Diguanidinocalix[4]arenes as effective and selective catalysts of the cleavage of diribonucleoside monophosphates

Article abstract of DOI:10.1039/c4ra05751a

Calix[4]arenes derivatives 1 and 2, featuring two guanidine units at the upper rim, catalyze the transesterification of diribonucleoside monophosphates much more effectively than that of HPNP. Rate accelerations relative to the background range from 10

Full text of DOI:10.1039/c4ra05751a

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Diguanidinocalix[4]arenes as effective and selective
catalysts of the cleavage of diribonucleoside
Cite this: RSC Adv., 2014, 4, 34412
monophosphates†
a
Riccardo Salvio, Roberta Cacciapaglia,a Luigi Mandolini,a Francesco Sansoneb
*
and Alessandro Casnatib
Received 14th June 2014
Accepted 24th July 2014
Calix[4]arenes derivatives 1 and 2, featuring two guanidine units at the upper rim, catalyze the
transesterification of diribonucleoside monophosphates much more effectively than that of HPNP. Rate
accelerations relative to the background range from 103 to 104-fold, and approach 105-fold with the
most favorable substrate-catalyst combinations.
DOI: 10.1039/c4ra05751a
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Introduction
The inertness of phosphodiesters towards hydrolysis1 has
challenged many research groups to the design of articial
phosphodiesterases2–6 with the purpose of achieving efficient
and selective cleavage of polynucleotides and poly-
ribonucleotides. The potential application of these enzyme
mimics to health related goals is an attractive target of
investigation in this eld.7
Metal cations, notably copper(II) and zinc(II), are at the
core of most articial phosphodiesterases,2 but nonmetallic
active units as well have received some attention.2p,3–6 In a
recent work5 we found that upper rim diguanidino-cone-calix
[4]arenes 1 and 2 catalyze the cleavage of the RNA model
Fig.
1 General-base/general-acid mechanism of HPNP cleavage
catalyzed by monoprotonated diguanidino compounds.
compound
2-hydroxypropyl-p-nitrophenyl
phosphate
Results and discussion
(HPNP). It was shown that the catalysts are active in their
protonated forms 1H+ and 2H+, in which the guanidine–
guanidinium dyad combines the general base action of the
neutral guanidine with the electrophilic/electrostatic activa-
tion of the protonated guanidine,4–6 (Fig. 1).
Since conclusions drawn from the cleavage of activated
phosphodiesters do not necessarily apply to the cleavage of
unactivated phoshodiesters,8 it seemed worthwhile to inves-
tigate the catalytic activity of 1 and 2 in the transesterication
of a series of diribonucleoside 30,50-monophosphates NpN0,
eqn (1), as more appropriate RNA models. The results of such
an investigation are reported herein.
Catalytic runs were carried out under the same conditions
used for the cleavage of HPNP,5 namely, pH 10.4, 10 mM
Me4NClO4, DMSO–H2O 80 : 20 (v/v), hereaer referred to as
80% DMSO. The sole difference is the higher temperature
50 ꢀC rather than 25 ꢀC, dictated by the lower reactivity
of diribonucleoside monophosphates compared to HPNP.
Solutions of precatalyst – 1$2HCl or 2$2HCl – were exactly
half neutralized with 1 mol equiv. of Me4NOH to give
buffer solutions at pH 10.4 (pOH 8.0)9 that were used for
the catalytic experiments. According to the previously
reported distribution diagrams of the species as a function of
pH,5 these solutions contain the maximum concentration of
monoprotonated species 1H+ or 2H+, amounting to a mole
fraction of 0.86 in both cases. The kinetics were monitored
by HPLC analysis of aliquots of the reaction mixture with-
drawn at time intervals in the early stages of the reaction,
as previously described.2d Initial rates of nucleoside N0
formation were translated into pseudo-rst-order specic
a
`
Dipartimento di Chimica and IMC – CNR Sezione Meccanismi di Reazione, Universita
La Sapienza, 00185 Roma, Italy. E-mail: riccardo.salvio@uniroma1.it
b
`
Dipartimento di Chimica, Universita degli Studi di Parma, Parco Area delle Scienze
17/A, 43124 Parma, Italy
† Electronic supplementary information (ESI) available: HPLC chromatograms for
the monitoring of NpN0 cleavage. See DOI: 10.1039/c4ra05751a
rates kobs
.
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Typical plots of reaction percentage vs. time related to the
cleavage of GpU catalyzed by 1 are shown in Fig. 2a. The
diagram of kobs vs. buffer concentration (Fig. 2b) shows a good
adherence of data points to a straight line with zero intercept,
and this indicates (i) that the contribution of background
hydrolysis to the overall rate is negligibly small and (ii) that the
catalyst works under subsaturating conditions, i.e. binding of
the catalyst to the substrate is too low to affect the kinetics in
the investigated concentration range (K < 25 Mꢁ1). An analogous
result was obtained in our previous studies of the cleavage of
HPNP.5,6 This nding not only conrms that in the reactant
state binding of guanidinium to the negatively charged phos-
phate moiety is insignicant, but also indicates that any
possible stabilizing interaction between guanidine/guanidi-
nium units and nucleobases B and B0 is negligibly small.
Having established that the kinetics of the reaction of GpU
catalyzed by 1 were not complicated by a pre-association equi-
librium, the whole set of catalytic runs listed in Table 1 were
carried out using a xed precatalyst concentration of 2.0 mM,
under the assumption that subsaturating conditions applies to
all runs. Table 1 shows that both catalysts effectively cleave all of
the investigated substrates, with a marked preference for GpU,
GpG and UpU (entries 1–3), while the reactions of the remaining
substrates experience a much lower nucleobase selectivity
(entries 4–8). The nding that UpG is much less reactive than
GpU, and that both ApG and GpA react slowly, clearly indicates
that catalytic efficiency depends in a very critical way on the
identity of both nucleobases B and B0. Interestingly, literature
data on the cleavage of diribonucleoside monophosphates cata-
lyzed by di- and trinuclear metal complexes2d,10 show that in many
cases in which high nucleobase selectivity is observed, at least
one of the two nucleobases is either uracyl or guanine.2d,10a,e–g In
the latter cases enhanced rates of cleavage have been ascribed
Fig. 2 Cleavage of 0.10 mM GpU catalyzed by 1.0–4.0 mM 1 (80%
DMSO, pH 10.4, 10 mM Me4NClO4, 50.0 ꢀC). (a) Reaction percent vs.
time, for experiments carried out at the given catalyst concentration.
(b) Plot of kobs vs. total catalyst concentration ccat
.
to a pre-association mechanism, supported by spectroscopic10f,g
and kinetic10a,e–g data, in which the deprotonated amidic N–H of a
uracyl or guanine serves as a site for anchoring to a ligated zinc(II)
or copper(II) unit of the catalyst. In the present work GpU, GpG,
and UpU turn out to be the most reactive substrates in the lot, but
Table 1 Cleavage of diribonucleoside 30,50-monophosphates NpN0 in
the presence of guanidinocalix[4]arenes 1 and 2a,b
1 (1,2-vicinal)
2 (1,3-distal)
Entry NpN0 106 ꢂ kobs (sꢁ1
)
krel 106 ꢂ kobs (sꢁ1
) krel kvicinal/kdistal
c
1
2
3
4
5
6
7
8
GpU
GpG
UpU
ApG
GpA
CpC
CpA
UpG
65
62
14
2.4
1.0
2.2
0.74
1.9
88
84
19
3.2
1.3
3.0
1.0
2.6
24
14
38
22
16
2.1
2.1
1.9
1.5
1.0
2.7
4.4
1.4
1.8
0.8
1.7
0.8
3.0
9.8
1.3
1.3
1.2
0.92
0.63
a
2.0 mM precatalyst, 0.10 mM NpN0, 10 mM Me4NClO4; 80% DMSO, pH
b
10.4, 50.0 ꢀC. Pseudo-rst order specic rates kobs calculated from
initial rates of HPLC monitored nucleoside liberation. Error limits on
the order of ꢃ10%. c For HPNP kvicinal/kdistal ¼ 0.5, see ref. 5.
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Table 2 Cleavage of CpA, GpU, and HPNP catalyzed by 1 and 2 in 80%
DMSO, pH 10.4. Catalytic rate enhancements relative to background,
in the absence of metal ions, deprotonation of the amidic N–H
bond is unlikely at the pH value of our measurements. No
signicant interaction between substrate and catalyst occurs in
the reactant state, as clearly demonstrated by the kinetics
(Fig. 2b). As suggested by CPK molecular models, interaction
between the relatively large and conformationally mobile
catalyst and substrate molecules generates a large number of
potentially attractive and repulsive secondary interactions
involving distal part structures of the reactants. In the lack of
information of spectroscopic nature, a rational interpretation of
the role played by uracyl and guanine bases appears to be out of
reach.
kobs/kbg
Catalyst
CpAa,b
GpUa,c
HPNPd
1
2
1.3 ꢂ 103
6.6 ꢂ 104
1.4 ꢂ 102
1.6 ꢂ 103
2.4 ꢂ 104
2.9 ꢂ 102
a
b
At 50.0 ꢀC. kobs values from Table 1. kbg ¼ (5.7 ꢃ 0.4) ꢂ 10ꢁ10 sꢁ1
.
c
d
kbg ¼ (9.8 ꢃ 0.4) ꢂ 10ꢁ10 sꢁ1
.
At 25.0 ꢀC; calculated from data in
ref. 5.
There is a denite tendency for the 1,2-vicinal regioisomer 1
to be a better catalyst than its 1,3-distal regioisomer 2 in the
reaction of most substrates, but the range of kvicinal/kdistal ratios
(Table 1) is within a factor of 5. Modest inversions are observed
for the reaction of GpA and CpA (entries 5 and 7), for which the
1,3-distal regioisomer is a slightly better catalyst, as it is for the
cleavage of HPNP,5 for which kvicinal/kdistal ¼ 0.5.
group with a poor one the transition state becomes tighter, i.e.
more associative in character and, consequently, bears a close
resemblance to a pentavalent phosphorane dianion, indepen-
dent of whether the mechanism is (AN + DN) or (ANDN).
Accordingly, the larger rate enhancements experienced by the
reactions of NpN0 substrates are understood as arising from a
stronger electrophilic/electrostatic stabilization of the transi-
tion state by the guanidinium unit of the bifunctional catalysts.
Interestingly, the kvicinal/kdistal ratio for the cleavage of CpA,
UpU, and HPNP catalyzed by bimetallic complexes 3-Cu2 and 4-
Cu2 (H2O, pH 7.0) are 2.8, 114, and 28, in the given order.2d
Although the comparison is based on a limited set of substrates,
it appears that the relative position of the two catalytic units in
the diguanidinocalix[4]arenes has but a moderate inuence on
catalytic efficiency. This suggests the existence of a certain
degree of exibility in transition states in which contacts
between catalyst and substrate are primarily provided by proton
bridges (Fig. 1). In contrast, the marked preference exhibited by
UpU and HPNP for the 1,2-vicinal catalyst 3-Cu2 is most likely
ascribable to the more stringent geometrical requirements of
As a nal comment, the close similarity of kbg values
coordinative bonds to copper(II) involved in the double Lewis measured for CpA and GpU is consistent with the fact that rates
acid activation.2d
of background cleavage of the phosphodiester bond of diribo-
nucleoside monophosphates are affected by nucleobase identity
to a moderate extent.8a,13 It seems therefore reasonable to
assume that the kbg values actually measured for two members
of the series are representative of the whole series and, conse-
quently, that rate enhancements as high as 103–105 characterize
In order to compare the catalytic efficiency of 1 and 2 in the
cleavage of diribonucleoside monophosphates vs. HPNP, cata-
lytic rates relative to background (kobs/kbg) are required. Initial
rates of the hydroxide catalyzed cleavage of CpA and GpU,
measured in the presence of 1.0 mM Me4NOH (pH 15.4), gave
kbg values of 5.7 ꢂ 10ꢁ5 sꢁ1 and 9.4 ꢂ 10ꢁ5 sꢁ1, respectively. the performances of catalysts 1 and 2 in the cleavage of the eight
These values were extrapolated to pH 10.4 under the assump- NpN0 investigated substrates. These values are comparable in
tion that the reaction is specic base catalyzed, on the analogy magnitude with rate accelerations reported for articial di- and
of the corresponding reaction of HPNP, that was found to be
strictly rst order in hydroxide concentration in the pH range
9.3–13.0.5 The results listed in Table 2 show that both 1 and 2
are from 1 to 2 orders of magnitude more effective in the
trimetallic phosphodiesterases.2d,2j,10,14
Conclusions
cleavage of CpA and GpU than in the cleavage of HPNP. Thus, To sum up, we have shown that diguanidinocalix[4]arenes 1 and
replacement of a good leaving group with a bad leaving group 2 catalyze the cleavage of diribonucleoside monophosphates
has a favorable effect on catalytic efficiency.
Both an associative two step (AN + DN) mechanism involving account of a greater negative charge development on the non-
pentavalent phosphorane dianion intermediate, and
bridging oxygen atoms in the transition state of the reaction of
much more efficiently than the cleavage of HPNP, most likely on
a
a
concerted (ANDN) mechanism in which no intermediate is phosphodiesters with bad leaving groups. To the best of our
involved, are likely possibilities for the hydrolysis of phosphate knowledge, reactivity data related to the cleavage of a series of
diesters.2o The reactions of phosphate diesters with good diribonucleoside monophosphates catalyzed by nonmetallic
leaving groups are believed to proceed via a one step (ANDN) synthetic catalysts are here presented for the rst time. When
mechanism,11 also labeled as SN2(P).12 When the leaving group combined with data from previous studies, the data reported in
is poor the question of mechanism is still under debate, but this work reinforce the notion that cone-calix[4]arenes are useful
there is little doubt that upon replacement of a good leaving platforms for the design of efficient bifunctional catalysts.15 The
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good catalytic efficiency and selectivity of the diguanidino
derivatives described in this work encourages further studies in
the cleavage of RNA polynucleotides.
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Experimental
Instruments and general methods
Materials. DMSO, purged 30 min with argon, and mQ water
were used in the preparation of 80% DMSO. Calixarenes 1 and 2
were available from previous investigations.5 Diribonucleoside
30,50-monophosphates UpU, GpG, and ApG were purchased
from Sigma-Aldrich, while UpG, CpC, GpA, GpU, and CpA were
from Dharmacon Research (Lafayette, CO). Pure samples of
ꢀ
NpN0 and their aqueous solutions were stored at ꢁ20 C.
Warning! Care was taken when handling tetramethylam-
monium perchlorate because it is potentially explosive. No
accident occurred in the course of the present work.
Kinetic measurements. Cleavage of diribonucleoside 30,50-
monophosphates NpN0 was monitored by HPLC analyses of
aliquots of the reaction mixture withdrawn at appropriate time
intervals. Reactions were carried out at 50.0 ꢀC, pH 10.4, on 0.10
mM NpN0, and 1–4 mM catalyst solutions in 80% DMSO, 10 mM
Me4NClO4. The pH of the solution was measured by a micro-
glass pH electrode. Experimental details and procedures for the
electrode calibration were as previously reported.5 In a typical
experiment, half-neutralization of precatalyst was carried out by
addition of a solution of Me4NOH in 80% DMSO until pH 10.4
was reached. The mixture was thermostated at 50.0 ꢀC for 30
min and the reaction was started by addition of a calculated
small volume of a 5.0 mM solution of NpN0 in water. At proper
time intervals, aliquots (80 mL) of the reaction mixture were
withdrawn and quenched with 80 mL of a 10 mM solution of
HClO4 in 80% DMSO. Aer addition of p-hydroxybenzoic acid
(internal standard) in 80% DMSO, the solution was ltered and
subjected to HPLC analysis by elution with H2O (0.1% tri-
uoroacetic acid)/MeCN, linear gradient from 100 : 0 to 85 : 15
in 25 min, ow 0.9 mL minꢁ1. The pseudo-rst-order rate
constant for the hydroxide catalyzed cleavage of CpA and GpU
¨
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ꢀ
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