Phosphoryl Transfer Processes Promoted by a Trifunctional Calix[4]arene Inspired by DNA Topoisomerase i

Article abstract of DOI:10.1021/acs.joc.6b01643

The cone-calix[4]arene derivative (1H₃)²⁺, decorated at the upper rim with two guanidinium units and a phenolic hydroxyl in an ABAH functionalization pattern, effectively promotes the cleavage of the DNA model compound bis(p-nitrophe

Full text of DOI:10.1021/acs.joc.6b01643

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Article
Phosphoryl Transfer Processes Promoted by a Trifunctional
Calix[4]arene inspired by DNA – Topoisomerase I
Riccardo Salvio, Stefano Volpi, Roberta Cacciapaglia,
Francesco Sansone, Luigi Mandolini, and Alessandro Casnati
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01643 • Publication Date (Web): 31 Aug 2016
Downloaded from http://pubs.acs.org on September 5, 2016
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Phosphoryl Transfer Processes Promoted by a Trifunctional
Calix[4]arene inspired by DNA Topoisomerase I
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Riccardo Salvio,*a Stefano Volpi,b Roberta Cacciapaglia,a Francesco Sansone,b Luigi Mandolini,a and
Alessandro Casnatib
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a Dipartimento di Chimica and IMC - CNR Sezione Meccanismi di Reazione, Università La Sapienza,
00185 Roma, Italy
b Dipartimento di Chimica, Università degli Studi di Parma, Viale delle Scienze 17/A, 43124 Parma,
Italy
Corresponding Author email: riccardo.salvio@uniroma1.it
Keywords: reaction mechanisms • enzyme mimics • supramolecular catalysis • artificial nucleases
• transphosphorylation
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ABSTRACT
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The cone-calix[4]arene derivative (1H3)2+, decorated at the upper rim with two guanidinium units
and a phenolic hydroxyl in an ABAH functionalization pattern, effectively promotes the cleavage of
the DNA model compound bis(p-nitrophenyl) phosphate (BNPP) in 80% DMSO solution, at pH
values in the range 8.5 – 12.0. The pH-dependence of the kinetics was found to be fully consistent
with the results of the potentiometric titration of the triprotic acid (1H3)2+. At pH 9.5 the rate
enhancement of p-nitrophenol liberation from BNPP relative to background hydrolysis is
6.5 × 104-fold at 1 mM concentration of the calix[4]arene derivative. Experimental data clearly
point to the effective cooperation of the three active units and to the involvement of the
phenolate moiety as a nucleophile in the phosphoryl transfer step. Subsequent liberation of a
second equivalent of p-nitrophenol from the phosphorylated calixarene intermediate is
conceivably promoted by the “built-in” guanidine/guanidinium catalytic dyad.
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INTRODUCTION
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Design of effective catalysts of the cleavage of the highly inert phosphodiester function of nucleic
acids and model compounds has been a challenging target since many decades.1 The strategic
presence of arginine residues in the active site of many nucleases has inspired the design of
artificial phosphodiesterases endowed with one or more guanidinium/guanidine units that proved
crucial in the catalytic mechanism.2 Among systems based on the guanidinium motif, many
examples of metal-free organocatalysts,3,4 as well as of mimics of metalloenzymes such as
staphylococcal nuclease, have been reported.5 Still quite rare, on the other hand, are examples of
mimics of the catalytic triad at the active site of Human DNA topoisomerase I6 composed of a
tyrosine and two arginine residues.4 It was found that effective cleavage of DNA substrates
proceeds via a transphosphorylation mechanism that involves the hydroxyl of the tyrosine unit,
and activation by the guanidinium moiety of the two arginine residues.
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The upper rim of cone-calix[4]arenes has been widely exploited as a suitable platform for the
design of supramolecular catalysts.7 As a convenient strategy for the development of trifunctional
mimics of the active site of Human DNA topoisomerase I, here we focus on the preorganization of
the target catalytic triad on a calix[4]arene scaffold. The development of synthetic strategies for
the heterofunctionalization of the upper rim of calix[4]arenes is of crucial importance for the
design of enzyme mimics featuring the cooperation of different active units. We report herein the
synthesis of calix[4]arene 1H, decorated at the upper rim with two guanidinium units and a
phenolic hydroxyl group in a ABAH pattern of functionalization, and a kinetic investigation of the
activity of this system in the cleavage of the DNA model compound bis(p-nitrophenyl) phosphate
(BNPP).
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RESULTS AND DISCUSSION
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Calixarene Synthesis Compound 1H was synthesized as the corresponding bis(hydrochloride)
starting from 5,17-dinitro-25,26,27,28-tetrakis(2-ethoxyethoxy)calix[4]arene 2,8 according to
Scheme 1.
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In the first step of the synthesis, the dinitroformyl derivative 3 was obtained from calixarene 2
by Gross formylation under controlled conditions (-10 °C, 2 h).9 Interestingly, 3 was obtained as
the main product (59% isolated yield), in an amount larger than the expected statistical
distribution, yet lower than the 84% yield obtained in the monoformylation of the analogous
dinitrotetrapropoxycalix[4]arene derivative under similar conditions.10 This finding can be ascribed
to the presence of the ethoxyethyl chains at the lower rim of 2 that can complex the Lewis acid
and thus influence the electrophilic aromatic substitution at the upper rim. Subsequent Baeyer-
Villiger oxidation,11 followed by the hydrolysis of the resulting formate ester (not isolated), led to
the dinitrohydroxy derivative 4 (81% yield). In the following step calixarene 4 was reduced12 to 5
in quantitative yield. Compound 5 was converted into the Boc-protected diguanidino derivative 6
(42% yield) according to a published procedure3a,3c,3d by using [N,N’–bis(Boc)]thiourea and HgCl2.
The target compound 1H was obtained as bis(hydrochloride) (93% yield) after removal of the Boc
1
protecting groups. All these newly synthesized ABAH derivatives show HR-MS, H and 13C NMR in
agreement with their structures. The ABAH substitution pattern is clearly supported by the
symmetry of the NMR spectra in the aromatic and methylene bridge regions (see SI, pp. S2-S6). In
1
particular, the ArCH2Ar H NMR signals displayed the two expected AX systems (in a 1:1 ratio)
around 4.5 and 3.2 ppm for the axial and equatorial protons, respectively.
Scheme 1. Synthesis of 1H.a
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aa: SnCl4, Cl2CHOCH3; dry CHCl3. b: (1) m-CPBA; DCM. (2) NaOH 2M, MeOH-H2O 4:1. c: NaBH4,
NiCl2∙6H2O; MeOH. d: [N,N’–bis(Boc)]thiourea, HgCl2, NEt3; dry DMF. e: (1) TFA, TES; DCM. (2) HCl
1M; EtOH.
Acid-base Titrations The determination of acidity constants of calixarene (1H3)2+ is a prerequisite
for a rational investigation of its activity in phosphoryl transfer reactions. The medium used in the
titration experiments was a mixture of DMSO/H2O 80:20 (v/v), hereafter referred to as 80%
DMSO. This mixture was successfully used for pKa determination,13,14 for the investigation of the
hydrolysis of phosphodiesters,3a-3d,5a,5b,15,16 and for the dephosphorylation of the terminal
phosphate of ATP.17 The autoprotolysis constant of water (pKw) in 80% DMSO is as high as 18.4.18
This implies that the water dissociation is significantly inhibited and that the pH value of a neutral
solution is 9.2 in this medium.
The dihydrochloride of 1H (2.0 mM) was potentiometrically titrated with a standard solution of
Me4NOH in the same solvent mixture. Figure 1 shows the titration plot and the distribution
diagram calculated on the basis of the given pKa values.
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Figure 1. Titration of (1H3)2+ (2.0 m
Me4NClO4 (a), and distribution diagram of the species as a function of pH (b).
M
) with Me4NOH in 80% DMSO, 25 °C, in the presence of 10 m
M
Titration of (1H3)2+ with Me4NOH showed, as expected, the presence of three titratable
protons. Comparison with the pKa values of model compounds (Chart 1) would suggest that the
most acidic proton (pKa1=9.1) belongs to a guanidinium unit, whose acidity is strongly enhanced by
electrostatic repulsion with the other guanidinium unit, and presumably unaffected by the
presence of the neutral phenolic hydroxyl. Another possibility, however, is that the most acidic
proton belongs to the phenolic hydroxyl, whose acidity is expected to be much higher than that of
the model compound p-methoxyphenol (Chart 1) on account of the strong electrostatic
stabilization of the phenolate ion provided by the neighboring guanidinium units. The above
hypotheses do not represent mutually exclusive alternatives, as it might well be that (1H2)+ is
actually a mixture of tautomeric forms A and B (Figure 2).19
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H2N
H2N
NH2
NH2
NH
HN
OH
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O
O
7
8
9
O
O
O
O
O
OCH3
O
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9H
+
pKa = 12.7
2
7
( H2)
pKa1 = 9.3
pKa2 = 11.5
Chart 1. pKa values of model compounds in 80% DMSO at 25 C (data from ref. 3d for (7H2)2+; for
o
compound 9H see SI, p. S9).
Figure 2. Possible tautomeric structures of (1H2)+, (top), and 1H, (bottom).
As to the second deprotonation step leading to the neutral species 1H, comparison of the pKa2
value of 9.9 with the corresponding value of the model compound lacking the phenolic hydroxyl,
pKa=11.5 (Chart 1), rules out the possibility that the phenolic hydroxyl acts as an innocent
spectator also in this deprotonation step. It seems more likely that ionization of the phenolic
hydroxyl is extensive and, consequently, that the equilibrium between tautomers C and D
(Figure 2) is mostly shifted towards the zwitterionic form D.
The question of the involvement of the phenolic hydroxyl during the first and second titration
step was decided by UV-Vis spectrophotometric titrations of (1H3)2+ with Me4NOH (Figure 3).
Whereas addition of 1 mol equiv of base caused a complete conversion of p-methoxyphenol into
p-methoxyphenolate ion, (Figure 3a), the same amount of base transformed only ca. 55% of the
phenolic hydroxyl of (1H3)2+ into the corresponding phenolate ion, (Figure 3b). Proton removal
from the phenolic hydroxyl was complete, or very nearly so, on addition of a second equivalent of
base. Thus, combination of potentiometric and spectrophotometric titration data indicates that
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the equilibrium between the tautomers A and B is well balanced, with only a slight prevalence of
B, while the equilibrium between C and D is strongly biased towards zwitterion D.
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Figure 3. UV-Vis titration of 0.2 m
M p-methoxyphenol (a) and 0.2 mM calixarene 1H∙2HCl (b) with
Me4NOH (25 oC, DMSO 80%). The full lines are calculated (see SI p. S7).
Cleavage of BNPP A set of kinetic experiments was carried out with the DNA model
bis(p-nitrophenyl) phosphate (BNPP) substrate. Compound (1H3)2+ was added to the reaction
medium buffered in the pH range 8.5
ethanolamine/perchlorate salt buffer.
–
12.0 with 0.10
M
N,N-diisopropyl
O N
2
NO
2
O
O
O
O
P
BNPP
The results of the kinetic experiments are listed in Table 1 (entries 1 – 7) as pseudo-first-order
specific rates kobs = v0/[BNPP], where v0 is the spectrophotometrically determined initial rate of
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p-nitrophenol (pNPOH) liberation. The pseudo-first- order rate constants for the cleavage of the
same substrate in the presence of the diguanidinocalixarene (7H2)2+, and phenol 9H are also
reported for comparison, (entries 8 and 9, respectively).
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Table 1. Cleavage of BNPP in the Presence of Additives, (80% DMSO, 50 oC).
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c
106 × kobs (s−1) a,b
Entry
Additive
pH
kobs /kbg
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2
3
4
5
6
7
8
9
1H∙2HCl
8.5
9.0
8.8
23
1.3 × 105
1.1 × 105
6.5 × 104
1.9 × 104
5.1 × 103
9.6 × 102
8.8 × 10
1.2 × 102
9.5
44
10.0
10.5
11.1
12.0
10.4
12.7
42
35
26
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7∙2HCl
9H
0.64
d
0.50
a From initial rate of PNPOH liberation measured in 0.20 m
0.10 N,N’-diisopropyl ethanolamine buffer, 10 m Me4NClO4 solutions. Calculated as
vo/[BNPP]; maximum error = ±10%. c The background rate constant (kbg, s−1) for the
M BNPP, 1.0 mM additive,
b
M
M
hydroxide-catalyzed cleavage has been calculated by the expression kbg = 10(pH−18.67)
,
obtained from data reported in ref. 5a. d Corrected for the contribution of the background
reaction OH− + BNPP: kbg = 1.1 × 10−6 s−1 at pH 12.7.
The trifunctional calixarene scaffold of (1H3)2+ turned out to be very effective in the cleavage of
BNPP in the investigated pH range (Table 1, entries 1 – 7), with accelerations over background
(kobs/kbg) ranging from 105- to 102-fold. Comparison with the control experiments in entries 8 and 9
indicates that the phenolic hydroxyl of the trifunctional calix[4]arene is directly involved in the
cleavage
of
BNPP,
presumably
via
a
transphosphorylation
process,
electrophilically/electrostatically assisted by the neighboring guanidinium units.20
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Figure 4. pH−rate profiles for the cleavage of 0.20 m
M
BNPP in the presence of 1.0 m
M
of (1H3)2+,
0.10 N,N-diisopropyl ethanolamine buffer, 10 mM Me4NClO4, (data points from Table 1). The
M
solid line is the plot of eqn (1) with best fit values of k1 and k2. The dotted and dashed lines are the
plot of the individual contribution of the species (1H2)+ and 1H, respectively, to the overall
reactivity.
A plot of kobs values in Table 1 (entries 1 – 7) vs pH (Figure 4) reveals a nonsymmetrical
bell-shaped profile, which indicates that more than one species is kinetically active. Comparison
with the distribution diagram in Figure 1 suggests that the active species are (1H2)+ and 1H ,
(eqn (1)),
kobs = k1[(1H2)+ ] + k2[1H]
(1)
whose pH-dependent concentrations are given by standard equations for a triprotic acid, (eqns (2)
and (3)), respectively, where CT is the total concentration of 1H⋅2HCl, and Ka1, Ka2, and Ka3 are the
acidity constants of the triprotic acid (1H3)2+ (see SI, p. S8).
ꢀ
+ꢁ2
CT H Ka1
ꢀ
ꢀ
+ꢁ
(1H2) =
(2)
(3)
+
ꢀ
+ꢁ3 ꢀ ꢁ2
ꢀ
+ꢁ
H
+ H Ka1+ H Ka1Ka2+Ka1Ka2 Ka3
ꢀ
+ꢁ
CT H Ka1Ka2
ꢁ
1H =
+
ꢀ
+ꢁ3 ꢀ ꢁ2
ꢀ
+ꢁ
H
+ H Ka1+ H Ka1Ka2+Ka1Ka2 Ka3
The potentiometrically determined Ka values (Figure 1) were used as known quantities in a
nonlinear least-squares fit of kinetic data, in which k1 and k2 were treated as adjustable
parameters. Best fit values of k1 = (6.2 ± 0.4) × 10-2
M M
-1s-1, and k2 = (2.8 ± 0.2) × 10-2 -1s-1, were
used to plot the overall pH-rate profile and the individual contributions of the species in Figure 4.
Notably, the monocationic species (1H2)+ is about twice as reactive as the neutral species 1H.
Remembering that about 1/2 of (1H2)+ is in the tautomeric form B (Figure 2), whereas 1H is almost
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exclusively in form D, it turns out that tautomer B, featuring two guanidinium units, is about 4
times more reactive than tautomer D, where only one of the two guanidines is protonated. This
observation clearly indicates that both guanidinium units in B cooperate in the stabilization of the
dianionic transition state (Figure 5).
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H
H
N
Ar
N
H
O
O
NH
H
P
H
HN
HN
O
O
O
H
N
H
NO2
Figure 5. Suggested mechanism of BNPP cleavage promoted by (1H2)+ involving two guanidinium
units as electrophilic activators and a phenolate moiety acting as a nucleophile.
Time-course kinetics. To get further insights into the mechanism of action of calix[4]arene (1H3)2+
as a phosphoryl transfer agent, pNPOH liberation was monitored by UV spectrophotometry in a
time-course experiment (Figure 6) carried out under substrate-excess conditions on a 5.0 m
BNPP and 0.1 m
pNPOH release is observed, followed by a much slower phase.
M
M
trifunctional calixarene 1H⋅2HCl solution buffered at pH 9.5. An initial burst of
Figure 6. Liberation of pNPOH in a 5.0 m
M
solution of BNPP upon addition of 0.10 m
M
calixarene
(1H3)2+ (10 m
M
Me4NClO4, 0.10 M N,N-diisopropyl ethanolamine buffer, pH 9.5; 80% DMSO,
50 oC). Data points are experimental and the solid line is the plot of eqn (4) with best fit
parameters k’ = 2.0 × 10-4 s-1, and k ’’= 5.4 × 10-6 s-1. The gray solid line corresponds to background
pNPOH liberation calculated at pH 9.5.
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This is consistent with the reaction sequence in Scheme 2, featuring a fast step of phosphorylation
of the calixarene scaffold with the liberation of a first molar equivalent of pNPOH, followed by a
much slower step with the liberation of a second equivalent of pNPOH from phosphodiester 10.
The latter step is presumably assisted by the neighboring guanidine/guanidinium catalytic
dyad.3c,16,21
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H2N
HN
H2N
HN
HO
O
NH2
O
O
O
O
O
H2N
HN
NH2
NH
H2N
NH
NH2
H2N
NH2
H2N
P
P
NH2
NH2
OR
O
O
NH
k'=k [BNPP]
k''
RO
RO
OR
RO
RO
RO
OR
OR
BNPP
RO
OR
OR
pNPOH
pNPOH
+
1
( H )
10
11
2
Scheme 2. BNPP cleavage promoted by (1H2)+.
This kinetic scheme corresponds to the case of two consecutive irreversible first-order
reactions.22 From standard integrated equations, properly adapted to the investigated reaction
system, eqn (4) follows for the time-dependent concentration of liberated pNPOH, where CT is the
total concentration of the calixarene derivative, k’ = k[BNPP], and τ = (k’+k’’)-1, (see Scheme 2).
t
pNPOH =CT τ k'
ꢂτ k'
ꢃ
1
−
e−
ꢄ
+
(1−
e−
)
ꢅ
(4)
k'' t
τ
ꢀ
ꢁ
When the first exponential term dies out, the second exponential decay becomes apparent and
eqn (4) reduces to eqn (5).
''
pNPOH =CT τ 2 k' 2
+
CT
ꢃ
1
−
e−k tꢄ
(5)
ꢀ
ꢁ
A nonlinear least-squares fit of experimental data to eqn (4) gave the following values: k ’= (2.0
± 0.2) × 10-4 s-1, and k ’’= (5.4 ± 0.2) × 10-6 s-1.
It is interesting to compare the pseudo-first-order rate constants k’ with the kobs value of 4.4
× 10-5 s-1 (Table 1, entry 3) measured at the same pH, but with different reactant concentrations.
Whereas the former corresponds to a second-order rate constant of 4.1 × 10-2
M
-1s-1, the latter
corresponds to a second-order rate constant of 4.4 × 10-2 -1s-1. The good agreement between the
M
two values shows that the rate constants are concentration-independent quantities and,
consequently, that association between reactants is too weak to affect the kinetics, as previously
found in related studies.3c,3d,5b In other words, there is no significant binding in the reactant state
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(subsaturating conditions) and all of the available binding energy arising from the interaction of
the guanidinium unit(s) with the phosphate group along the activation process is selectively
utilized in transition state stabilization.
7
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9
The operation of the mechanism presented in Scheme 2, with nucleophilic assistance in the
cleavage of BNPP is also supported by ES-MS analysis of the reaction mixture of the experiment
reported in Figure 6. The ES-MS spectrum of an aliquot of the reaction mixture withdrawn after 15
minutes confirmed the formation of a p-nitrophenylphosphoryl derivative, that we ascribe to the
O-phosphorylated product 10 (Figure S2 (top), SI). A similar analysis carried out on a sample
withdrawn after 3 days confirmed the presence of 10, whose peak has in this sample an higher
intensity than in the sample withdrawn after 15 minutes, and of the phosphoryl derivative 11
(Figure S2 (bottom), SI).
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The whole set of experimental data points to the effective cooperation of the three functional
groups of (1H2)+ or 1H in the promotion of BNPP cleavage, and to the crucial role of the
nucleophilic phenolate unit (Figure 5), with the formation of a key O-phosphorylated intermediate.
CONCLUSIONS
To sum up, we have shown that the diguanidino derivative of a calix[4]arene featuring a phenolic
hydroxyl at the upper rim effectively promotes the cleavage of the DNA model compound BNPP in
80% DMSO solution at pH values ranging from weakly acidic (8.5) to moderately basic (12.0)
values. Comparison of the pH-dependent initial rates of pNPOH liberation with the results of
potentiometric and spectrophotometric acid-base titrations, clearly indicates the involvement of
two distinct kinetically active species, differing in composition for the presence of one acidic
proton in one species, and lack in the other. The more reactive species was inferred to be the
positively charged tautomer B (Figure 2), in which phosphorylation of the aryloxide oxygen is
strongly assisted by the synergic action of the two neighboring guanidinium units, acting as
electrophilic/electrostatic activators. Less active is the neutral species D, in which activation is
provided by one guanidinium moiety only. Investigation of time-course kinetics, coupled with
mass spectrometric analysis, confirmed that exhaustive cleavage of BNPP is paralleled by
phosphorylation of the aryloxide oxygen of the calix[4]arene derivative. It was found that overall
two equivalents of pNPOH were liberated in subsequent steps occurring on widely different time
scales. We suggest that liberation of the second equivalent of pNPOH from the phosphorylated
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intermediate 10 is assisted by the built-in guanidine/guanidinium catalytic dyad. On the other
hand, the efficiency of the latter in the final liberation of the active form (1H2)+ by
dephosphorylation of 11 is still too low to assure turnover. This is of course at variance with the
catalytic cycle of DNA Topoisomerase I,6 and a key step to be improved in forthcoming mimics of
this class of enzymes.
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EXPERIMENTAL SECTION
Instruments. NMR spectra were recorded on either 400 or 300 MHz spectrometers. Partially
deuterated solvents were used as internal standards to calculate the chemical shifts (δ values in
ppm). High resolution mass spectra were obtained by an electrospray ionization (ES-MS) 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.23 Spectrophotometric
measurements were carried out at 400 nm on a double beam spectrophotometer.
Materials and general procedures. Syntheses of compounds 3 and 6, were carried out under a
nitrogen atmosphere. Flash chromatography was carried out on 230-240 mesh silica gel.
Anhydrous CHCl3 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 acid-base titration
experiments. HPNP,24 5,17-dinitro-25,26,27,28-tetrakis(2-ethoxyethoxy)-calix[4]arene 2,8 and the
bis(hydrochloride) diguanidino derivative 7∙2HCl,3d were prepared according to literature
procedures. All other solvents and reagents were commercial samples and used as such.
Warning! Care was taken when handling tetramethylammonium perchlorate because it is
potentially explosive.25 No accident occurred in the course of the present work.
5,17-Dinitro-11-formyl-25,26,27,28-tetrakis(2-ethoxyethoxy) calix[4]arene (3): To a solution of 2
(0.22 g, 0.27 mmol) in dry CHCl3 (20 mL) cooled at -10°C, SnCl4 (1.6 mL, 13.92 mmol) and
Cl2CHOCH3 (1.26 mL, 13.81 mmol) were added. The reaction mixture was stirred for 2 hours at -
10°C, then it was quenched with distilled water (30 mL) and vigorously stirred for additional 30
minutes. The organic layer was washed with a saturated solution of NaHCO3 (2 x 20 mL) and brine
(20 mL), dried over anhydrous Na2SO4 and evaporated under reduced pressure. The crude
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material was purified by flash chromatography (hexane/AcOEt 5.5:4.5 – hexane/AcOEt 1:1) to give
3 as a white solid (0.13 g, 0.16 mmol; 59% yield) mp 65–67 °C.: 1H NMR (300 MHz, CDCl3) δ (ppm):
9.61 (s, 1H), 7.75 (s, 4H), 7.03 (s, 2H), 6.48 (m, 3H), 4.70 (d, 2H, J =12.6 Hz), 4.61 (d, 2H, J =13.8 Hz),
4.35-4.26 (m, 4H), 4.17 (t, 2H, J=4.8 Hz), 4.07 (t, 2H, J=4.8 Hz), 3.85-3.76 (m, 8H), 3.55-3.47 (m, 8H),
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3.38 (d, 2H, J=12.6 Hz), 3.31 (d, 2H, J=13.8 Hz), 1.23-1.15 (m, 12H). C NMR (100 MHz, CDCl3) δ
(ppm): 191.1, 162.7, 161.2, 155.6, 142.6, 137.1, 136.3, 134.8, 133.3, 131.6, 130.2, 128.5, 124.2,
123.6, 123.1, 74.1, 74.0, 73.7, 69.8, 69.6, 69.5, 66.6, 66.5, 66.4, 30.9, 15.3. . HR ES-MS: m/z Calcd
for C45H55O13N2 [(3+H)+] 831.36987, found 831.36950.
5,17-Dinitro-11-hydroxy-25,26,27,28-tetrakis(2-ethoxyethoxy)calix[4]arene (4): To a solution of 3
(0.13 g, 0.16 mmol) in DCM (15 mL), m-CPBA (0.19 g, 1.10 mmol) was added. The reaction mixture
was stirred for 5 days at room temperature, then was quenched with a solution of NaHSO3 0.2
M
and vigorously stirred for additional 30 minutes. The organic layer was washed with brine (2 x 20
mL), dried over anhydrous Na2SO4 and evaporated under reduced pressure. The resulting material
was taken with a solution of NaOH 2
M in MeOH:H2O 4:1 (5 mL) and was stirred for 2 hours at
room temperature, then the mixture was concentrated by evaporation of the MeOH under
reduced pressure. The remaining aqueous layer was neutralized with a solution of 1M HCl and
extracted with DCM (2 x 15 mL), then the combined organic layers were dried on anhydrous
Na2SO4 and evaporated under reduced pressure. The residue was triturated in refluxing hexane
1
overnight, for three times, to give 4 as a pale yellow oil (0.11 g, 0.13 mmol; 81% yield): H NMR
(300 MHz, CD3OD) δ (ppm): 7.38 (d, 2H, J=2.7 Hz), 7.34 (d, 2H, J=2.7 Hz), 6.91 (dd, 2H, J1=7.6 Hz,
J2=2.1 Hz), 6.86 (td, 1H, J1=7.6 Hz, J2=2.1 Hz), 6.36 (s, 2H), 4.67 (d, 2H, J=13.8 Hz), 4.60 (d, 2H,
J=13.8 Hz), 4.20 (t, 6H, J=5.1 Hz), 4.14 (t, 2H, J=5.4 Hz), 3.90 (t, 8H, J=5.4 Hz), 3.60-3.55 (m, 8H),
13
3.33 (d, 2H, J=13.8 Hz), 3.22 (d, 2H, J=13.8 Hz), 1.24-1.17 (m, 12H). C NMR (75 MHz, CD3OD) δ
(ppm): 161.6, 156.7, 152.0, 149.7, 142.4, 136.5, 136.4, 135.3, 134.7, 128.7, 122.8, 122.7, 115.1,
74.1, 73.1, 73.0, 69.7, 69.6, 60.1, 66.0, 65.9, 30.6, 30.5, 14.3. HR ES-MS: m/z Calcd for C44H55N2O13
[(4+H)+] 819.36987, found 819.36987; m/z Calcd for C44H54N2O13Na [(4+Na)+] 841.35181, found
841.35284.
5,17-Diamino-11-hydroxy-25,26,27,28-tetrakis(2-ethoxyethoxy)calix[4]arene (5): To a solution of
4 (0.053 g, 0.065 mmol) in MeOH (10 mL), NiCl2∙6H2O (0.062 g, 0.072 mmol) and NaBH4 (0.025 g,
0.66 mmol) were added. The reaction mixture was stirred for 3 hours at room temperature, then
was quenched with a solution of 1
M HCl (15 mL) and the pH raised to 8-9 with a solution of 1 M
NaOH. The resulting mixture was extracted with AcOEt (3 x 15 mL) and the combined organic
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layers were washed with distilled water (2 x 20 mL), dried over anhydrous Na2SO4 and evaporated
under reduced pressure. Compound 5 was obtained as an orange to brown oil (0.049 g, 0.065
mmol; quantitative yield), pure enough to avoid further purifications. 1H NMR (400 MHz, CD3OD) δ
(ppm): 7.10 (d, 2H, J=7.6 Hz), 6.92 (t, 1H, J=7.6 Hz), 6.56 (s, 2H), 5.99 (s, 2H), 5.89 (s, 2H), 4.58 (d,
2H, J=12.8 Hz), 4.52 (d, 2H, J=12.8 Hz), 4.26 (m, 2H), 4.17 (m, 2H), 3.94-3.83 (m, 12H), 3.63-3.55
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(m, 8H), 3.17 (d, 2H, J=12.8 Hz), 3.06 (d, 2H, J=12.8 Hz), 1.26-1.19 (m, 12H). C NMR (75 MHz,
CD3OD) δ (ppm): 151.3, 140.7, 135.5, 135.4, 134.9, 127.9, 116.2, 114.3, 73.1, 69.6, 66.0, 30.6, 30.5,
14.3. HR ES-MS: m/z Calcd for C44H59N2O9 [(5+H)+] 759.42151, found 759.42188.
5,17–Bis-[N,N’–bis(tert-butoxycarbonyl)guanidine]-11-hydroxy-25,26,27,28-tetrakis(2-
ethoxyethoxy)calix[4]arene (6): To a solution of 5 (0.049 g, 0.065 mmol) and triethylamine (72 μL,
0.039 mmol) in dry DMF (5 mL), N,N’-bis(tert-butoxycarbonyl)thiourea (0.015 g, 0.039 mmol) and
HgCl2 (0.070 g, 0.26 mmol) were added. The reaction mixture was stirred for 2 days at room
temperature, then was quenched by adding AcOEt (15 mL) and the precipitated HgS was filtered
off. The filtrate was washed with brine (3 x 20 mL) and the organic layer was dried over anhydrous
Na2SO4 and evaporated under reduced pressure. The crude material was purified by flash
chromatography (hexane/AcOEt 7:3) to give 6 as a colorless oil (0.034 g, 0.027 mmol; 42% yield).
1H NMR (300 MHz, CDCl3) δ (ppm): 11.62 (s, 2H), 10.18 (s, 2H), 7.25 (s, 4H), 6.38 (t, 1H, J=6.9 Hz),
6.28 (d, 2H, J=6.9 Hz), 5.68 (s, 2H), 4.49 (d, 2H, J=12.6 Hz), 4.42 (d, 2H, J=13.5 Hz), 4.24 (3 t, 4H,
J=6.6 Hz), 4.22-4.04 (m, 8H), 3.90-3.84 (m, 8H), 3.78-3.72 (m, 4H), 3.60-3.48 (m, 8H), 3.15 (d, 2H,
J=12.6 Hz), 3.09 (d, 2H, J=13.5 Hz), 1.60-1.49 (m, 36H), 1.25-1.15 (m, 12H). 13C NMR (75 MHz,
CDCl3) δ (ppm): 163.6, 155.5, 155.0, 153.9, 153.4, 150.7, 137.1, 137.0, 133.9, 133.0, 127.8, 123.6,
123.3, 122.5, 114.7, 83.6, 79.5, 74.0, 72.5, 69.6, 66.5, 66.2, 30.9, 30.8, 29.7, 28.2, 28.1, 15.4, 15.3.
HR ES-MS: m/z Calcd for C66H95N6O17 [(6+H)+] 1243.67482, found 1243.67444; m/z Calcd for
C61H87N6O15 [(6-Boc+H)+] 1143.62239, found 1143.62122.
5,17–Diguanidine-11-hydroxy-25,26,27,28-tetrakis(2-ethoxyethoxy)calix[4]arene
bis(hydrochloride) (1·2HCl). In a mixture of DCM/TFA/TES 95:2.5:2.5 (10 mL), 6 (0.034 g, 0.027
mmol) was dissolved. The reaction mixture was stirred overnight at room temperature and
quenched by removal of the solvent under reduced pressure. The residue was taken into a 1
M HCl
EtOH solution (3 mL), vigorously stirred for 30 min and then the solvent removed under reduced
pressure. This procedure was repeated three times to exchange the CF3COO- anion to chloride.
Then 1∙2HCl was obtained as a colorless oil (0.021 g, 0.025 mmol; 93% yield), pure enough to avoid
1
further purifications. H NMR (400 MHz, CD3OD) δ (ppm): 6.89 (d, 2H, J=7.2 Hz), 6.77 (t, 1H, J=7.2
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Hz), 6.55 (s, 2H), 6.51 (s, 2H), 6.32 (s, 2H), 4.63 (d, 2H, J=13.2 Hz), 4.57 (d, 2H, J=13.2 Hz), 4.60 (d,
2H, J=12.8 Hz), 4.26 (t, 2H, J=5.6 Hz), 4.18 (t, 2H, J=5.2 Hz), 4.12 (m, 4H), 3.94-3.91 (m, 8H), 3.67-
6
7
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3.56 (m, 8H), 3.26 (d, 2H, J=13.6 Hz), 3.15 (d, 2H, J=12.8 Hz), 1.27-1.18 (m, 12H). C NMR (100
8
9
MHz, CD3OD) δ (ppm): 156.6, 155.5, 151.8, 149.6, 136.6, 136.5, 135.5, 135.0, 128.4, 128.1, 124.9,
124.8, 122.5, 114.8, 73.9, 73.0, 72.9, 69.9, 69.8, 69.7, 66.2, 66.1, 66.0, 30.5, 30.4, 14.3 HR ES-MS:
m/z Calcd for C46H63N6O9 [(1+H)+] 843.46510, found 843.46593.
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Acid-base titrations
Potentiometric acid-base titrations of 5mL solutions of 2.0 – 3.0 m
Me4NClO4 was carried out according to a previously reported procedure,3d by addition in small
increments under argon atmosphere of a freshly prepared solution of 50 – 100 m Me4NOH, (80%
DMSO, 25 °C). Elaboration of the titration plot was carried out with the software HYPERQUAD
2000.26 UV-Vis spectrophotometric titrations of a 0.2 m
p-methoxyphenol or of 0.2 m
M M
(1H3)2+ (or 9H) and 10 m
M
M
M
calixarene (1H3)2+ solution with Me4NOH was carried out analogously under argon atmosphere,
(DMSO 80%, at 25 oC).
Kinetic measurements
Liberation of pNPOH was spectrophotometrically monitored at 400 nm. Initial-rate measurements
(data in Table 1) were carried out on 0.20 m
0.10 N,N-diisopropyl ethanolamine buffer, 10 m
The pH of the solution was adjusted at the selected pH value with a 50−100 m
in 80% DMSO. The time-course experiment was carried out on a 5.0 m BNPP, 0.1 m
Me4NClO4, 0.10 N,N-diisopropyl ethanolamine buffer solution, (pH 9.5; 80% DMSO, 50.0 °C).
The pH of the solution was adjusted at pH 9.5 by addition of the proper volume of the 50 – 100
solution of HClO4 in 80% DMSO. Non-linear least-square fit of experimental data to eqn (4)
M
BNPP, 1.0 m
Me4NClO4 solutions, (80% DMSO, 50.0 °C).
solution of HClO4
(1H3)2+, 10
M
additive ((1H3)2+, or (7H2)2+, or 9H),
M
M
M
M
M
m
M
M
m
M
was carried out with the software SigmaPlot 12.0 (Systat Software, Inc.).
ACKNOWLEDGMENTS
Thanks are due to the Ministero dell’Istruzione e dell’Università e della Ricerca (PRIN 2010JMAZML
MultiNanoIta), Ateneo 2015, and Consiglio Nazionale delle Ricerche (CNR) for financial support.
Thanks are also due to CIM (Parma University) for the use of NMR and mass spectrometry
facilities.
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ASSOCIATED CONTENT
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Supporting information
1H and 13C NMR spectra of compounds, potentiometric acid-base titration of compound 9H,
spectrophotometric acid-base titrations of (1H3)2+ and 9H (equations for the calculated lines in
Figure 3 of main text), standard equations for the distribution diagram of the species of a triprotic
acid, ESI-MS spectra of the reaction mixture in the time-course experiment in Figure 6 of main text
(BNPP cleavage in the presence of (1H3)2+).
REFERENCES AND NOTES
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(19) It can be shown that Ka1 = (Ka1)A + (Ka1)B, , where and (Ka1)A and (Ka1)B are the acidity constants
for the deprotonation equilibrium producing tautomer A or tautomer B, respectively.
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(20) The rates of cleavage of BNPP by the trifunctional calix[4]arene (entries 1 – 7) are from more
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p-methoxyphenoxide ion (entry 9). These rate enhancements strongly underestimate the effect of
the neighboring guanidinium units because p-methoxyphenoxide ion is a much stronger base and,
conceivably, a stronger nucleophile than the phenoxide unit in (1H2)+ and 1H.
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