is in its active form.
3.0
2.5
2.0
1.5
1.0
0.5
0.0
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pH
Figure 5. pH-rate profile for the cleavage of 0.20 mM HPNP catalyzed by 0.50 mM 2H+-Cu in 80%
DMSO, 25 oC. Data points are experimental and the line is the plot of eq 3.
3. Cleavage of diribonucleoside monophosphates. Independent of whether the cleavage
of phosphate diesters occurs via a two step (AN+DN) or a concerted (ANDN) mechanism,1b there
seems to be little doubt that upon replacement of a good leaving group with a poor one the
transition state bears a closer resemblance to a pentavalent phosphorane dianion. Consequently,
larger rate enhancements are expected for the reactions of phosphodiesters less activated than
HPNP, arising from a stronger electrostatic/electrophilic interaction with the neighboring
guanidinium of the bifunctional catalyst. Accordingly, the high catalytic performance of 2H+-Cu in
the cleavage of HPNP prompted us to expand the study to the cleavage of
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diribonucleoside-3’,5’-monophosphates NpN’ (eq 2). A set of four NpN’ substrates, namely GpA,
GpU, UpU, and CpA were subjected to catalytic cleavage under the same conditions adopted for
HPNP, with the sole difference that the temperature was raised from 25.0 to 50.0 °C because
diribonucleoside monophosphates are much more reluctant than HPNP to undergo hydrolytic
cleavage. The reaction progress was monitored as before13 by HPLC analysis of periodically
withdrawn samples of the reaction mixtures. Pseudo-first-order specific rates kobs listed in Table 3
were calculated as kobs= vo/[NpN’], where vo is the initial rate of nucleoside N’ formation (eq 2).
Table 3 shows that 2H+-Cu effectively cleaves the investigated substrates, with catalytic rates
decreasing in the order GpA>GpU>UpU>>CpA. Because of the different temperatures at which the
kinetics were carried out, namely, 50 °C vs 25 °C, a convenient way to compare catalytic
efficiencies in the cleavage of diribonucleoside monophosphates vs HPNP is to resort to catalytic
rates relative to background (kobs/kbg), under the assumption that the temperature coefficients of
the kobs/kbg ratios are not too different. In line with expectations, comparison of the kobs/kbg values
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Table 3. Cleavage of diribonucleoside 3',5'-monophosphates
NpN' by 2H+-Cu.a
106 × kobs (s−1) b 1011 × kbg (s−1)
entry NpN’
kobs / kbg
1.3 × 107
3.4× 106
1.3 × 106
8.6 × 104
1
2
3
4
GpA
GpU
UpU
CpA
160
85
1.2 c
2.5 d
2.0 e
1.4 d
26
1.2
a 0.50 mM precatalyst 2∙4HCl; 0.50 mM CuCl2; 0.10 mM NpN';
0.10 M N,N’-diisopropyl-N-ethanolamine buffer; 80% DMSO, pH
o
c
8.8, 50.0 C. b Error limits on the order of ±10%. Extrapolated to
pH 8.8, from data measured in the presence of 1.0 mM Me4NOH
in 80% DMSO, 50.0 °C. d Calculated at pH 8.8 from data in ref 13a.
e Calculated at pH 8.8 from data in ref 13b.
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in Table 3 with the corresponding value for HPNP (kobs/kbg = 3.2 × 105 in Table 2) shows that 2H+-
Cu catalyzes the cleavage of GpA, GpU, and UpU much more efficiently than the cleavage of HPNP,
but the reverse holds for CpA.14 Since the value of kbg of the latter is similar to that of the other
diribonucleoside monophosphates, the low rate of catalytic cleavage cannot be ascribed to the
inherently low reactivity of CpA, but presumably to unfavorable interactions between the catalyst
and the altered substrate in the transition state, that are either absent or less important for the
more reactive diribonucleoside monophosphates.
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Attempts at a quantitative comparison of the catalytic efficiency of artificial
phosphodiesterases in the cleavage of diribonucleoside monophosphates are hampered by the
fact that available data often consist of erratic examples, related to different substrates and
scattered over diverse experimental conditions.15 Another difficulty arises from the paucity of
reliable kbg data required for the calculation of kobs/kbg values. The only sets of catalytic data that
are homogeneous enough in terms of solvent and temperature for a meaningful comparison with
data from the present work are listed in Table 4. All these systems are based on the guanidine-
Table 4. Phosphodiesterase activity of guanidino-based catalytic systems (I – IV) in 80%
DMSO; 106 × kobs (s−1) and kobs/kbg values (in parentheses) normalized to 1 mM catalyst
concentration
entry substrate
I a
II b
III c
IV d
1
2
3
4
5
GpA e
GpU e
UpU e
CpA e
320 (2.7 × 107)
0.50 (1.0 × 103)
n.d.f
n.d.f
170 (6.8 × 106) 33
(3.4 × 104)
14 (2.3 × 104)
22 (4.3 × 104)
0.92 (2.6 × 103)
(9.2 × 102)
8.0 (2.6 × 104)
12 (4.6 × 104)
30 (1.7 × 105)
(5.7 × 103)
52 (2.6 × 106)
2.4 (1.7 × 105)
(6.4 × 105)
7.0 (8.6 × 103)
0.37 (6.5 × 102)
(70)
HPNPg
a
b
Catalyst: 2H+-Cu; pH 8.8, (this work). Catalyst: 1,2-vicinal diguanidinocalix[4]arene; pH 10.4,
c
(data from ref 13c). Catalyst: guanidine based self-assembled monolayers on Au nanoparticles;
d
pH 10.2, (data from ref 13b). Catalyst: guanidine-based polymer brushes grafted onto silica
nanoparticles; pH 9.9, (data from ref 13a). e Data at 50.0 °C. f Not determined. g Data at 25.0 °C.
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guanidinium catalytic dyad, held in close proximity by means of different strategies, i.e. using a
cone-calix[4]arene spacer or nanostructured supports, see footnotes c−e to Table 4. It is apparent
that catalytic rates critically depend on the identity of the catalyst-substrate pair. For example,
CpA is the worst substrate for catalyst 2H+-Cu, but turns out to be the most reactive with catalytic
system IV in Table 4. In spite of these limitations, the data point to a marked superiority of catalyst
2H+-Cu, both in terms of absolute catalytic rates and rates relative to background hydrolysis, at
1 mM catalyst concentration.
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To sum up, we have found that catalyst 2H+-Cu is 40-fold more active than 1H+-Cu in the
transesterification of HPNP, which indicates that the calix[4]arene linker in 2H+-Cu strongly
enhances the extent of cooperation between guanidinium and the ligated CuII ion. Catalyst 2H+-Cu
promotes the cleavage of GpA, GpU, and UpU much more effectively than that of HPNP, but the
reverse holds for CpA. The rate enhancement relative to the background hydroxide reaction is 105
for the latter substrate, but unprecedented values as high as 107 are found in the reactions of the
best substrates.
Combination of catalytic data for the present work with analogous data from previous works
reinforces the view that the upper rim of cone-calix[4]arenes is a very convenient platform for the
construction of bifunctional catalysts. Inspection of CPK molecular models suggests that the
strength of the interaction between the conformationally mobile substrates and catalyst critically
depends on a large number of attractive and repulsive secondary interactions, that are difficult to
rationalize with a computational approach. Thus the question of catalytic effectiveness does not
resolve itself into a mere question of “distance” between catalytic groups. As a matter of fact, the
novel arrangement obtained through the implantation of two different functionalities at the upper
rim of a calix[4]arene, now easily achievable via the synthetic strategy herein reported, can give
rise to remarkable cooperation factors and might therefore play a strategic role in the
preparation of further catalysts and receptors with increased molecular diversity.
EXPERIMENTAL SECTION
Instruments. NMR spectra were recorded on either a 400- or 300-MHz spectrometer.
Partially deuterated solvents were used as internal standards to calculate the chemical shifts (
δ
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values in ppm). High resolution mass spectra were obtained by an electrospray ionization (ESI)
single-quadrupole spectrometer. Potentiometric titrations were performed by an automatic
titrator equipped with a combined microglass pH electrode. The experimental details and the
procedure for the electrode calibration were the same as previously reported.3c
Spectrophotometric measurements were carried out at 400 nm on either a double beam or on
a diode array spectrophotometer. HPLC analyses were performed on a liquid chromatograph fitted
with a UV-vis detector operating at 254 nm.
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Materials and general procedures. All reactions, with the sole exception of the
deprotection of 7, were carried out under a nitrogen atmosphere. Flash chromatography was
carried out on 230-240 mesh silica gel. Anhydrous CH2Cl2 (DCM) was obtained by distillation over
CaCl2. DMSO, purged 30 min with argon, and mQ water were used in the preparation of 80%
DMSO used in kinetic and potentiometric experiments. HPNP,16 and 5,17-bis(chloromethyl)-
25,26,27,28-tetrakis(2-ethoxyethoxy)calix[4]arene 3
8 were prepared according to literature
procedures. All other solvents and reagents were commercial samples and used as such.
Commercial samples of NpN’13 and their aqueous solutions were stored at −20 °C.
Warning! Care was taken when handling tetramethylammonium perchlorate because it is
potentially explosive.17 No accident occurred in the course of the present work.
5-(N-phthalimidomethyl)-17-(chloromethyl)-25,26,27,28-tetrakis(2-ethoxy-
ethoxy)calix[4]arene (4). To a solution of 3 (0.242 g, 0.30 mmol) in dry toluene (20 mL),
potassium phthalimide (0.061 g, 0.30 mmol) and dicyclohexyl-18-crown-6 (0.11 g, 0.30 mmol)
were added. The reaction mixture was heated to 110°C and stirred for 6 h, then was quenched by
adding a 1M HCl solution (25 mL) and vigorously stirred for additional 30 min at room
temperature. The organic phase was separated, washed with distilled water (2 x 20 mL), dried
over anhydrous Na2SO4 and evaporated under reduced pressure. The crude was purified by flash
chromatography (hexane/AcOEt 6:4) to give 4 as a white solid foam (0.105 g, 0.114 mmol; 38%
1
yield): mp 68-72°C. H NMR (300 MHz, CDCl3) δ (ppm): 7.87-7.82 (m, 2H), 7.70-7.69 (m, 2H), 6.80
(s, 2H), 6.77 (s, 2H), 6.51 (m, 6H), 4.54 (s, 2H), 4.51 (d, 2H, J = 13.4 Hz), 4.44 (d, 2H, J = 13.4 Hz),
4.40 (s, 4H), 4.15 (t, 4H, J = 5.6, Hz), 4.12-4.03 (m, 4H), 3.85-3.78 (m, 8H), 3.56-3.45 (m, 8H), 3.16
(d, 2H, J = 13.4 Hz), 3.11 (d, 2H, J = 13.4 Hz), 1.28-1.11 (m, 12H). 13C NMR (75 MHz, CDCl3) δ (ppm):
168.1, 157.1, 153.5, 155.8, 135.9, 135.6, 134.4, 134.2, 133.9, 132.2, 130.9, 129.7, 128.7, 128.2,
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128.0, 123.2, 122.5, 73.3, 73.1, 72.9, 69.7, 69.6, 66.4, 66.3, 46.8, 41.3, 30.8, 30.7, 15.3. HR ES-MS:
m/z Calcd for C54H62O10ClNNa [(4+Na)+] 942.39599, found 942.39545; m/z Calcd for C54H66ClN2O10,
[(4+NH4)+] 937.44060, found 937.44005.
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5-(N-phthalimidomethyl)-17-[N,N’-bis(tert-butoxycarbonyl)-1,4,7-triazacyclo-non-
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1-yl]methyl-25,26,27,28-tetrakis(2-ethoxyethoxy)calix[4]arene (5). To a solution of 4
(0.105 g, 0.114 mmol) in dry CH3CN (4 mL), 1,4-bis(tert-butoxycarbonyl)-1,4,7-triazacyclononane
(0.055 g, 0.17 mmol) and K2CO3 (0.021 g, 0.15 mmol) were added. The reaction mixture was
stirred for 4 days at room temperature, during which additional 1,4-bis-(tert-butoxycarbonyl)-
1,4,7-triazacyclononane (2 x 0.015 g, 0.046 mmol) was added. The reaction was then quenched by
evaporating the solvent under reduced pressure. The residue was taken with DCM (20 mL) and the
organic phase was washed with a saturated solution of NaHCO3 (20 mL), dried over anhydrous
Na2SO4 and evaporated under reduced pressure. The crude was purified by flash chromatography
(hexane/AcOEt 6:4 – AcOEt/MeOH 7:3) to give 5 as a colorless oil (0.11 g; 0.080 mmol; 70% yield)
and recover unreacted 1,4-bis-(tert-butoxycarbonyl)-1,4,7-triazacyclononane (0.066 g , 0.055
1
mmol; 48% yield): H NMR (300 MHz, CDCl3) δ (ppm): 7.83-7.82 (m, 2H), 7.70-7.67 (m, 2H), 7.08,
6.99, 6.97 (3 s, 2H), 6.89, 6.85-6.75 (3 s, 2H), 6.33-6.30 (2 m, 6H), 4.68 (4 s, 2H), 4.55-4.40 (m, 4H),
4.18-3.92 (m, 8H), 3.86-3.76 (m, 8H), 3.57-3.45 (m, 14H), 3.23-3.08 (m, 8H), 2.36 (m, 4H), 1.47-1.44
13
(m, 18H), 1.28-1.11 (m, 12H). C NMR (75 MHz, CDCl3) δ (ppm): 168.1, 157.6, 155.8-155.3, 136.2-
135.7, 133.9, 132.5-132.0, 129.8, 129.2, 127.8, 123.3, 122.4, 73.7, 73.3, 72.7, 69.6, 66.4, 60.7, 52.6,
52.4, 50.3, 49.9, 49.4, 41.6, 30.8, 28.6, 15.3. HR ES-MS: m/z Calcd for C70H93N4O14 [(5+H)+]
1213.66883, found 1213.66828; m/z Calcd for C70H92N4O14Na, [(5+Na)+] 1235.65078, found
1235.65453.
5-(Aminomethyl)-17-[N,N’-bis(tert-butoxycarbonyl)-1,4,7-triazacyclonon-1-yl]-
methyl-25,26,27,28-tetrakis(2-ethoxyethoxy)calix[4]arene (6). To a solution of 5 (0.043
g, 0.035 mmol) in dry MeOH (4 mL), N2H4∙H2O (44 μL, 1.42 mmol) was added. The reaction mixture
was heated to 60°C and stirred for 3 h, then was quenched by evaporating the solvent under
reduced pressure. The residue was taken with DCM (20 mL) and the organic phase was washed
with a solution of NaOH 1M (20 mL), dried over anhydrous Na2SO4 and evaporated under reduced
pressure. 6 was obtained as a colorless oil (0.038 g, 0.035 mmol; quantitative yield), pure enough
1
to avoid further purifications. H NMR (300 MHz, CDCl3) δ (ppm): 6.83, 6.75, 6.67 (3 s, 2H), 6.79,
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6.69, 6.64 (3 s, 2H), 6.53-6.33 (3 m, 6H, ArH), 4.48-4.43 (m, 4H), 4.17-4.01 (m, 8H), 3.86-3.81 (m,
4
5
8H), 3.69-3.41 (m, 16H), 3.13-3.08 (m, 8H), 2.60-2.46 (m, 4H), 1.47-1.44 (m, 18H), 1.28-1.17 (m,
6
7
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12H). C NMR (75 MHz, CDCl3) δ (ppm): 157.1, 155.9, 155.8, 155.7, 155.6, 155.5, 155.3, 136.7,
8
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135.9-134.0, 133.2, 133.0, 132.8, 129.2, 128.9, 128.5, 128.1, 127.9, 127.8, 127.8, 127.1, 127.0,
122.3, 79.4, 79.3, 79.3, 73.5-72.6, 69.7, 69.6, 66.4-66.2, 60.5, 53.6, 53.4, 50.3, 49.9, 49.6, 49.3,
46.1, 30.9, 28.6, 15.3. HR ES-MS: m/z Calcd for C62H91N4O12 [(6+H)+] 1083.66335, found
1083.66638; m/z Calcd for C62H90N4O12Na, [(6+Na)+] 1105.64530, found 1105.64758.
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5–N-[N’,N’’–bis(tert-butoxycarbonyl)guanidine]methyl-17-[4,7-bis(tert-
butoxycarbonyl)-1,4,7-triazacyclonon-1-yl]methyl-25,26,27,28-tetrakis(2-
ethoxyethoxy)calix[4]arene (7). To a solution of 6 (0.040 g, 0.037 mmol) in dry DCM (4 mL),
N,N’-bis(tert-butoxycarbonyl)-N’’-triflylguanidine (0.017 g, 0.044 mmol) and triethylamine (5 μL,
0.037 mmol) were added. The reaction mixture was stirred overnight at room temperature, then
was quenched by adding distilled water (20 mL) and was vigorously stirred for an additional 30
min. The aqueous phase was separated and washed with DCM (2 x 20 mL), then the combined
organic phases were dried over anhydrous Na2SO4 and evaporated under reduced pressure. The
crude was purified by preparative TLC plates (hexane/AcOEt 6.5:3.5) to give 7 as a light yellow oil
1
(0.033 g, 0.025 mmol; 68% yield). H NMR (400 MHz, CDCl3) δ (ppm): 11.52 (s, 1H), 8.50 (t, 1H,
J=4.8 Hz), 6.96, 6.87, 6.79 (3 s, 2H), 6.93, 6.85, 6.78 (3 s, 2H), 6.40-6.20 (m, 6H), 4.52-4.39 (m, 6H),
4.21-4.10 (m, 4H), 4.03-3.94 (m, 4H), 3.87-3.81 (m, 8H), 3.56-3.46 (m, 14H), 3.23-3.09 (m, 8H),
2.65-2.55 (m, 4H), 1.51-1.44 (m, 36H), 1.26-1.22 (m, 12H). 13C NMR (75 MHz, CDCl3) δ (ppm):
163.6, 157.7, 156.9, 156.5, 155.9, 155.7, 155.5, 155.1, 154.8, 136.7-135.3, 133.9-133.0, 130.5,
129.6, 129.3, 128.8, 128.0, 127.8, 122.3, 83.0, 79.5-79.2, 73.5-72.3, 69.7, 69.6, 66.4-66.2, 60.5,
53.9-53.2, 51.0-49.0, 44.2, 30.9, 28.6, 28.3, 28.1, 15.3. HR ES-MS: m/z Calcd for C73H109N6O16
[(7+H)+] 1325.79001, found 1325.78946; m/z Calcd for C73H108N6O16Na [(7+Na)+] 1347.77195,
found 1347.77140.
5–N-guanidinomethyl-17-N-[1,4,7-triazacyclonon-1-yl]methyl-25,26,27,28-
tetrakis(2-ethoxyethoxy)calix[4]arene Tetrahydrochloride (2·4HCl). In a mixture of
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DCM/TFA/TES 95:2.5:2.5 (10 mL), 7 (0.040 g, 0.030 mmol) was dissolved. The reaction mixture was
stirred over night at room temperature and quenched by evaporating the solvent under reduced
pressure. The residue was first taken in a solution of HCl 1 M in EtOH (3mL) and vigorously stirred
for 30 min three times, to exchange the TFA anion, then was recrystallized from DCM with hexane
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to give 2∙4HCl as a light gray solid (0.032 g, 0.030 mmol; 98% yield): mp 123-130 °C. H NMR (300
MHz, CD3OD) δ (ppm): 7.56 (t, J=5.0Hz, 1H), 7.08 (s, 1H), 6.89 (s, 2H), 6.80 (s, 2H), 6.53 (d, 4H,
J=6.6 Hz), 6.49 (t, 4H, J=6.6 Hz), 4.59 (d, 4H), 4.19-4.15 (m, 6H), 4.08-4.05 (m, 4H), 3.92-3.85 (m,
13
8H), 3.70 (s, 2H), 3.60-3.54 (m, 12H), 3.22-2.97 (m, 8H), 2.69 (m, 4H), 1.23-1.16 (m, 12H). C NMR
(75 MHz, CD3OD) δ (ppm): 157.1, 156.9, 156.8, 155.8, 136.1, 135.8, 134.2, 134.1, 130.2, 129.4,
129.4, 127.9, 127.8, 127.4, 122.0, 73.3, 72.1, 69.9, 69.7, 69.6, 66.0, 58.8, 47.4, 44.8, 43.3, 42.1,
30.5, 30.4, 14.3. ES-MS: m/z 925.9 [(2+H)+]. HR ES-MS: m/z Calcd for C53H77N6O8 [(2+H)+]
925.58029, found 925.58091.
Potentiometric Titrations. Potentiometric titrations were carried out as previously
reported.3
c,13 A solution of 70 mM Me4NOH in 80% DMSO was added in small increments under a
nitrogen atmosphere to 6 mL of a 1 mM solution (80% DMSO) of 2(H+)4, 10 mM Me4NClO4, in the
absence or presence of stoichiometric 1 mM ZnCl2 or CuCl2, (80% DMSO, 25 °C). Analysis of the
titration plots was carried out by using the programm HYPERQUAD 2000.18
Kinetic Measurements. Metal complexes were formed in situ by addition of the calculated
stoichiometric amount of the aqueous solution of the metal salt (ZnCl2 or CuCl2) to the buffered
reaction mixture. The solutions were incubated for 1h before the start of the kinetic run by fast
addition of a small volume of the substrate solution.
Cleavage of HPNP. Rate constants were obtained by UV−vis monitoring of p-nitrophenol
liberation at 400 nm (initial rate method). Experiments were carried out in the presence of 0.50
mM precatalyst 2∙4HCl, 0.50 mM ZnCl2 or CuCl2, and 0.10 M N,N’-diisopropyl-N-ethanolamine
buffer, (80% DMSO, 25.0 oC).
Cleavage of diribonucleoside monophosphates. HPLC monitoring of nucleoside liberation
(initial rate method) in the 2H+-Cu promoted NpN' cleavage was carried out on 0.50 mM solution
of precatalyst 2∙4HCl, 0.50 mM CuCl2, 0.10 mM NpN', 0.10 M N,N’-diisopropyl-N-ethanolamine
o
buffer, (80% DMSO, pH 8.8, 50.0 C). Aliquots of the reaction mixture (80 ꢀL) were withdrawn at
appropriate time intervals and quenched with 80 ꢀL of a 10 mM solution of HClO4 in 80% DMSO.
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After addition of p-hydroxybenzoic acid (internal standard) in 80% DMSO, the solution was filtered
and subjected to HPLC analysis on a Supelcosil C-18 DB column (25 cm × 4.6 mm I.D., particle size 5
ꢀm) by elution with H2O (0.1% trifluoroacetic acid)/MeCN, linear gradient from 100 : 0 to 85 : 15 in
25 min, flow 0.9 mL min−1.
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The pseudo-first order specific rate for background reaction (kbg) of GpA was extrapolated at
pH 8.8, from the initial rate of hydroxide catalyzed nucleoside liberation in the presence of 1.0 mM
Me4NOH (pH 15.4), 10 mM Me4NClO4, 80% DMSO, 50.0 °C.
ACKNOWLEDGEMENTS
Thanks are due to the Ministero dell'Istruzione e dell'Università e della Ricerca (MIUR, PRIN 2010
JMAZML, MultiNanoIta), Sapienza Università di Roma, and Consiglio Nazionale delle Ricerche
(CNR) for financial support.
ASSOCIATED CONTENT
*S Supporting Information
NMR spectra for the products. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: riccardo.salvio@uniroma1.it
Notes
The authors declare no competing financial interest.
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REFERENCES
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48, 5545 – 5559. (c) Lönnberg, H. Org. Biomol. Chem. 2011, 9, 1687 – 1703. (d) Aiba, Y.; Sumaoka,
J.; Komiyama M. Chem. Soc. Rev. 2011, 40, 5657 – 5668. (e) Morrow, J. R.; Amyest, T. L.; Richard, J.
P. Acc. Chem. Res. 2008, 41, 539 – 548. (f) Niittymäki, T.; Lönnberg, H. Org. Biomol. Chem. 2006, 4,
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2 For metal-based systems see: (a) Diez-Castellnou, M.; Mancin, F.; Scrimin, P. J. Am. Chem. Soc.
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50 °C) with a kobs value of 2.2 × 10−4 s−1 at 2.0 mM catalyst concentration. The kobs values reported
in Table 3 for GpA , GpU, and UpU compare well with the above value when allowance is made for
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