A calix[4]arene with acylguanidine units as an efficient catalyst for phosphodiester bond cleavage in RNA and DNA model compounds

Article abstract of DOI:10.1039/c9ob01141b

A calix[4]arene scaffold, blocked in the cone conformation and decorated at the upper rim with two acylguanidine units, effectively catalyzes the cleavage of phosphodiester bonds of HPNP and BNPP under neutral pH conditions. The catalyst performance is di

Full text of DOI:10.1039/c9ob01141b

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DOI: 10.1039/C9OB01141B
ARTICLE
A Calix[4]arene with Acylguanidine Units as an Efficient Catalyst for
Phosphodiester Bond Cleavage in RNA and DNA Model Compounds
ꝉ
Received 00th January 20xx,
Accepted 00th January 20xx
Riccardo Salvio,*a,b Stefano Volpi,c Tommaso Folcarelli,d Alessandro Casnatic and Roberta
Cacciapaglia*b,d
DOI: 10.1039/x0xx00000x
A calix[4]arene scaffold, blocked in the cone conformation and decorated at the upper rim with two acylguanidine units,
effectively catalyzes the cleavage of phosphodiester bonds of HPNP and BNPP under neutral pH conditions. The catalyst
performance is discussed in terms of acceleration over background hydrolysis and effective molarity (EM). Combination of
potentiometric acid-base titrations with pH-rate profiles for HPNP and BNPP cleavage in the presence of 2·2HCl additive
points to a marked synergic action of an acylguanidine/acylguanidinium catalytic dyad in 2H+, via general base - electrophilic
bifunctional catalysis. Acceleration factors over background larger than 3 orders of magnitude are obtained. Connection of
the guanidine/guanidinium dyad to the calixarene scaffold by means of carbonyl joints has a double advantage: i) the acidity
of the guanidinium moiety is enhanced by the electron-withdrawing carbonyl group and maximum conversion into the
catalytically active form 2H+ occurs at almost neutral pH, lower than pH needed for the monoprotonated form 1H+ devoid
of carbonyl groups; ii) the EM value for HPNP cleavage with 2H+ is definitely higher than with 1H+, suggesting a highly
preorganized catalyst that perfectly fits in a strainless ring-shaped transition state in the catalyzed process. DFT calculations
also provide useful insights on the reaction mechanisms and transition states.
phosphates and other oxoanions,7,8 plays a crucial role in the
activity of several phosphodiesterases.9,10
Introduction
The importance of phosphodiester bonds in living systems and
its reluctance to spontaneous hydrolysis1 has prompted many
researchers to develop enzyme mimics based either on
molecular platforms2 and nanostructured supports.3 In these
systems a number of active functions are organized onto a
scaffold in order to induce a cooperative action of the catalytic
groups. Typically, these functions are metal cations that play the
role of nucleophile carriers, binding sites, Lewis acid activators,
or promoter of leaving group departure.4 However, in a number
of artificial nucleases and ribonucleases, the guanidinium units
are employed as active functions on their own,5 or in
combination with other active components.6 The use of
guanidinium in these systems has been definitely inspired by
nature since this unit, thanks to its ability to interact with
+
+
+
NH2
NH2
H2N
H
N
+
H
N
O
HN
O
NH
H2N
+
H
N
NH2
NH2
H2N
O
NH2
H2N
NH2
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OCH3
+
+
2
+
2
1
H2
2
3
H2
H
Calix[4]arenes in cone conformation, notably functionalized at the
upper rim, have been employed as a convenient platform for the
design of supramolecular catalysts.11 In a study from our group we
reported on the synthesis and the catalytic activity of
cone-calix[4]arenes decorated at the upper rim with two to four
guanidinium
units.5e
The
1,3-distal
arrangement
of
diguanidinocalixarene 1 turned out to have the distance and
a. Dipartimento di Scienze e Tecnologie Chimiche, Università “Tor Vergata”, Via della
Ricerca Scientifica, 1 I-00133 Roma, Italy.
b. ISB - CNR Sezione Meccanismi di Reazione, Università La Sapienza, 00185 Roma, Italy
c. Dipartimento di Scienze Chimiche, della Vita e della Sostenibilità Ambientale,
Università degli Studi di Parma, Viale delle Scienze, 17/A, 43124 Parma, Italy
d. Dipartimento di Chimica, Università La Sapienza, 00185 Roma, Italy
*To whom the correspondence should be addressed:
roberta.cacciapaglia@uniroma1.it, riccardo.salvio@uniroma2.it.
ꝉElectronic Supplementary Information (ESI) available: potentiometric titrations, NMR
spectra, coordinates and energies of DFT calculations. See DOI: 10.1039/x0xx00000x
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DOI: 10.1039/C9OB01141B
Scheme 1 Synthesis of the Diacylguanidinylated Calix[4]arene 2.a
NH
HN
NH
H
HN
H
O
H
H
CO2H
HO2C
O
O
N
O
N
CHO
OHC
N
HN
H2N
N
NH2
NH
Boc
Boc
c,d
b
a
O
O
O
O
O
O
2 HCl
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
5
6
4
2·
2HCl
a Reagents and conditions: (a) NaClO2, H2NSO3H in acetone/CHCl3 3:1; (b) PyBOP, NMM, N-Boc-guanidine in dry DMF; (c) DCM/TFA/TES
95:2.5:2.5; (d) HCl, EtOH.
calculations provide indications about the nature of the transition
orientation of the active functions more appropriate to achieve high
states of the investigated reactions.
catalytic efficiency.5e However, calixarene 1 experiences a limited
mobility of the guanidinium units that are directly linked onto the
aromatic rings. This limitation can prevent the catalyst from an
optimal adaptation to the transition state of the catalyzed reaction.
Furthermore, the best catalytic activity of this compound was
Results and Discussion
Synthesis
of
the
Catalyst
The
synthesis
of
the
achieved around pH 10.4, because of the relatively high pKa values
of the phenylguanidinium units. At this pH the rate of the background
reaction for the hydroxide catalyzed hydrolysis of phosphodiesters is
much lower compared to the catalyzed reaction, but still significant.
In the light of all this, in an attempt to improve the performance of
such artificial phosphodiesterases and gain further insights into the
mechanistic aspects of this reaction, we designed and synthesized
the bifunctional calix[4]arene system 2, which features an upper rim
diacylguanidinium decoration with a 1,3-distal arrangement. The two
carbonyl groups have the function to slightly increase the
conformational mobility of the catalyst with the purpose to reach the
best compromise between preorganization and flexibility, which is
an essential requisite to obtain high catalytic activity. Looking for fine
tuning in the search of the best compromise between
preorganization and flexibility, the insertion of two carbonyl groups
may result to be particularly convenient since, due to extensive
conjugation of the carbonyl groups with the aromatic ring and the
guanidine nitrogen, it corresponds to the introduction of significantly
less than two freely rotatable bonds. The electron withdrawing effect
of the carbonyl unit is expected to increase the acidity constants of
the guanidinium groups. The highest concentration of the active
form of the catalyst, i.e. the calix[4]arene scaffold provided with the
acylguanidine/acylguanidinium catalytic dyad, will be achieved at a
pH lower than that needed for the system devoid of carbonyl groups.
This will translate into milder reaction conditions for effective
phosphodiester cleavage and higher rate enhancement factors over
the background spontaneous hydrolysis. Although there are
different examples of calixarenes functionalized at the upper or
lower rim with guanidine moieties which also show important
binding abilities towards natural12 and artificial13 nucleic acids, to the
best of our knowledge, calixarenes functionalized with acylguanidine
are very rare. There is only an example of a study where
acylguanidinium calixarenes were reported,14 but they were never
used as acid-base catalysts.
dicarbonylguanidinylated
tetrakis(2-ethoxyethoxy)calix[4]arene
2·2HCl was carried out according to Scheme 1. The 1,3-diformyl
derivative 4 was synthesized by Gross formylation15 of tetrakis(2-
ethoxyethoxy)calix[4]arene following a procedure reported in the
literature.16 An unresolved 10:1 mixture of the 1,3-distal (4) and 1,2-
vicinal bisformylated regioisomers (58% overall yield) was actually
obtained by column chromatography purification of the reaction
mixture.16b By adaptation of a literature procedure,17 this mixture
was here subjected to oxidation with NaClO2 in the presence of
amidosulfonic acid, and the 1,3-distal dicarboxylic acid 5 was isolated
as a pure compound (56% yield) by recrystallization from methanol
of the mixture of regioisomeric 1,3- and 1,2-products.
The condensation reaction between 5 and N-Boc-guanidine was
carried
out
in
the
presence
of
(benzotriazol-1-
yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) and
N-methyl-morpholine (NMM) in anhydrous DMF, and afforded the
di-Boc-protected derivative 6. The removal of protecting groups was
accomplished with an equimolar mixture of trifluoroacetic acid and
triethylsilane
(TES)
in
dichloromethane.
The
trifluoroacetate/chloride counterion exchange, carried out in the last
step of the reaction scheme, led to the dihydrochloride 2·2HCl that
was purified by trituration in CH2Cl2/hexane/EtOH (50:49:1).
The monofunctional model compound 3·HCl was synthesized
starting from 4-methoxybenzoic acid following an analogous
reaction pathway.
Potentiometric Investigation The determination of the
acid-base properties of catalyst 2 and the model compound 3 is
essential for an exhaustive investigation of their activity as
phosphodiesterases. A 80:20 (v/v) mixture of DMSO and water
(hereafter referred to as 80% DMSO) was chosen as a
convenient medium for the titration experiments18,19 and for
the kinetic investigations of phosphoryl transfer processes.3c,20
This medium has the advantage to increase the strength of the
interaction of guanidinium with phosphate ions compared with
pure water. This effect, experienced in less aqueous solvent
In this paper we report the synthesis of the novel
diacylguanidinocalix[4]arene
2
and the study of its
phosphodiesterase catalytic activity, together with the
potentiometric investigation of its acid-base properties. The
monofunctional N-(4-methoxybenzoyl) guanidine (3) was also
synthesized and investigated as a model compound. In addition, DFT
2 | J. Name., 2012, 00, 1-3
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mixture, might result in
ARTICLE
a
higher catalytic activity.20,21 apparently overwhelms the acidity-depressing effect due to a
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23,24
Furthermore, in this reaction medium the pKw of water possible intramolecular hydrogen bonding iDntOeIr:a1c0t.1io0n39/C9ObeB0tw11e4e1Bn
autoprotolysis increases from 14.0 to 18.4.22 This implies that the carbonyl oxygen and the guanidine or guanidinium hydrogens
the neutrality condition is satisfied at pH 9.2.
The electrostatic repulsion between the two positively charged
2+
The dihydrochloride of calixarene 2 was titrated with a standard acylguanidinium groups in 2H2 promotes the removal of a proton
solution of tetramethylammonium hydroxide in 80% DMSO, in the from one of the two units, and, as a result, the pK1 value (8.9) is
presence of 10 mM tetramethylammonium perchlorate as ion significantly lower than the pK2 value (9.8) in 2H22+. The latter is on
strength buffer. The titration plot indicates the presence of two the other hand not far from that of the monofunctional model 3H+,
titratable protons (see Fig. 1a). A best-fitting procedure of the (10.1).
titration data (see the experimental section for further details) The acidity of guanidinium units has thus been successfully increased
afforded the following values of acidity constants: pK1=8.9 and by connection to the aromatic rings via carbonyl groups. The largest
pK2=9.8, to be compared with pK1=9.3 and pK2=11.5, previously conversion into the active form of the bifunctional catalyst 2H+ is
5e
obtained for the system devoid of carbonyl groups, 1·2HCl. The obtained at pH 9.3, pretty close to neutrality, while the conversion
species distribution diagram for calixarene 2, calculated on the basis into the monoprotonated active form 1H+ reached its maximum at
of the acidity constant values measured in the titration experiment, pH 10.4.5e The simple structural variation here introduced widens the
is reported in Fig. 1b.
scope of these bifunctional catalysts towards milder reaction
conditions, namely towards neutral pH reaction media.
Kinetic Measurements
The phosphodiesterase activity of the diacylguanidinocalixarene 2 in
the reaction of the RNA model compound 2-hydroxypropyl
p-nitrophenyl phosphate (HPNP), (eq 1), was evaluated in a set of
kinetic experiments carried out at different pH values on 0.2 mM
substrate solutions, in 80% DMSO, at 25 °C. The reaction medium
was buffered in the pH range 7.9−11.0 with 0.10 M N,N-diisopropyl
ethanolamine and its perchlorate salt. The pseudo-first-order rate
constants for the transesterification reaction of HPNP carried out in
the presence of 1.0 mM 2·2HCl are reported in Table 1 (entries 1-5),
together with the rate acceleration over the background reaction at
the given pH value.
OH
NO2
O
O
O
O
O
O
O
O
P
(1)
P
OH
HPNP
NO2
Table 1 HPNP Transesterification in the Presence of Additives, (80%
DMSO, 25 oC).a
106 × kobs (s-1) a,b
kobs /kbg
c
Entry
Additive
pH
Fig. 1 (a) Titration plot of a 1.0 mM solution of 2·2HCl with Me4NOH in 80%
DMSO (10 mM Me4NClO4; 80% DMSO, 25 oC). Best fit pK values are reported
in the plot; (b) distribution diagram of the indicated species vs pH, calculated
on the basis of the acidity constants determined in the potentiometric
experiment.
2 ·2HCl
1
2
3
4
5
6
7.9
8.8
3.6
9.0
15
7300
2300
1200
100
9.3
10.1
11.0
10.1
8.3
1.6
0.01
2.6
3 ·2HCl
1.0
The same titration experiment was carried out on the
monofunctional model compound 3·HCl (see p. S2 in Electronic
supplementary information) and showed a single titratable proton
with a pK value of 10.1. This number turns out to be significantly
lower whenever compared with the one of N-(4-
methoxyphenyl)guanidinium (pK=11.5),5e the corresponding
monofunctional model compound devoid of the carbonyl unit. The
whole picture confirms the expected acidity-enhancing effect of the
carbonyl group. The electron-withdrawing contribution thus
a kobs values (initial rate method) by UV-Vis monitoring of p-nitrophenol
liberation at 0.20 mM HPNP, 1.0 mM additive, 0.10 M N,N’-diisopropyl
ethanolamine buffer; 10 mM Me4NClO4. The values of kobs reported in the
Table are not corrected for the background contribution. b Calculated as
vo/[HPNP]; error limit = ±10%. c The background rate constant (kbg, s-1) for the
hydroxide-catalyzed cleavage has been calculated by the expression
kbg = 10(pH−17.2), obtained from kinetic data reported in ref. 5e.
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DOI: 10.1039/C9OB01141B
Calixarene 2 turns out to be an efficient catalyst for this phosphoryl
transfer process, with the best acceleration observed at lower pH,
due to the slower spontaneous hydroxide catalyzed cleavage. At
higher pH values the acceleration over the background
transesterification declines until the catalyzed cleavage becomes
comparable to the uncatalyzed process (entry 5, Table 1). The
absolute catalytic efficiency, in terms of kobs values, shows a
maximum around pH 9.3 (entry 3). This experimental evidence, in
the light of the distribution diagram in Fig. 1b, suggests that the
active species in the catalytic process is 2H+, endowed with the
Calixarene 2 exhibits a remarkable catalytic activity also in the
reaction of this substrate, with the highest specific rate of catalytic
BNPP cleavage around pH 9.3. The activity, both in terms of
acceleration and kobs , decreases at higher pH values and becomes
significantly affected by background at pH 11. The model compound
3, tested at the pH value corresponding to equimolar concentration
of protonated and deprotonated forms, was found to be completely
uneffective (Table 2, entry 6). Comparison of the distribution
diagram of protonated and unprotonated species vs pH reported in
Figure 1b with the pH-rate profile of Figure 2b, suggests that also in
the reaction of BNPP the monoprotonated form 2H+ is the
catalytically active species.
acylguanidine-acylguanidinium catalytic dyad. As
a control
experiment, in order to evaluate the effectiveness of the
intermolecular version of the acylguanidine-acylguanidinium
couple, the activity of the monofunctional compound 3 was
measured at the pH value corresponding to equimolar
concentration of the protonated and deprotonated forms, i.e.
pH=10.1 (Table 1, entry 6). In the latter experiment kobs turned out
to be basically undistinguishable from the spontaneous hydrolysis at
that pH, thus indicating the remarkable cooperativity between the
acylguanidine-acylguanidinium units preorganized on the
calix[4]arene molecular scaffold in the bifunctional catalyst 2H+.
A similar set of catalytic measurements was carried out with the DNA
model compound BNPP (eq 2).
The efficiency of the present 2H+ catalyst is higher than that of the
previously reported system devoid of carbonyl groups 1H+, both in
the HPNP5e and BNPP reaction.6d
In the HPNP cleavage, at pH 9.3, where maximum conversion
(ca 60%; Fig. 1b) into the monoprotonated active form 2H+ is
observed, relative acceleration over background is 1200, definitely
higher than the acceleration factor of 440 reported for 1H+ at pH
10.4,5e in spite of the fact that in the latter case the experiments
were carried out at a precatalyst concentration 3 times higher (3 mM
1·2HCl) and, incidentally, the maximum conversion into the
monoprotonated active form 1H+ was higher (ca 85% at pH 10.4), due
to a larger difference between pK1 and pK2 values for 1H+.
The highest efficiency of 2H+ catalyst is evident also in BNPP
cleavage. With this substrate both sets of experiments were carried
out at 1 mM additive concentration. A kobs/kbg value of 1100 is here
observed at pH 9.3 with 2H+ catalyst vs a kobs/kbg value of 120
OPO3H
OH
O2N
NO2
O
O
O
O
H2O
(2)
P
NO2
NO2
BNPP
This substrate exhibits a much lower reactivity compared to HPNP,
both in the presence and in the absence of the catalyst, due to the reported for 1H+ catalyst at pH 10.4.6d
lack of an intramolecular nucleophilic group. In consideration of this
lower reactivity the measurements were carried out at higher
temperature, i.e. at 50 oC. Reactivity data for the cleavage of 0.2 mM
BNPP carried out at different pH values and in the presence of 1.0
mM 2·2HCl or of 1.0 mM of the monofunctional control 3·HCl are
reported in Table 2.
The advantages in kobs/kbg values here commented largely depend on
the higher acidity of 2·2HCl compared to 1·2HCl and on the
consequent possibility to work at lower pH with 2·2HCl additive.
Plots of kobs values vs pH for the cleavage for both substrates in the
presence of 2·2HCl precatalyst are given in Fig. 2. Whenever the
contribution of background reaction is not completely negligible
(pH>10), corrected kobs data have been plotted in Figure 2 (filled
circles) together with uncorrected ones (empty circles) for reaction
of both substrates.
Table 2 Cleavage of BNPP in the Presence of Additives, (80% DMSO,
50 oC). a
As previously suggested, according to the observation of a maximum
of catalyst activity around pH 9, and in view of the distribution
diagram in Fig. 1b, we reasonably assume that the monoprotonated
form 2H+ is the only catalytically active species.25 Taking also into
account that the association of the substrate to the catalyst is
negligibly small in the explored concentration range (vide infra), eq
3 follows, where v is the reaction rate, that is proportional to the
concentration of 2H+, and kcat is the specific rate of the reaction of
the substrate with 2H+ .
c
Entry
Additive
pH
108 × kobs (s-1) a,b
kobs /kbg
2 ·2HCl
1
2
3
4
5
6
7.9
5.0
24
2900
1800
1100
120
8.8
9.3
47
10.1
11.0
10.1
31
7.1
0.32
3.3
3 ·2HCl
1.2
a kobs values (initial rate method) by UV-Vis monitoring of p-nitrophenol
liberation at 0.20 mM BNPP, 1.0 mM additive, 0.10 M N,N’-diisopropyl
ethanolamine buffer, 10 mM Me4NClO4. The values of kobs reported in the
Table are not corrected for the background contribution. bCalculated as
vo/[BNPP]; error limit= ±10%. c The background rate constant (kbg, s-1) for
the hydroxide-catalyzed cleavage has been calculated by the expression
kbg = 10(pH−18.67), obtained from kinetic data reported in ref. 6b.
v=kcat[2H+][Substrate]
(3)
(4)
푘푐푎푡 퐶푇
+
[
]
=
푘
obs = 푘푐푎푡 ퟐ퐻
+
]
퐾
2
1 + [퐻
+
퐾
+
]
1
[퐻
4 | J. Name., 2012, 00, 1-3
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The observed rate constant kobs is given by eq 4, that is easily
obtained from eq 3 when [2H+] is calculated as a function of the
analytical concentration of added 2·HCl (CT), and of the
potentiometrically determined acidity constants K1 and K2.
The pH-rate profiles in Fig. 2 were used in a nonlinear least-square
fitting procedure to eq 4, in which CT is the total concentration of 2,
K1 and K2 are the acidity constants independently determined by the
titration experiment (vide supra), and kcat is the only adjustable
parameter, as defined in eq 3.
View Article Online
DOI: 10.1039/C9OB01141B
Fig. 3 Plot of kobs for the transesterification of HPNP catalyzed by 2H+ vs the
o
concentration of added 22HCl (CT) in 80% DMSO buffered at pH 9.3, 25 C.
From the slope of the straight line, the the second order rate constant
k2 = (1.50 ± 0.04) × 10−2 M−1 s−1 was calculated. The value plotted at CT=0 is the
value of the background reaction at pH 9.3 (kbg=1.3×10−8 s−1, see footnote c to
Table 1).
In order to evaluate the extent of the potential binding between the
catalyst and the substrate, measurements at different catalyst
concentration were performed in the cleavage of HPNP at the
optimal pH value, i.e. at pH=9.3. A linear dependence of kobs vs
catalyst concentration was observed (see Fig. 3). This evidence
indicates that, in the explored concentration range, the catalyst
works under subsaturating conditions (K < 50 M-1), in line with the
very low binding constant values reported in the literature for the
guanidinium-phosphate interaction.3c,20
The k2 value, calculated as the slope of the straigt line in Fig. 3, is
lower compared to the kcat(HPNP) value reported above. This finding
is consistent with a calculated conversion lower than 60% into the
monoprotonated active form 2H+ at pH 9.3, as clearly shown in the
distribution diagram in Fig. 1b.
The transesterification of HPNP (eq 1) in the presence of N-(4-
methoxybenzoyl) guanidine buffer (3/3H+) was investigated in a wide
concentration range, (Fig. 4).
Fig. 2 Cleavage of HPNP at 25 oC (a), and BNPP at 50 oC (b), catalyzed by 1.0
mM 2·2HCl vs pH, in 80% DMSO. Data from Tables 1 and 2. The kobs values
measured at pH>10 were corrected for the background contribution (filled
circles). The empty circles represent the uncorrected kobs values. The solid
lines are the plot of eq 4 with the best fit values of kcat reported in the text. In
the fitting procedure only the corrected values were taken into account.
The fitting procedure for the cleavage of BNPP at 50 oC was carried
out under the approximation that the value of the acidity constants
K1 and K2 of 2H22+, experimentally obtained at 25 °C, do not
o
significantly change in the temperature range 25-50 C, or at least
may be taken as representative values also at 50 °C.
Best fit values of kcat (HPNP) = (2.4 ± 0.2) × 10−2 M−1 s−1 and kcat (BNPP)
= (7.6 ± 0.2) × 10−4 M−1s−1 were used to plot the overall pH-rate
profile (solid lines in Fig. 2). The good agreement between calculated
lines and experimental data confirms the soundness of the
assumptions in eq 3, featuring 2H+ as the only catalytically active
Fig. 4 Transesterification of 0.2 mM HPNP catalyzed by an equimolar buffer
of 3 and 3H+ (pH 10.1), (80% DMSO, 25 oC). The value plotted at zero buffer
concentration is the value of the background reaction at pH 10.1 (kbg=7.9×10−8
s−1; see footnote c to Table 1). The solid line is the plot of eq 5 with the best
fit values of k3 and k3/3H reported in the text.
species, that profits of
a synergic intramolecular action of
acylguanidinium-acylguanidine moieties in the catalytic mechanism.
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The experiments were carried out in solutions buffered at pH=10.1 mol-1.16b On the basis of these considerations a 1V0iewMArteicnletOronpliniec
(pH=pKa of N-(4-methoxybenzoyl)guanidinium) by addition of component EMS (eq 6) of the effective molarDitOyIc: o10u.l1d03b9e/C9OB01141B
equimolar 3 and 3H+. The kobs value increases with the buffer
≠
푖푛푡푟푎 ― ∆퐻푖≠푛푡푒푟
∆푆푖≠푛푡푟푎 ― ∆푆푖≠푛푡푒푟
푒푥푝 ― ∆퐻
∙ 푒푥푝
concentration. Furthermore a marked deviation from a linear trend
is apparent (Fig. 4), which is consistent with a second-order
contribution of the buffer concentration to the reaction rate. At low
buffer concentration, the parabolic profile approaches the value of
spontaneous hydrolysis at pH 10.1 (kbg; see footnote c to Table 1).
The overall contribution to the reaction rate can be expressed by eq
5, where k0 is the background contribution at the buffer pH, k3 is the
general-base contribution of N-(4-methoxybenzoyl)guanidine, and
k3/3H refers to the second order contribution due to the
EM = EMH  EMS =
(6)
(
)
(
)
푅푇
푅
estimated for the reaction system involving 1H+ catalyst.28d From the
observed value of EM = 2.3 M, we could also estimate a 0.9 kcal mol-1
enthalpic penalty, i.e. strain at the pseudo-cyclic transition state of
HPNP cleavage catalyzed by 1H+.28d The attempt to remove such an
enthalpic penalty by a slight increase of the conformational mobility
of the catalytic system has provided notably good results in the
present investigation. With the introduction of additional 2 freely
rotatable bonds, an EMS value as low as 0.2 M should have been
actually estimated in the absence of enthalpic penalties for the
diacylguanidino catalyst.28d Possible reasons of the notably high
value of observed EM, considerably higher than expected, are as
follows: i) due to extensive conjugation, the C(O)-N and C(O)-CAr
bonds are significantly blocked, and taking into account the
introduction of additional 2 rotors upon introduction of 2 α-carbonyl
groups, definitely overestimates the number of rotors to be blocked
in the productive transition state, i.e. largely underestimates the EMS
value; ii) complete freezing of the residual skeletal motions of a
calix[4]arene blocked in a cone conformation is not stringent in the
investigated system, i.e. in the relatively large pseudo-cyclic
transition state of the catalyzed reaction an entropic contribution of
9-10 cal K-1 mol-1 in the estimate of EMS is exceedingly high; iii)
possible intramolecular hydrogen bonding interaction(s)
conveniently rigidify and preorganize the system in a productive
form for catalysis.
intermolecular
synergic
action
of
the
N-(4-methoxybenzoyl)guanidine/guanidinium catalytic dyad.
+
[
]
푘
표푏푠 = 푘표 +푘ퟑ[퐵] + 푘ퟑ/ퟑ퐻[퐵] 퐵퐻
(5)
A nonlinear least-squares procedure was performed to fit the data to
the parabolic eq 5, using the k0 value determined in an independent
experiment (footnote c to Table 1). Best fit values of the two
adjustable parameters were as follows: k3=(2.0 ± 0.2) × 10−4 M-1 s−1
and k3/3H=(3.1 ± 0.2) × 10−3 M-2 s−1.
Concerning the simple general-base catalyzed mechanism, it should
be noted that the second-order rate constant k3 for the contribution
given by 3 (eq 5) is 120-fold lower than kcat (HPNP), in spite of the fact
that 3 is more basic than 2H+ (10.1 (pK 3H+) vs 8.9 (pK1 2(H+)2)). We
can therefore conclude that the catalytic process promoted by 2H+ is
entirely
due
to
a
mechanism
involving
an
acylguanidinium/acylguanidine catalytic dyad, the contribution of
any possible simple general-base catalyzed mechanism being
definitely negligible. The mechanism of the HPNP transesterification
catalyzed by 2H+ is the intramolecular counterpart of the general-
base/electrophilic-activation mechanism (or in alternative
general-base/general-acid)26,27 operated by 3/3H+.
Ab initio Calculations The general-base/electrophilic-activation
mechanism, which has been proposed on the basis of the
potentiometric and kinetic data reported in the present study, was
also investigated in silico with the Gaussian 09 package29 on a
simplified model of calixarene 2H+ (the ethoxyethyl chains were
replaced with methyl groups). The alternative hypothesis in which
the acylguanidine acts as a nucleophile can be reasonably discarded
on the basis of 31P NMR experiments and computational studies
carried out in our previous investigations.30
The initial structures of calculations were selected by a preliminary
screening based on molecular dynamics (see the Experimental
Section for details). After this step, the structures were optimized at
b3lyp/6–31g(d)//b3lyp/6–31g(d,p) level of theory. The solvent effect
was taken into account with the use of the polarized continuum
model, setting the dielectric constant measured in bulk for an 80%
DMSO aqueous solution, i.e. ε=72.20 The transition state (TS)
searches for the HPNP and BNPP cleavage were carried out with the
QST3 algorithm. The two optimized TS structures, for the HPNP and
BNPP cleavage (Fig. 5a and 5b, respectively), show a single imaginary
frequency. The animation of the normal mode of vibrations with
imaginary frequencies confirms that the saddle points, obtained
from this optimization procedure, are actually the transition states
of the investigated reactions. In both the TS structures it is evident a
phosphate-guanidinium interaction, with an average length of the
non-covalent O·H bond of 1.75 Å. This distance is significantly shorter
than the typical distance interval reported between the acceptor
The ratio kcat(HPNP)/k3/3H = 7.7 M is the effective molarity
28
(EM = kinra/kinter
)
of the intramolecular reaction catalyzed by 2H+
and can be taken as a measure of the high degree of synergism
displayed by the active units in the bifunctional catalyst and,
therefore, a measure of the efficiency of this system. Ideally, it means
that 7.7 M concentrations of 3 and its conjugated acid 3H+ exhibit the
same reaction rate of HPNP cleavage as that of a solution of 2H+ at
the same concentration. In other words, since the use of such a high
concentration of 3/3H+ is not realistic, an EM value of 7.7 M indicates
that at millimolar catalyst concentrations the reaction catalyzed by
2H+ is nearly 4 orders of magnitude faster than the intermolecular
version.
An EM value as high as 7.7 M for the HPNP cleavage in the presence
of the bifunctional catalyst of 2H+ is definitely an excellent result,
attesting a high level of preorganization of the catalyst with a very
good fit and the absence of relevant enthalpic penalties in the
pseudo-cyclic transition state of the catalyzed reaction. This result is
remarkable when we compare it with the EM value of 2.3 M obtained
with the bifunctional catalyst 1H+. We had to block in that case 4
rotatable bonds and a presumably significant fraction of residual
skeletal motions of a calix[4]arene blocked in a cone conformation.
The latter contribution was estimated to amount to up 9-10 cal K-1
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model of monoprotonated calix[4]arene 2H+. In the structures a
atom and the hydrogen in a single hydrogen bond (2.0-2.2 Å).31 This
is an indication that such a binding has a double contribution from a
chelate hydrogen bond and an electrostatic interaction.
number of hydrogen atoms are omitted for tDhOeIs:a1k0e.10o3Vf9iec/wlCaA9rriOttiycBl.e01O1n4li1nBe
The bond between the hydroxyl hydrogen atom of the HPNP (H1) and
the oxygen atom O1 is breaking and a new bond is forming between
the hydrogen and the guanidine nitrogen N1 as indicated by the bond
lengths (O1–H1 = 1.41 Å, H1–N1 = 1.14 Å, Fig. 5a). The distances O1-P
(1.96 Å) and O2–P (1.99 Å), compared with the typical P–O distance
in stable phosphodiesters (1.62-1.65 Å),24 indicate the forthcoming
formation of new phosphorus-oxygen bond and the breaking of the
O-P bond of the leaving group. Furthermore, the phosphorus atom
shows a pentacoordination and a geometry similar to a trigonal
bipyramid, with an O3–P–O4 angle of 122°, therefore significantly
different from the tetrahedral geometry of phosphorus atom in
phosphodiesters. This computational evidence suggests the
Experimental Section
Instruments and General Methods. NMR spectra were recorded on
a 300 MHz Bruker spectrometer. Chemical shifts are reported as δ
values in ppm from tetramethylsilane added as an internal standard.
Mass spectra analyses were performed by an electrospray ionization
time-of-flight spectrometer. The automatic titrator was equipped
with a combined microglass pH electrode.
Materials. DMSO was purified from volatile impurities by bubbling
argon for 30 min. Ultrapure mQ water was used for the preparation
of 80% DMSO. HPNP33 and 416b were prepared as reported in the
literature. Other solvents and reagents were commercially available
and used without any further purification. Warning!
Tetramethylammonium perchlorate is potentially explosive, so it has
been handled with care.34 No accident occurred in the course of the
present work.
5,17-Dicarboxy-25,26,27,28-tetrakis(2-ethoxyethoxy)calix[4]arene
(5). The diformylderivative 4 (178 mg, 0.231 mmol), contaminated by
ca. 10% of its 1,2-vicinal regioisomer,16b was dissolved in
acetone/CHCl3 3:1 (v/v) (30 mL). A solution of amidosulfonic acid
(120 mg, 1.24 mmol) in water (0.9 mL), and a solution of NaClO2 (102
mg, 1.15 mmol) in water (0.9 mL), were rapidly added to the solution
of the substrate. The mixture was stirred for 12 h at room
temperature. After a further addition of the same amounts of
amidosulfonic acid and NaClO2 , the reaction mixture was stirred for
12 more hours at room temperature. The solvent was removed
under reduced pressure and the residue dissolved in DCM (90 mL).
The organic phase was washed with 1 M aqueous HCl (3 × 45 mL) and
with a saturated NaCl water solution (45 mL). The organic phase was
dried over anhydrous MgSO4. After removal of the solid, the solvent
was evaporated under reduced pressure. Recrystallization from
methanol of the solid residue, afforded the diacid 5 as a pure
2d
operation of a concerted mechanism, labeled as ANDN or SN2(P),32
rather than a AN+DN,2d,27 the latter involving the formation of a
pentacoordinated phosphorus species as an intermediate instead of
a transition state. Similar considerations apply to for the calculated
TS for the BNPP cleavage (Fig. 5b). The most likely scenario for the
cleavage of this substrate involves a water molecule5f,27 which
attacks the phosphorus as a nucleophile while is undergoing
deprotonation by the guanidine unit acting as a general base (see
also note 30). The distances between the atoms involved in the
reaction (O5-P2=2.25 Å, O6-P2=1.93, H2-O5=1.25, H2-N2=1.41),
together with the evident phosphorus pentacoordination, indicate,
as in the case of HPNP, the operation of a concerted ANDN
mechanism.
o
compound (104 mg, 0.13 mmol, 56% yield); m.p. 277-278 C. The
NMR data are in agreement with those reported in the
literature:17 1H NMR (300 MHz, CDCl3) δ 7.16 (d, 4H, J=7.5 Hz), 7.03
(t, 2H, J=7.5 Hz), 6.78 (s, 4H), 4.48 (d, 4H, J=13.5 Hz), 4.27 (t, 4H, J=6
Hz), 3.94 (t, 4H, J=4 Hz), 3.84 (t, 4H, J=6 Hz), 3.75 (t, 4H, J=4 Hz), 3.55
(q, 4H, J=7 Hz), 3.48 (q, 4H, J=7 Hz), 3.14 (d, 4H, J=13.5 Hz), 1.23 (t,
6H, J=7 Hz), 1.16 (t, 6H, J=7 Hz). 13C NMR (75 MHz, CDCl3) δ 171.9,
159.2, 157.7, 136.3, 133.8, 129.8, 129.3, 123.4, 123.0, 73.8, 72.4,
69.6, 69.4, 66.6, 66.2, 30.7, 15.3, 15.2.
5,17-Bis-[N-(tert-butoxycarbonyl)carbonylguanidine]-25,26,27,28-
tetrakis(2-ethoxyethoxy)calix[4]arene (6). The diacid 5 (104 mg,
0.13 mmol), N-(tert-butoxycarbonyl)guanidine (42 mg, 0.26 mmol)
and N-methylmorpholine (0.032 mL, 0.29 mmol) were dissolved in
anhydrous DMF (1.6 mL). A solution of benzotriazole-1-yl-oxy-tris-
pyrrolidino-phosphonium hexafluoro-phosphate (PyBOP; 134 mg,
0.26 mmol) and N-methylmorpholine (0.032 mL, 0.29 mmol) in
anhydrous DMF (1.6 mL) was added to the reaction vessel and the
mixture was stirred at room temperature for 6 days under nitrogen
atmosphere. The mixture was poured in water (40 mL). The
precipitated was filtrated and purified by silica gel chromatography
(eluent: hexane/ethyl ether 1:1). Compound 6 was obtained as a
white solid (41 mg, 0.038 mmol; 29% yield). 1H NMR (300 MHz, CDCl3)
Fig. 5 Transition state structures, determined by DFT calculations, for
the cleavage of HPNP (a) and BNPP (b) catalyzed by a simplified
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J. Name., 2013, 00, 1-3 | 7
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δ 8.90 (br. s, 2H), 7.59 (app. s, 4H), 6.38 (app. s, 6H), 4.53 (d, 4H, J=15 described in previously reported procedures.6b,6d Elaboration of the
View Article Online
35
DOI: 10.1039/C9OB01141B
Hz), 4.28 (t, 4H, J=5 Hz), 4.02 (t, 4H, J=4 Hz), 3.84 (t, 4H, J=5 Hz), 3.78 titration plot was carried out with the software HYPERQUAD 2000.
(t, 4H, J=4 Hz), 3.55 (q, 4H, J=7 Hz), 3.49 (q, 4H, J=7 Hz), 3.21 (d, 4H, Kinetic Measurements. Cleavage of HPNP and BNPP was monitored
J=15 Hz), 1.49 (s, 18H), 1.21 (t, 6H, J=7 Hz), 1.15 (t, 6H, J=7 Hz). spectrophotometrically following the liberation of p-nitrophenol at
13C NMR (75 MHz, CDCl3) δ 161.7, 158.5, 155.2, 136.4, 133.3, 128.9, 400 nm. Initial rate measurements reported in Tables 1 and 2 were
128.2, 122.8, 81.6, 73.6, 73.1, 69.7, 69.5, 66.4, 66.2, 30.9, 28.1, 15.25, carried out in 80% DMSO solutions thermostated at 25 oC or 50 oC for
15.23.
5,17-Bis-[carbonylguanidine]-25,26,27,28-tetrakis(2-ethoxyethoxy) species in solutions are reported in the footnotes of the tables.
calix[4]arene Bis(hydrochloride) (2·2HCl). The di-Boc-protected The solutions were buffered with 0.10 N,N-diisopropyl
HPNP or BNPP cleavage, respectively. The concentrations of the
M
derivative 6 (41 mg, 0.038 mmol) was dissolved in DCM/TFA/TES ethanolamine and the pH was adjusted with 50-100 mM solutions of
95:2.5:2.5 (14 mL). The reaction mixture was stirred at room HClO4 in 80% DMSO. The elaboration of the experimental data and
temperature for 12 hours. The solvent was removed under reduced the nonlinear least-square fitting procedures described in the text
pressure and the residue dissolved in a 1 M HCl ethanolic solution were carried out with the software SigmaPlot 12.0 (Systat Software,
(4.5 mL). After 30 minutes vigourous stirring the solvent was Inc.).
evaporated and the same dissolution-evaporation procedure was In a paper36 the authors indicate that the HPNP prepared as
repeated two more times. Finally the solid was triturated in a described by Brown33 might contain up to 5% of 1-hydroxyl-2-propyl
DCM/hexane/ethanol 50:49:1 mixture, affording compound 2 as isomer, which is, according to the authors, ca. 10 times more
dihydrochloride (20 mg, 0.021 mmol; 55% yield).
reactive than the main 2-hydroxypropyl isomer.36,37 The presence of
1H NMR (300 MHz, CDCl3) δ 7.54 (s, 4H), 6.58 (d, 4H, J=6 Hz), 6.48 (t, this minor isomer could affect the initial reaction rate used for
2H, J=6 Hz), 4.65 (d, 4H, J=12 Hz), 4.33 (t, 4H, J=5 Hz), 4.08 (t, 4H, J=4 studies of slow kinetics. For this reason it is appropriate to check the
Hz), 3.92 (t, 4H, J=5 Hz), 3.85 (t, 4H, J=4 Hz), 3.57 (q, 4H, J=7 Hz), 3.54 purity of the substrate and, in case it is necessary, to carry out a
(q, 4H, J=7 Hz), 3.30 (d, 4H, J=12 Hz; superimp. to CD2HOD), 1.21 (t, double recrystallization of the HPNP. In the present study we
6H, J=7 Hz), 1.16 (t, 6H, J=7 Hz). 13C NMR (75 MHz, CD3OD) δ 169.9, checked the rate profiles at higher reaction percentage (40-50%) and
164.8, 158.3, 158.0, 139.1, 135.8, 130.7, 130.5, 126.8, 124.7, 75.89, we made sure the profile are compatible wit a first order reaction
75.86, 72.2, 71.9, 68.3, 68.2, 32.7, 16.57, 16.56. HRMS (ESI-TOF): m/z (see Figure 2S in ESI): in the case there is a significant amount of more
Calcd for C48H63N6O10 [2+H]+ 883.4606; found 883.4587.
N-(4-methoxybenzoyl)-N’-(tert-butoxycarbonyl)guanidine
reactive containant an initial burst of pNPOH release followed by a
(7) much slower phase would be observed. Alternatively, it is possible to
p-Methoxybenzoic acid (236 mg, 1.55 mmol), N-Boc-guanidine (247 use in the kinetic experiments stock solutions of HPNP prehydrolyzed
mg, 1.55 mmol) and N-methylmorpholine (0.180 mL, 1.64 mmol) by ca. 30% to reduce the content of the rapidly reacting isomer to a
were dissolved in anhydrous DMF (10 mL). A solution of PyBOP (780 negligible amount.36
mg, 1.58 mmol) and N-methylmorpholine (0.180 mL, 1.64 mmol) in In silico Calculations. The preliminary screening of the starting
anhydrous DMF (10 mL) was added to the reaction vessel and the structures was carried out with molecular mechanics using MM+ as
mixture was stirred at room temperature, under nitrogen force field with the software HyperChem 8.0.6. Details of the MM
atmosphere, for 6 days. The reaction mixture was poured into water simulation are as follows: run time= 10ps, step size= 0.001 ps, T=330
(40 mL) and the precipitate was purified by silica gel chromatography K. A number of structures for the initial states of the cleavage
(eluent: hexane/ethyl ether 1:1). This procedure afforded compound reactions of HPNP and BNPP were collected with a snapshot
1
7 as a white solid (202 mg, 0.689 mmol; 44% yield). H NMR (300 frequency of 20 data steps. The saved structures were optimized
MHz, CDCl3) δ 8.80 (br. s, 1H), 8.06 (d, 2H, J=9 Hz), 6.92 (d, 2H, J=9 with molecular mechanics with the same force field. The structures
Hz), 3.84 (s, 3H), 1.38 (s, 9H). 13C NMR (75 MHz, CDCl3) δ 176.6, 162.9, showing the lowest energies were used as initial structure of the
159.2, 154.9, 130.8, 128.9, 113.5, 82.3, 55.4, 27.8.
more computational demanding DFT calculations. DFT calculations
N-(4-methoxybenzoyl)guanidine hydrochloride (3·HCl) Compound were carried out at the b3lyp/6–31 g(d)//b3lyp/6–31g(d,p) level of
7 (200 mg, 0.68 mmol) was dissolved in DCM/TFA/TES 95:2.5:2.5 (30 theory (GAUSSIAN-09 package).29 The polarized continuum model
mL). The reaction mixture was stirred at room temperature for 24 was used to take into account the solvent effect. In the case of BNPP
hours. The solvent was removed under reduced pressure and the cleavage an explicit water molecule was used in the calculation since
residue dissolved in a 1 M HCl ethanolic solution (35 mL). After 30 previously reported evidences indicate the operation of this
minutes vigourous stirring the solvent was evaporated and the same mechanism27,30 as argumented in the main text. The solvent
dissolution-evaporation procedure was repeated two more times. parameters were set using the following keyword and options: scrf
Finally the solid was triturated in DCM/hexane 1:1 affording =(pcm, read). eps=72 was used in the separate PCM input section to
1
compound 3 as hydrochloride (155 mg, 0.675 mmol; 99% yield). H define dielectric constant of the solvent mixture. The command
NMR (300 MHz, CD3OD) δ 8.03 (d, 2H, J=9 Hz), 7.09 (d, 2H, J=9 Hz), AddSphereonH = N was used in the PCM input section in order to
3.88 (s, 3H). 13C NMR (75 MHz, CD3OD) δ 167.2, 164.6, 156.1, 130.2, place an individual sphere on the guanidinium hydrogen atoms
122.9, 113.9, 54.8. HRMS (ESI-TOF): m/z Calcd for C9H12N3O2 [3+H]+ directly involved in the hydrogen bonding interaction with the
194.0924; found 194.0908.
phosphate and on the hydrogen atom of the HPNP hydroxyl group.
Titrations. Acid-base potentiometric titrations of 1 mL solutions of The transition state searches of the cleavage reaction of the two
2·2HCl and 3·HCl (5-8 mL) were carried out on an automatic titrator substrates were carried out with the QST3 algorithm. The keywords
equipped with a pH microelectrode. The calibration of the electrode cartesian, calcfc and noeigentest were used in the optimization of
was carried out with standard solutions of HClO4 and Me4NOH as the transition state structures to avoid errors and accelerate the
8 | J. Name., 2012, 00, 1-3
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4
5
(a) E. Salvadeo, L. Dubois and J.-M. Latour, Coord. Chem. Rev.,
conversion. The following keywords synthax was used: opt=(QST3,
calcfc,cartesian). All the energy determined with the calculations
were corrected for the zero point vibrational energy. Vibrational
analysis confirmed that the TS structures of the two cleavage
reactions are first-order saddle points (one imaginary frequency).
The animation of the normal modes of vibration with negative spring
constant confirmed that the saddle points resulting from the
transition state searching procedure are actually the TS of the HPNP
and BNPP cleavage. The coordinates and the energies of the
calculated structures are repored in the Supporting Informations.
View Article Online
2018, 374, 345375; (b) H. Daver, B. Das, E. Nordlander and F.
DOI: 10.1039/C9OB01141B
Himo, Inorg. Chem., 2016, 55, 18721882; (c) M. Ruiz Kubli
and A. K. Yatsimirsky, Inorg. Chim. Acta, 2016, 440, 915; (d)
M. F. Mohamed and R. S. Brown, J. Org. Chem., 2010, 75,
84718477; (e) M. Subat, K. Woinaroschy, C. Gerstl, B. Sarkar,
W. Kaim and B. Konig, Inorg. Chem., 2008, 47, 46614668; (f)
D. Bím, E. Svobodová, V. Eigner, L. Rulíšek and J. Hodačová,
Chem. Eur. J., 2016, 22, 1042610437.
(a) J. Smith, K. Ariga and E. V. Anslyn, J. Am. Chem. Soc., 1993,
115, 362364; (b) U. Scheffer, A. Strick, V. Ludwig, S. Peter, E.
Kalden and M. W. Göbel, J. Am. Chem. Soc., 2005, 127,
22112217; (c) C. Gnaccarini, S. Peter, U. Scheffer, S. Vonhoff,
S. Klussmann and M. W. Göbel, J. Am. Chem. Soc., 2006, 128,
80638067; (d) M. Hollenstein, C. J. Hipolito, C. H. Lam and D.
M. Perrin, ChemBioChem, 2009, 10, 19881992; (e) L. Baldini,
R. Cacciapaglia, A. Casnati, L. Mandolini, R. Salvio, F. Sansone
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Salvio, A. Casnati, L. Mandolini, F. Sansone and R. Ungaro, Org.
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Cacciapaglia, L. Mandolini, F. Sansone and A. Casnati, RSC
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(a) L. Tjioe, A. Meininger, T. Joshi, L. Spiccia and B. Graham,
Inorg. Chem., 2011, 50, 43274339; (b) R. Salvio, S. Volpi, R.
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Chem., 2016, 81, 47284735; (c) R. Salvio, M. Moliterno, D.
Caramelli, L. Pisciottani, A. Antenucci, M. D'Amico and M.
Bella, Catal. Sci. Technol., 2016, 6, 22802288; (d) R. Salvio, S.
Volpi, R. Cacciapaglia, F. Sansone, L. Mandolini and A. Casnati,
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Conclusions
In conclusion, we have successfully developed the novel
diacylguanidinocalix[4]arene system 2 that is an effective catalyst of
the phosphodiester bond cleavage of RNA and DNA model
compounds. The picture obtained from potentiometric and kinetic
investigations
acylguanidine/acylguanidinium
point
to
the
catalytic
operation
dyad
of
via
an
a
6
general-acid/electrophilic activation mechanism. Upon connection
of the active functions to the calix[4]arene scaffold through carbonyl
units, the maximum of catalytic activity is observed at essentially
neutral pH, and the acceleration over background hydrolysis is
enhanced. The high EM value observed in HPNP cleavage is
consistent with a notably preorganized catalyst and a strainless
ring-shaped transition state in the catalyzed process.
7
8
K. A. Schug and W. Lindner, Chem. Rev., 2005, 105, 67113.
P. Blondeau, M. Segura, R. Pérez-Fernández and J. de
Mendoza, Chem. Soc. Rev., 2007, 36, 198210.
F. A. Cotton, E. E. J. Hazen and M. J. Legg, PNAS, 1979, 76,
25512555.
Conflicts of interest
There are no conflicts to declare about the authors.
9
10 M. R. Redinbo, L. Stewart, P. Kuhn, J. J. Champoux and W. G.
J. Hol, Science, 1998, 279, 15041513.
11 (a) J.-N. Rebilly and O. Reinaud, Supramol. Chem., 2014, 26,
454479; (b) R. Cacciapaglia, S. Di Stefano, L. Mandolini and
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Acknowledgements
The authors thank the Sapienza University - Progetti di Ateneo
2016 – 2017, and “Ministero dell’Istruzione, dell’Università e
della Ricerca” - PRIN2017 n. 2017E44A9P (BacHound) for
financial support.
12 Giuliani, M.; Morbioli, I.; Sansone, F.; Casnati, A. Chem. Comm.
2015, 51, 1414014159.
13 Gasparello, J.; Manicardi, A.; Casnati, A.; Corradini, R.;
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14 Martos, V.; Bell, S. C.; Santos, E.; Isacoff, E. Y.; Trauner, D.; de
Mendoza, J. PNAS 2009, 106, 1048210486.
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G. K. Schroeder, C. Lad, P. Wyman, N. H. Williams and R.
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For recent review articles see: (a) E. Kuah, S. Toh, J. Yee, Q.
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3
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Organic & Biomolecular Chemistry
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ARTICLE
Journal Name
23 R. P. Dixon, S. J. Geib and A. D. Hamilton, J. Am. Chem. Soc. ,
1992, 114, 365366.
View Article Online
DOI: 10.1039/C9OB01141B
24 A. E. Hargrove, S. Nieto, T. Zhang, J. L. Sessler and E. V. Anslyn,
Chem Rev, 2011, 111, 66036782.
25 In an alternative hypothesis the only catalytically active
species is the doubly protonated form 2H22+ which accelerates
the reaction by stabilizing the dianionic transition state of the
hydrolysis. In this case the rate law would be as follows:
v=kcat’[2H22+][OH][Substrate]. This possibility is kinetically
indistinguishable from that proposed in the present work. In
this regard see: (a) M. W. Gobel, Angew. Chem. Int. Ed., 1994,
33, 11411143; (b) M. S. Muche and M. W. Göbel, Angew.
Chem. Int. Ed., 1996, 35, 21262129.
26 In principle, the two mechanisms could be distinguished by a
proton inventory study. In fact, the thermodynamic isotope
effect (K. Swiderek and P. Paneth, Chem. Rev., 2013, 113,
78517879) in guanidinium-phosphate binding can affect the
reliability of proton inventory studies dealing with hydrolysis
mechanism of phosphodiesters, as pointed out by Yatsimirsky
and by us, see refs. 20 and 27. On account of this effect the
computational approach could be the only possibility to figure
out whether the guanidinium performs its activity delivering
a
proton, i.e., acting as
a
general acid, or as
electrophilic/electrostatic activator. The calculations
reported in the present work and previously reported by us
(ref. 27), point to a general-base/electrophilic activation
mechanism.
27 R. Salvio and A. Casnati, J. Org. Chem., 2017, 82,
1046110469.
28 (a) L. Mandolini, Adv. Phys. Org., 1980, 17, 183278; (b) C.
Galli and L. Mandolini, Eur. J. Org. Chem., 2014, 73047315;
(c) R. Cacciapaglia, S. Di Stefano and L. Mandolini, Acc. Chem.
Res., 2004, 37, 113122; (d) S. Di Stefano, R. Cacciapaglia, and
L. Mandolini, Eur. J. Org. Chem., 2014, 73047315.
29 M. J. Frisch et al. Gaussian 09, Revision D.01, Gaussian, Inc.,
Wallingford CT, 2009.
30 The possibility of a mechanism that involves the guanidine as
nucleophile was carefully considered in our previous research
work on guanidinium-based systems. 31P NMR experiments
were carried out in several phosphoryl transfer reactions
previously investigated (see ref. 5f and ref. 27). No
accumulation of phosphoryl-guanidine adduct was ever
detected. This experimental evidence, reinforced also by in
silico experiments (ref. 27), points to the operation of a
mechanism in which the guanidine is acting as a general base.
Since the acylguanidine unit is even less nucleophilic than
guanidine, we are inclined to postulate a general base
mechanism also in the present case.
31 E. V. Anslyn, D. A. Dougherty Modern Physical Organic
Chemistry, University Science Books. Sausalito California,
2005.
32 A. J. Kirby, M. Medeiros, J. R. Mora, P. S. M. Oliveira, A. Amer,
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33 D. M. Brown and D. A. Usher, J. Chem. Soc., 1965, 65586564.
34 Hazards in the Chemical Laboratory, 5th ed.; Luxon, S. G., Ed.;
The Royal Society of Chemistry: Cambridge, UK, 1992; p 524.
35 (a) P. Gans, A. Sabatini and A. Vacca, Talanta, 1996, 43,
17391753; (b) L. Alderighi, P. Gans, A. Ienco, D. Peters, A.
Sabatini and A. Vacca, Coord. Chem. Rev., 1999, 184, 311318.
36 D. O. Corona-Martinez, P. Gomez-Tagle, A. K. Yatsimirsky, J.
Org. Chem. 2012, 77, 9110-9119.
37 Williams, N. H. unpublished results
10 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C9OB01141B
Conjugated carbonyl units in a calixarene scaffold provide the right amount of flexibility for catalysis
with a minimum entropic cost.

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