General base-guanidinium cooperation in bifunctional artificial phosphodiesterases

Article abstract of DOI:10.1021/jo2004007

Artificial phosphodiesterases that combine a guanidinium unit with a general base connected by a m-xylylene linker catalyze the transesterification of the RNA model compound 2-hydroxypropyl p-nitrophenyl phosphate (HPNP). The bifunctional catalysts presented in this work show varying extents of cooperation between catalytic units and a rate enhancement of 4 ×10 ⁴ in the most favorable case.

Full text of DOI:10.1021/jo2004007

NOTE
pubs.acs.org/joc
General BaseꢀGuanidinium Cooperation in Bifunctional Artificial
Phosphodiesterases
Riccardo Salvio,* Roberta Cacciapaglia, and Luigi Mandolini
Dipartimento di Chimica and IMC - CNR Sezione Meccanismi di Reazione, Universitꢀa La Sapienza, P.le Aldo Moro 5,
00185 Roma, Italy
S Supporting Information
b
ABSTRACT: Artificial phosphodiesterases that combine a guanidinium unit with a general
base connected by a m-xylylene linker catalyze the transesterification of the RNA model
compound 2-hydroxypropyl p-nitrophenyl phosphate (HPNP). The bifunctional catalysts
presented in this work show varying extents of cooperation between catalytic units and a rate
enhancement of 4 ꢁ 104 in the most favorable case.
n the past two decades, simple, nonpeptidic molecules that
mimic structural and functional aspects of natural enzymes
highly suitable for potentiometric pKa determination32 and for
investigation of the kinetics of hydrolytic reactions of phospho-
diesters.22,33,34 Furthermore, the interaction of guanidinium with
phosphate via a two-point hydrogen bonding motif is stronger in
DMSO/water mixtures than in pure water.35,36 As a consequence,
a catalyst featuring a guanidinium unit as an anchoring and/or
activating group is potentially favored in a less aqueous solvent
mixture.
I
have received considerable attention.1ꢀ3 The extreme reluctance
of phosphodiester bonds to undergo hydrolysis4 has challenged
many research groups to design and synthesize artificial catalysts
capable of cleaving DNA, RNA, and their model compounds.5ꢀ15
These studies are interesting not only in their own right but also
in the prospect of health-related goals, such as the antisense
therapy based on the coupling of RNA hydrolytic agents to
antisense DNA.16
The imidazole in catalyst 1Hþ is expected to act as a general
base, whereas intramolecular attack of the HPNP activated
hydroxyl on phosphate should benefit from electrophilic activa-
tion promoted by the neighboring guanidinium. As for the metal-
based catalysts, there are strong indications that the cleavage
of HPNP and related compounds catalyzed by metal complexes
in water involves reversible deprotonation of the substrate
hydroxyl, followed by rate-limiting intramolecular nucleophilic
attack.37ꢀ39 However, Sꢁanchez-Lombardo and Yatsimirsky40
convincingly argued that in 80% DMSO the general base-assisted
intramolecular cyclization of HPNP is more favorable because
of the extremely low acidity of the substrate hydroxyl in this
medium. Although a specific base catalytic mechanism cannot
be ruled out with any certainty, we assume that the hydroxo
forms of CuII and ZnII complexes of 2Hþ will act as general
bases, in keeping with Sꢁanchez-Lombardo and Yatsimirsky’s
conclusions.
The guanidinium unit, which plays a key role in enzymes such as
staphylococcal nuclease,17 has been extensively used as an
activating and/or anchoring group in the design of hydrolytic
catalysts.9,18ꢀ28 These catalysts contain one or more guanidi-
nium units, or a guanidinium unit in conjunction with another
active unit such as a metal center,23ꢀ25 a hydroxyl group,26 or a
free base in solution.27,28
As a first part of a program devoted to the investigation of
guanidinium units in the development of artificial phosphodies-
terases, we report here a kinetic investigation of the cleavage of
the RNA model compound HPNP (eq 1) in the presence of 1Hþ
and of the CuII and ZnII complexes of 2Hþ. The use of a m-xylylene
spacer as a convenient platform for the connection of catalytic
groups is well precedented.21,29ꢀ31 Compounds 1 and 2 were
synthesized as described in Scheme 1 and Scheme 2, respectively.
Catalytic reactions were carried out in DMSO/H2O 4:1 (v/v),
hereafter referred to as 80% DMSO, which is a solvent mixture
Received: February 22, 2011
Published: May 25, 2011
r
2011 American Chemical Society
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|

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NOTE
Scheme 1. Synthesis of 1a
a a: Imidazole, K2CO3; CH3CN, reflux. b: H2, PdꢀC; MeOH, rt. c: N,N0-Bis(tert-butoxycarbonyl)-N00-triflylguanidine; CH2Cl2, rt. d: 0.1 M aqueous
HCl/dioxane 1:1, rt.
Scheme 2. Synthesis of 2a
a a: LiAlH4; THF, reflux. b: N,N0-Bis(tert-butoxycarbonyl)-N00-triflylguanidine; CH2Cl2, rt. c: PBr3, 2,6-di-tert-butylpyridine; toluene, rt. d: N,N0-Bis(tert-
butoxycarbonyl)-1,4,7-triazacyclononane, 2,6-di-tert-butylpyridine; CH3CN, rt. e: 0.5 M aqueous HCl/dioxane 1:1, rt.
Table 1. Pseudo-First-Order Rate Constantsaand Accelera-
tion Factors Relative to Background for the HPNP Cleavage
in 80% DMSO in the Presence of Additives
kobs (sꢀ1
)
kobs/kbkg
b
conditions
entry
additives
pH 7.0, 50 °Cc,d
1
2
3
4
5
6
7
8
9
guanidine HCl
4.8 ꢁ 10ꢀ8
8.2 ꢁ 10ꢀ8
5.2 ꢁ 10ꢀ7
1.4 ꢁ 10ꢀ8d
1.3 ꢁ 10ꢀ5
5.1 ꢁ 10ꢀ4
8.1 ꢁ 10ꢀ8d
1.8 ꢁ 10ꢀ4
1.0 ꢁ 10ꢀ3
0.94
1.6
3
12
1Hþ
10
pH 9.0, 25 °Ce
pH 9.8, 25 °Ce
guanidine HCl
1.1
3
TACN þ CuCl2
980
39000
1.0
2Hþ þ CuCl2
guanidine HCl
3
TACN þ ZnCl2
2300
13000
Figure 1. Transesterification of 3.0 mM HPNP catalyzed by 1Hþ in
80% DMSO, pH 7.0, 50 °C. Pseudo-first-order rate constant vs catalyst
concentration.
2Hþ þ ZnCl2
a kobs calculated as vo/[HPNP], where vo is the initial rate of p-
nitrophenol liberation; [additive] = 5.0 mM; [HPNP] = 0.20 mM,
unless otherwise stated. b kbkg = 5.1 ꢁ 10ꢀ8 sꢀ1 at pH 7.0, 50 °C; kbkg
=
1.3ꢁ 10ꢀ8 sꢀ1 at pH 9.0, 25 °C; kbkg = 7.9 ꢁ 10ꢀ8 sꢀ1 at pH 9.8, 25 °C.
c 0.10 M HEPES buffer. d [HPNP] = 3.0 mM. e 0.10 M N,N0-diisopropyl-
N-ethanolamine buffer.
catalyst 1Hþ (entry 3) compared with monofunctional mod-
els reveals a modest, but clearly detectable, degree of syner-
gism between functional groups. These data are consistent
with the expected mechanism in which the imidazole unit
acts as a general base and the negative charge developing
on the altered phosphate in the transition state is stabilized by
the neighboring guanidinium, possibly via a two-point hydro-
gen bonding motif. Figure 1 shows that kobs values for HPNP
transesterification exhibit a strictly linear dependence on
catalyst
In 80% DMSO, the autoprotolysis of water is strongly
suppressed (pKw = 18.4 at 25 °C),41 but the pKa values of
nitrogen bases are hardly affected.42 For guanidinium chloride,
we measured a pKa value of 13.7 at 25 °C (Figure 2S, Supporting
Information), very close to the value of 13.6 at the same
temperature in water.43 Titration of 1•2HCl revealed two pKa
values of 4.844 and 13.4 (Figure 3S, Supporting Information),
which were assigned to the imidazole and guanidine groups in the
given order. The influence of 1Hþ on rates of transesterification
of HPNP was investigated at pH 7.0, 50 °C. At this pH, the
imidazole unit is >99% in the neutral form, whereas the guanidine
unit is fully protonated. Rate data listed in Table 1 show an
insignificant influence of guanidinium (entry 1) and a very
modest rate enhancement brought about by 12 (entry 2) relative
to background hydrolysis (kbkg). The superiority of the bifunctional
concentration, which implies that K in eq 2 is too small to affect
the kinetics (K < 10 Mꢀ1). In other words, there is significant
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NOTE
Figure 3. Suggested mechanism of HPNP cleavage catalyzed by the
metal complexes of 2Hþ (MII = CuII, ZnII).
Figure 2. Titration of 2.0 mM 2 4HCl with Me4NOH in 80% DMSO
3
in the absence (a) and presence (b) of 1 equiv of CuII. Data points are
experimental, and the lines are calculated.
binding of HPNP to 1Hþ in the transition state, but not in the
reactant state.
K
cat þ HPNP a cat HPNP f products
ð2Þ
3
Figure 4. Plot of kobs for the cleavage of 0.20 mM HPNP in 80% DMSO
vs the concentration of the CuII complex of 2Hþ, pH 9.0, 25 °C. From
Titration of 2 4HCl (Figure 2, curve a) reveals, as expected,
3
the slope of the straight line k2 = 9.5 ꢁ 10ꢀ2 sꢀ1 Mꢀ1
.
four titratable protons (pKa values: <2, 4.9, 9.8, 13.1). The largest
pKa value belongs to the guanidinium moiety, whereas the other
three values compare well with the pKa values of TACN 3HCl
3
(<2, 6.0, 10.6; Figure 4S, Supporting Information), when allow-
ance is made for the acidity enhancing effect of the positively
charged guanidinium. Addition of 1 mol equiv of CuCl2 caused a
deep modification of the titration curve (Figure 2, curve b). The
number of titratable protons was raised to five, with pKa values of
<2, <2, 6.5, 9.0, and 13.4. The three most acidic protons were
assigned to the fully protonated triazacyclononane unit. The
increased apparent acidity is a result of the strong binding of CuII
to the triazacyclononane ligand (log K 8.6), whereas the acidity of
the guanidinium moiety is affected to a moderate extent. The pKa
value of 9.0 was attributed to a CuII-bound water molecule that
turns out to be 10.4 pKa units more acidic than bulk water in 80%
DMSO. Quite unexpectedly, the metal-bound water molecule in
the complex between CuII and TACN turned out to be more
acidic (pKa = 8.4; Figure 6S, Supporting Information) than in the
corresponding complex with 2Hþ, but the reason for this
enhanced acidity is unclear.
Figure 5. pH-rate profile for the cleavage of 0.2 mM HPNP catalyzed by
1.0 mM 2HþCuII in 80% DMSO, 25.0 °C. The data at pH 12 and 13 are
corrected for the background hydrolysis (kbkg = 5.0 ꢁ 10ꢀ6 sꢀ1 at pH 12
and kbkg = 7.6 ꢁ 10ꢀ5 sꢀ1 at pH 13).
Catalytic experiments in the presence of equimolar amounts
(5.0 mM) of 2Hþ and CuCl2 were carried out at pH 9.0. At the
given pH, the triazacyclononane ligand is fully bound to CuII; the
guanidine unit is still fully protonated; and the metal-bound
water molecule is 50% deprotonated.
Control experiments (Table 1) confirmed the insignificant
influence of guanidinium (entry 4) but showed that the CuII
complex of TACN, that is 80% in the hydroxo form at pH 9.0,
strongly enhances the transesterification rate of HPNP compared
with background (entry 5). Still larger was the rate enhancement
brought about by the CuII complex of the bifunctional catalyst
2Hþ (entry 6). Its catalytic efficiency is 40 times higher than that
of the monofunctional control45 and indicates a large extent of
cooperation of the hydroxo complex of the ligated CuII cation
with the neighboring guanidinium (Figure 3). Here again a linear
dependence of kobs vs catalyst concentration was observed
(Figure 4), showing that in the investigated concentration range
the catalyst works under subsaturating conditions, with a second-
order rate constant k2 = 9.5 ꢁ 10ꢀ2 sꢀ1 Mꢀ1, at pH 9.0.
The catalytic efficiency of the CuII complexes of 2Hþ was also
investigated at different pH values. The sigmoidal shape of the
pH-rate profile (Figure 5) is typical of a kinetic titration in
which an unreactive species is transformed into a reactive one
at higher pH values. Data points nicely fit to eq 3, where k2
is the second-order rate constant for the reaction of the
conjugate base of an acid whose acidity constant is Ka. A
least-squares fitting procedure gave pKa = 8.94 ( 0.12 and
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NOTE
k2 = (1.97 ( 0.06) ꢁ 10ꢀ1 Mꢀ1 sꢀ1
.
reported in the literature.47 Other solvents and reagents were commer-
cially available and used without any further purification.
vo
k2Ka
½Hþꢂ þ Ka
3-((Imidazol-1-yl)methyl)benzonitrile (4). Imidazole (2.95 g,
43.3 mmol) and anhydrous K2CO3 (8 g, 58 mmol) were added to a
solution of 3-(bromomethyl)benzonitrile (3) (2.26 g, 11.5 mmol) in
acetonitrile (100 mL). The reaction mixture was kept at reflux under
stirring for 30 min and then, after cooling, filtered to eliminate K2CO3
and evaporated under reduced pressure. A saturated Na2CO3 aqueous
solution (50 mL) and dichloromethane (50 mL) were added to the
residue. The organic phase was separated, washed with a saturated
Na2CO3 aqueous solution (50 mL ꢁ 3), and dried over MgSO4. The salt
was separated by filtration and the solvent evaporated under reduced
pressure to give 4 as a pale yellow solid (1.52 g, 8.30 mmol; 72% yield):
¼
ð3Þ
½catꢂ½HPNPꢂ
The very good agreement with the potentiometric pKa value
of 9.0 (Figure 2, curve b) strongly reinforces the view that the
reactive species is the hydroxo complex of the ligated CuII cation.
Furthermore the limiting value of the second-order rate constant
is almost exactly twice as large as the value of 9.5 ꢁ 10ꢀ2 Mꢀ1sꢀ1
determined at pH 9.0 (Figure 4).
Titration of 2 4HCl in the presence of 1 mol equiv of ZnCl2
3
1
(Figure 5S, Supporting Information) showed similarities with the
analogous titration in the presence of CuCl2 (Figure 2, curve b)
but also significant differences. The first 3 equiv of added base
was used up as before for deprotonating the triprotonated ligand
unit. Addition of the fourth equivalent of base revealed a proton
with a pKa = 9.8 which was assigned to a ZnII complexed water
molecule, to be compared with the pKa value of 9.0 of the CuII
complex. Complexation of the triazacyclononane unit with ZnII
(log K = 6.8) is weaker than complexation with CuII (log K =
8.6), yet strong enough to ensure complete binding of the metal
under the conditions of the catalytic runs, namely, equimolar
amounts (5 mM) of 2Hþ and ZnCl2 at pH 9.8.46
mp 58ꢀ62 °C. H NMR (200 MHz; CDCl3): δ 5.12 (s, 2H), 6.84
(s, 1H), 7.04 (s, 1H), 7.21ꢀ7.59 (m, 5H). 13C NMR (50 MHz, CDCl3):
δ 49.6, 113.0, 118.0, 119.0, 129.8, 130.2, 130.3, 131.2, 131.7, 137.3,
137.8. ES-MS: m/z 184.12 (M þ Hþ), 206.14 (M þ Naþ). Anal. Calcd
for C11H9N3: C, 72.11; H, 4.95; N, 22.94. Found: C, 71.97; H, 5.09;
N, 22.79.
(3-((Imidazol-1-yl)methyl)phenyl)methylamine (5). 10% Pdꢀ
C (0.25 g) was added to a solution of the nitrile 4 (1.52 g, 8.31 mmol) in
methanol (50 mL). The resulting mixture was stirred for 12 h at room
temperature in a hydrogen atmosphere and then filtered through Celite
under reduced pressure. The solvent was evaporated, and the crude product
was purified by column chromatography (basic Al2O3; CH2Cl2/CH3OH
from 99:1 to 95:5), giving the amine 5as a colorless oil (0.212 g, 1.13 mmol;
14% yield). 1H NMR (200 MHz, CD3OD): δ 3.87 (s, 2H), 5.23 (s, 2H),
6.94ꢀ7.44 (m, 6H), 7.74 (s, 1H). 13C NMR (50 MHz, CD3OD): δ 45.7,
51.5, 120.9, 128.0, 128.3, 128.7, 129.3, 130.4, 138.6, 138.7, 141.7. ES-MS:
m/z 188.12 (M þ Hþ), 210.10 (M þ Naþ). Anal. Calcd for C11H13N3: C,
70.56; H, 7.00; N, 22.44. Found: C, 70.38; H, 7.15; N, 22.17.
(3-(Imidazol-1-yl)methyl)benzyl-N,N0-bis(tert-butoxycar-
bonyl)guanidine (6). N,N0-Bis(tert-butoxycarbonyl)-N00-triflylgua-
nidine (0.48 g, 1.23 mmol) was added under stirring to a solution of
the amine 5 (0.212 g, 1.13 mmol) in anhydrous dichloromethane
(30 mL). The reaction mixture was kept under stirring at room
temperature for 24 h and then extracted with a saturated NaHCO3
aqueous solution (50 mL). The organic phase was separated, washed
with a saturated NaCl aqueous solution, dried over anhydrous Na2SO4,
and evaporated. The crude product was purified by column chroma-
tography (SiO2; CH2Cl2/CH3OH from 99:1 to 95:5), giving the
compound 6 as a pale yellow oil (0.175 g, 0.407 mmol; 36% yield).
1H NMR (200 MHz, CD3OD): δ 1.44 (s, 9H), 1.49 (s, 9H), 4.53
(s, 2H), 5.17 (s, 2H), 6.97 (s, 1H), 7.05ꢀ7.38 (m, 5H), 7.72 (s, 1H). 13C
NMR (50 MHz, CD3OD): δ 28.1, 28.2, 45.0, 51.6, 80.5, 84.6, 121.0,
127.7, 127.8, 128.3, 128.9, 130.3, 138.5, 140.0, 152.5, 154.1, 157.5. Anal.
Calcd for C22H31N5O4: C, 61.52; H, 7.27; N, 16.31. Found: C, 61.62; H,
7.10; N, 16.20.
Here again, the metal-bound water molecule in the ZnII
complex with TACN turned out to be more acidic (pKa = 8.6;
Figure 7S, Supporting Information) than in the corresponding
complex with 2Hþ.
The results of the catalytic experiments (Table 1) confirm
once more the ineffectiveness of the guanidinium control
(entry 7) and show that the hydroxy derivative of the ZnII
complex of TACN is 14 times more effective a catalyst than the
corresponding CuII complex in terms of kobs and 2.3 times more
effective in terms of rate enhancement relative to background
(compare entry 8 with entry 5). Comparison of the catalytic
efficiency of the ZnII complex of the bifunctional catalyst 2Hþ
with that of the control complex with TACN (compare entry 9
with entry 8)45 reveals a modest degree of cooperation of the
guanidinium unit with the metal center, which is significantly
lower than that found in the corresponding CuII complex. The
net result is that the hydroxo derivative of the ZnII complex of
2Hþ is twice as effective as the corresponding CuII complex in
terms of absolute rates but three times less effective when rates
relative to background are compared.
In summary, the structurally simple m-xylylene spacer induces
fair to good contacts between catalytic groups and altered
substrate in the transition state. Investigations of more elaborate,
hopefully more efficient spacers, are in progress. The good
catalytic performance of the CuII containing bifunctional catalyst
encourages application to the cleavage of ribonucleotides.
(3-(Imidazol-1-yl)methyl)benzylguanidine (1). Compound 6
(0.175 g, 0.407 mmol) was dissolved in a 1:1 mixture (100 mL) of 0.1 M
aqueous HCl/dioxane. The reaction was kept under stirring at room
temperature for 70 h. Afterward the solvent was evaporated under
reduced pressure and the residue dried under high vacuum for 8 h to give
1
1 HCl as a colorless oil (0.10 g, 0.331 mmol; 81% yield). H NMR
’ EXPERIMENTAL SECTION
3
(200 MHz, D2O): δ 4.49 (s, 2H), 5.49 (s, 2H), 7.35ꢀ7.58 (m, 7H). 13
C
NMR (50 MHz, D2O): δ 44.8, 53.1, 120.7, 122.5, 127.4, 128.3, 128.6,
130.5, 135.1, 135.2, 137.9, 157.5. ES-MS: m/z 230.10 (1Hþ), 213.09
(1HþꢀNH3). Anal. Calcd for C12H17Cl2N5: C, 47.69; H, 5.67; N,
23.17. Found: C, 47.42; H, 5.79; N 23.12.
Instruments and General Methods. NMR spectra were re-
corded on either a 300 or 200 MHz spectrometer. Chemical shifts are
reported as δ values in parts per million from tetramethylsilane added as
an internal standard. Mass spectra were performed by an Electrospray
Ionization Time-of-Flight spectrometer.
Materials. Dry CH3CN and CH2Cl2 were obtained by distillation
over CaH2 and CaCl2, respectively. THF and toluene were dried by
distillation over Na. DMSO purged 30 min with argon and mQ water
were used in the preparation of 80% DMSO. HPNP was prepared as
3-(Aminomethyl)phenylmethanol (8). LiAlH4 1 M in THF
(62 mL, 62 mmol) was added dropwise under an argon atmosphere to a
solution of 3-formylbenzonitrile (7) (2.31 g, 17.6 mmol) in anhydrous
THF (50 mL). The mixture was stirred for 30 min at room temperature
and then heated up to reflux for 8 h. After cooling, the reaction was
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NOTE
quenched with NaOH 3 M in water (20 mL), and the mixture was
extracted with chloroform (90 mL ꢁ 3). The collected organic phases
were dried over MgSO4 anhydrous, and the solvent was removed at
reduced pressure to give the aminoalcohol 8 as a pale yellow oil (1.4 g,
10.2 mmol; 58% yield). 1H NMR (200 MHz, CD3OD): δ 3.78 (s, 2H),
4.60 (s, 2H), 7.15ꢀ7.36 (m, 4H). 13C NMR (50 MHz, CD3OD): δ 46.6,
65.2, 126.6, 127.0, 127.4, 129.6, 143.0, 143.6. ES-MS: m/z 138.11
(M þ Hþ), 160.09 (M þ Naþ). Anal. Calcd for C8H11NO: C, 70.04;
H, 8.08; N, 10.21; Found: C, 70.43; H, 7.72; N, 10.03.
3-(Hydroxymethyl)benzyl-N,N0-bis(tert-butoxycarbonyl)-
guanidine (9). The aminoalcohol 8 (0.390 g, 2.85 mmol) and N,N0-
bis(tert-butoxycarbonyl)-N00-triflylguanidine (0.55 g, 1.4 mmol) were
dissolved in anhydrous dichloromethane (100 mL). After 1 h a further
portion of 1.4 mmol of the latter reagent was added to the solution. The
reaction mixture was left under stirring for 16 h at room temperature,
extracted with a 2 M NaHSO4 aqueous solution (50 mL), separated, and
washed with a saturated NaHCO3 water solution (50 mL). The aqueous
phase was extracted with dichloromethane (30 mL ꢁ 3), and the
combined organic phases were washed with a saturated NaCl aqueous
solution, dried over MgSO4, and evaporated. The crude material was
purified by column chromatography (SiO2; Hex/AcOEt from 20:1 to
4:1), giving the alcohol 9 as a colorless oil (0.730 g, 1.92 mmol; 67%
yield). 1H NMR (200 MHz, CDCl3): δ 1.48 (s, 9H), 1.52 (s, 9H), 1.74
(br s, 1H), 4.63 (d, 2H, J = 5.2 Hz), 4.70 (s, 2H), 7.21ꢀ7.42 (m, 4H),
8.59 (s, 1H), 11.54 (s, 1H). 13C NMR (50 MHz, CDCl3): δ 28.2, 28.4,
45.0, 65.1, 79.6, 83.3, 126.3, 126.5, 127.1, 129.0, 137.6, 141.6, 153.2,
156.2, 163.6. ES-MS: m/z 380.17 (M þ Hþ). Anal. Calcd for
C19H29N3O5: C, 60.14; H, 7.70; N, 11.07. Found: C, 59.79; H, 7.58;
N, 11.37.
1:1 mixture of 0.5 M aqueous HCl/dioxane and stirred at room
temperature for 70 h. Afterward the solvent mixture was evaporated
under reduced pressure and the residue dried under high vacuum for 8 h,
giving the tetrahydrochloride 2 4HCl as a white solid (0.080 g, 0.183
3
mmol; 97% yield): mp 157ꢀ158 °C. 1H NMR (300 MHz, D2O): δ 2.95
(t, 4 H, J = 3.1 Hz), 3.15 (t, 4H, J = 3.1 Hz), 3.39 (s, 4H), 3.56 (s, 4H),
3.85 (s, 2H), 4.38 (s, 2H), 7.25ꢀ7.45 (m, 4H). 13C NMR (75 MHz,
D2O): δ 42.7, 44.2, 44.9, 48.1, 59.3, 127.4, 129.0, 129.9, 130.3, 136.5,
137.3, 157.5. ES-MS: m/z 291.41 (2Hþ). Anal. Calcd for C15H30Cl4N6:
C, 41.30; H, 6.93; N, 19.26. Found: C, 41.23; H, 7.18; N, 19.22.
N-Benzylimidazole (12). K2CO3 (1 g, 7.2 mmol) and imidazole
(1.25 g, 18.4 mmol) were added to a solution of benzyl bromide (0.3 mL,
2.35 mmol) in anhydrous acetonitrile (50 mL). The mixture was
refluxed for 30 min and then filtered. The solvent was removed under
vacuum and the residue dissolved in CH2Cl2 (60 mL). The organic
phase was washed with a saturated Na2CO3 aqueous solution (50 mL ꢁ 3),
dried over MgSO4, and evaporated to give 12 as a colorless oil (0.296 g,
1.87 mmol; 80% yield). 1H NMR (200 MHz, CDCl3): δ 5.10 (s, 2H),
6.89 (s, 1H), 7.08 (m, 1H), 7.09ꢀ7.19 (m, 2H), 7.27ꢀ7.37 (m, 3H),
7.57 (s, 1H). 13C NMR (50 MHz, CDCl3) δ: 50.7, 119.2, 127.1, 128.1,
128.8, 129.5, 136.0, 137.2. ES-MS: m/z 159.12 (M þ Hþ), 181.11 (M þ
Naþ). Anal. Calcd for C10H10N2: C, 75.92; H, 6.37; N, 17.71. Found: C,
75.45; H, 6.39; N, 17.82.
Potentiometric Titrations. Potentiometric titrations were per-
formed by an automatic titrator equipped with a combined microglass
pH electrode. The electrode was calibrated using standard HClO4 and
Me4NOH solutions at different concentrations, I = 0.1 (Et4NBr). The
time required to obtain a stable pH reading increased from 1 min in acid
mediumþup to 6 min at pH above 9. The calibration plot of calculated
ꢀlog cH values vs experimental pH readings was linear in the range
3-(Bromomethyl)benzyl-N,N0-bis(tert-butoxycarbonyl)-
guanidine (10). PBr3 (0.038 mL, 0.405 mmol) was added to a
solution cooled to ꢀ10 °C of the alcohol 9 (0.460 g, 1.21 mmol) and
2,6-di-tert-butylpyridine (0.272 mL, 1.21 mmol) in dry toluene (7 mL).
The reaction was stirred at room temperature for 16 h under an argon
atmosphere. The mixture was then filtered and the solvent removed by
evaporation at reduced pressure to give the bromide 10 as a yellow oil
(0.53 g, 1.20 mmol; 99% yield), pure enough to be used in the following
2ꢀ17, with ꢀlog cHþ = a þ b pHread, and best fit values a = ꢀ0.7852 (
3
1.5% and b = 0.965 ( 5%. The pKw values determined in several
titrations coincided, within experimental errors, with the value reported
in the literature.36,41 Potentiometric titrations were carried out under a
nitrogen atmosphere, on 6 mL of 1ꢀ3 mM solutions of the compound,
in the presence of 0.1 M Et4NBr (80% DMSO, 25 °C). A 0.05ꢀ0.2 M
Me4NOH solution in 80% DMSO was added to the titration vessel in
small increments. Analysis of titration plots was carried out by the
program HYPERQUAD 2000.48,49
1
step without any further purification. H NMR (200 MHz, CDCl3):
δ 1.49 (s, 9H), 1.53 (s, 9H), 4.49 (s, 2H), 4.72 (d, 2H, J = 5.3 Hz),
7.11ꢀ7.38 (m, 4H), 8.79 (s, 1H), 11.53 (s, 1H). ES-MS: m/z 442.18
(M þ Hþ), 464.18 (M þ Naþ).
Kinetic Measurements. Spectrophotometric measurements
were carried out on either a double beam or on a diode array
spectrophotometer.
Kinetic measurements of HPNP transesterification were carried out
by UVꢀvis monitoring of p-nitrophenol liberation at 400 nm. Rate
constants were obtained by an initial rate method, error limit on the
order of (10% for rate constants of 10ꢀ8ꢀ10ꢀ7 sꢀ1 and (5% for faster
reactions.
Hydrochlorides were neutralized, with the exception of the guanidi-
nium proton, by the addition to the reaction mixture of the calculated
amount of Me4NOH before kinetic runs.
Metal complexes were formed in situ by addition of the calculated
stoichiometric amount of ZnCl2 or CuCl2 to the buffered reaction
mixture. Due to the slow formation of the complexes,50 the solutions
were incubated for 1 h before the start of the kinetic run by fast addition
of a small volume of the substrate solution.
3-((N,N0-Bis(tert-butoxycarbonyl)triazacyclononane)methyl)-
benzyl-N,N0-bis(tert-butoxycarbonyl)guanidine (11). A solu-
tion of the bromide 10 (0.420 g, 0.949 mmol), N,N0-bis(tert-butox-
ycarbonyl)triazacyclononane (0.195 g, 0.592 mmol), and 2,6-di-tert-
butylpyridine (0.546 mL, 2.43 mmol) in anhydrous acetonitrile (5 mL)
was stirred at room temperature for 30 h under an argon atmosphere.
The solvent was then removed by evaporation at reduced pressure, and
the residue was dissolved in dichloromethane (20 mL). The mixture was
filtered, and the solution was washed with a saturated NaHCO3 aqueous
solution (30 mL). The organic phase was separated, dried over MgSO4,
and evaporated at reduced pressure. The crude product was purified by
column chromatography (SiO2; from Hex to Hex/AcOEt 4:1), to give
11 as a colorless oil (0.213 g, 0.308 mmol; 52% yield). 1H NMR
(200 MHz, CDCl3): δ 1.48 and 1.51 (s, 36H), 2.71 (m, 4H), 3.26ꢀ3.58
(m, 8H), 3.70 (s, 2H), 4.60 (d, 2H, J = 5.1 Hz), 7.15ꢀ7.33 (m, 4H), 8.51
(s, 1H), 11.51 (s, 1H). 13C NMR (50 MHz, CDCl3): δ 27.9, 28.2, 28.4,
28.5, 44.9, 48.6, 49.1, 49.7, 50.3, 51.5, 52.8, 53.6, 54.1, 60.6, 79.2, 83.0,
126.3, 128.1, 128.3, 128.5, 136.9, 140.5, 153.0, 155.3, 155.6, 155.9, 163.5.
ES-MS: m/z 691.46 (M þ Hþ), 713.47 (M þ Naþ). Anal. Calcd for
C35H58N6O8: C, 60.85; H, 8.46; N, 12.16. Found: C, 60.78; H, 8.31;
N, 12.29.
’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra,
S
b
plots of potentiometric acidꢀbase titrations, and kinetic data
on the effect of chloride on TACNꢀCuII catalyzed cleavage of
HPNP. This material is available free of charge via the Internet at
http://pubs.acs.org.
(3-(1,4,7-Triazacyclononan-1-yl)methyl)benzylguanidine
(2). An amount of 0.130 g (0.188 mmol) of 11 was dissolved in 6 mL of a
5442
dx.doi.org/10.1021/jo2004007 |J. Org. Chem. 2011, 76, 5438–5443

The Journal of Organic Chemistry
NOTE
’ AUTHOR INFORMATION
(29) Cacciapaglia, R.; Di Stefano, S.; Kelderman, E.; Mandolini, L.
Angew. Chem., Int. Ed. 1999, 38, 348–351.
(30) Cacciapaglia, R.; Di Stefano, S.; Mandolini, L. J. Org. Chem.
2001, 66, 5926–5928.
Corresponding Author
riccardo.salvio@uniroma1.it
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A. K. J. Org. Chem. 2006, 71, 9713–9722.
’ ACKNOWLEDGMENT
Financial contribution from MIUR/PRIN 2008 is acknowl-
edged. The authors also thank Ms. Gabriella Angiulli,
Mr. Antonio Cincotti, and Ms. Claudia Savelli for technical
assistance in the synthesis of the compounds and in the potentio-
metric experiments.
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