Synthesis of mono- and di-[12]aneN3 ligands and study on the catalytic cleavage of RNA model 2-hydroxypropyl-p-nitrophenyl phosphate with their metal complexes

Article abstract of DOI:10.1039/c1ob05942d

A series of mono- and di-[12]aneN₃ ligands 1-6, which contain different substituents on the coordinating backbone, different linkers between two [12]aneN₃ units and different N-methylation on the [12]aneN ₃ units, have been synthesized and fully characterized. The catalytic activities of their metal complexes on the cleavage of RNA model phosphate 2-hydroxypropyl-p-nitrophenyl phosphate (HPNPP) varied with the structures of the ligands and metal ions. Click reactions afforded an efficient method to prepare a series of [12]aneN₃ ligands, however, the incorporation of triazole moieties reduced the catalytic activities due to their coordination with metal ions and the strong inhibition from the triflate counter ion. Dinuclear zinc(ii) complexes containing an m-xylyl bridge showed higher catalytic activities with synergistic effects up to 700-fold. Copper(ii) complexes with the ligands without triazole moieties proved to be highly reactive and showed strong cooperativity between the two copper(ii) ions. In terms of k₂, dinuclear complexes Zn₂-3b, Zn ₂-3d, Zn₂-4b, and Cu₂-4b afforded activities of 7.9 × 10⁵, 3.9 × 10⁴, 9.0 × 10 ⁴, and 8.1 × 10⁴-fold higher than that of methoxide. The ortho arrangement of the two [12]aneN₃ units and the presence of 5- or 2-positioned substituents in the benzene ring as well as N-methylation of [12]aneN₃ units greatly reduced the catalytic activities due to the steric effects. These results clearly indicate that the structures of the linker between two [12]aneN₃ units play very important role in their catalytic synergistic effects.

Full text of DOI:10.1039/c1ob05942d

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PAPER
Synthesis of mono- and di-[12]aneN3 ligands and study on the catalytic
cleavage of RNA model 2-hydroxypropyl-p-nitrophenyl phosphate with their
metal complexes†
Zhi-Fo Guo, Hao Yan, Zhi-Fen Li and Zhong-Lin Lu*
Received 11th June 2011, Accepted 22nd July 2011
DOI: 10.1039/c1ob05942d
A series of mono- and di-[12]aneN3 ligands 1–6, which contain different substituents on the
coordinating backbone, different linkers between two [12]aneN3 units and different N-methylation on
the [12]aneN3 units, have been synthesized and fully characterized. The catalytic activities of their metal
complexes on the cleavage of RNA model phosphate 2-hydroxypropyl-p-nitrophenyl phosphate
(HPNPP) varied with the structures of the ligands and metal ions. Click reactions afforded an efficient
method to prepare a series of [12]aneN3 ligands, however, the incorporation of triazole moieties reduced
the catalytic activities due to their coordination with metal ions and the strong inhibition from the
triflate counter ion. Dinuclear zinc(II) complexes containing an m-xylyl bridge showed higher catalytic
activities with synergistic effects up to 700-fold. Copper(II) complexes with the ligands without triazole
moieties proved to be highly reactive and showed strong cooperativity between the two copper(II) ions.
In terms of k2, dinuclear complexes Zn2-3b, Zn2-3d, Zn2-4b, and Cu2-4b afforded activities of 7.9 ¥ 105,
3.9 ¥ 104, 9.0 ¥ 104, and 8.1 ¥ 104-fold higher than that of methoxide. The ortho arrangement of the two
[12]aneN3 units and the presence of 5- or 2-positioned substituents in the benzene ring as well as
N-methylation of [12]aneN3 units greatly reduced the catalytic activities due to the steric effects. These
results clearly indicate that the structures of the linker between two [12]aneN3 units play very important
role in their catalytic synergistic effects.
For these reasons, many studies have been devoted to the design
Introduction
and synthesis of dinuclear or polynuclear metal complexes.10–12
Intensive work has been focused on the development of small
Among the dinuclear systems developed by the spacer strategy,
ligands containing 1,4,7-triazacyclononane ([9]aneN3)1,13 1,5,9-
triazacyclododecane ([12]aneN3),14–16 1,4,7,10-tetraazacyclodo-
decane ([12]aneN4)17,18 units are very promising in the development
of artificial nucleases. Recently, Brown’s group has investigated
a system of dinuclear metal complexes of 1,3-bis-N1-(1,5,9-
triazacyclododecyl)propane (abbreviated as PP-Di-[12]aneN3, see
Scheme 1) in methanol and ethanol and found that they can ac-
celerate the cleavage of model phosphate diesters by a remarkable
factor of up to 1012–14-fold, when compared to the background
reactions.2,19 The excellent catalytic performance of the system
has been attributed to the medium effect with lower dielectric
constants and the synergistic effect between metal ions.
synthetic metallonucleases for the cleavage of the phosphodiester
bond in RNA, DNA, and their model compounds.1–6 These
studies are beneficial for shedding light on understanding the
chemistry of the corresponding hydrolytic enzymes, providing
artificial restriction enzymes for molecular biology which would
be highly valuable in the manipulation of DNA, and developing
antibiotic and chemotherapeutic drugs. It has been shown that
many of the nucleases have active sites comprising two or more
metal ions. For example, alkaline phosphatase possesses dinuclear
Zn2+ and Mg2+ centers, purple acid phosphatase employs a center
containing one Fe3+ and one Fe2+ or Zn2+ ions, while P1 nuclease
contains a trinuclear Zn2+ active site and ribonuclease H from
HIV reverse transcriptase contains a two-metal ion active site.7–9
College of Chemistry, Beijing Normal University, Xinjiekouwai Street 19,
Beijing, 100875, China. E-mail: luzl@bnu.edu.cn; Fax: +86-10-58801804;
Tel: +86-10-58801804
† Electronic supplementary information (ESI) available: Tables of kinetic
data and method for triflate ion inhibition, plots of kinetics data as
a function of metal complex concentrations and triflate; NMR and
mass spectroscopic data for ligands 1–6 and the intermediates. See DOI:
10.1039/c1ob05942d
Scheme
PP-Di-[12]aneN3.
1
Structures of [9]aneN3, [12]aneN3, [12]aneN4, and
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Previous work demonstrates that the distances between the
two coordinating moieties, the structures of the linkers, and the
secondary coordination spheres of the ligands all play important
roles in achieving good catalytic activity for the dinuclear metal
complexes. With the above considerations, we report here on
the synthesis and characterization of a series of mono- and
di-[12]aneN3 ligands containing different substituents on the
coordinating backbone, different linkers between two [12]aneN3
units as well as different steric coordinating spheres (see Scheme
2). The catalytic activities of their zinc(II) and copper(II) complexes
on the cleavage of RNA model phosphate 2-hydroxypropyl-4-
nitrophenyl phosphate (HPNPP) have been investigated and are
reported here.
Scheme 4 Syntheses of ligand 4–6: i) 13, CHCl3, reflux 2 h; ii) 3 M HCl,
12 h; iii) NaBH4, EtOH, overnight; iv) HCHO, HCOOH, reflux overnight.
method.21 The terminal alkyne, di-tert-butyl-1-propargyl-1,5,9-
triazacyclododecane-5,9-tricarboxylate 8, an important build-
ing block for the construction of multi-[12]aneN3 compounds,
was prepared from the reaction between di-tert-butyl-1,5,9-
triazacyclododecane-5,9-tricarboxylate 7 and propargyl bromide
in the presence of triethylamine. The click reactions between
alkyne 8 and azides afforded the Boc-protected products in high
yields. The removal of the Boc protecting groups was accomplished
in anhydrous methanol in the presence of acetyl chloride at room
temperature for 1 h. The resultant hydrochloride salts of ligands
2 and 3, which are stable at room temperature for long period
of time and are easily handled for their further use, were fully
characterized. In the 1H-NMR spectra, the protons from triazole
moieties generally appear at 8.10–8.19 ppm in a singlet, the protons
from the CH2 moieties at the 1-and 4- positions of the triazole
units appear at 4.18–4.37 (singlet) and 5.62–5.76 (singlet, CH2Ar)
ppm, respectively. The protons from the aryl moieties appear in
the range of 6.98–8.07 ppm and their splitting is consistent with
their substitution mode. The protons from the methylene groups
of the [12]aneN3 units generally appear around 2.20, 3.10 and 3.30
ppm as multiplets, and are consistent with those reported in the
literature.16,22,23
Scheme 2 Structures of the mono- and di-[12]aneN3 ligands.
Results and discussion
Syntheses and characterization of ligands 1–6
We synthesized new [12]aneN3 ligands for our artificial nucleases
study using two different strategies (Schemes 3 and 4).
The synthesis of ligands 4–6 began with the alkylation re-
actions between the [12]aneN3 precursor 13 and either 1,3-
bis(bromomethyl)benzene 10b or 5-substituted-1,3-bis- (bro-
momethyl)benzenes 10d/10f. Depending on the following work-
up procedure, the intermediates 12b/d/f were converted to non-
methyl, dimethyl, and tetramethyl substituted compounds 4b/d/f,
5b, and 6b. Although compounds 4b and 6b have been reported
previously,24,25 no detailed characterization data were provided. In
the 1H-NMR spectra, the protons from the CH2 group between the
aryl and [12]aneN3 units are located at 3.38–3.57 ppm. The methyl
groups on the [12]aneN3 unit appear at 2.08–2.20 ppm, and the
integration of the protons are consistent with the structure. The
chemical shifts of the protons on the aryl and [12]aneN3 groups
are consistent with those in the literature.16,22,23,26
Scheme 3 Syntheses of 2 and 3: i) propargyl bromide, THF, NEt3, reflux
72h; ii) NaN3, THF/H2O; iii) a) THF/H2O, CuSO4, Vc; b) CH3COCl,
CH3OH.
Spectroscopic study of the coordination behavior of ligand 2
Click reactions afforded the efficient preparation of various
[12]aneN3 ligands containing the triazole moieties in this work.
Since copper(II) complexes of the N-ligands are highly colored, the
changes in the immediate coordination environment surrounding
the metal ion can be inferred from the electronic spectra. To
get information about the coordination of the triazole moiety
Ligands 2 and 3 were prepared by using highly efficient Cu(I)-
catalyzed alkyne-azide cycloaddition reactions.20 The mono- and
bis-azido compounds were prepared from their corresponding
bromides, which were in turn prepared from the bromina-
tion of substituted xylenes following the modified literature
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to copper ion, the visible spectra of Cu-2 together with Cu-1
and Cu-[12]aneN3 were recorded in methanol and are shown in
Fig. 1. The maximum peaks of the absorption appear at 681,
669, and 659 nm for complexes Cu-[12]aneN3, Cu-1, and Cu-2,
respectively. The blue shift of the maximum absorption and the
appearance of a shoulder around near 780 nm clearly indicated
that the presence of the triazole moiety in ligand 2 resulted in the
change of coordination geometry of the copper(II) ion.27,28
Fig. 1 UV spectra of Cu-[12]aneN3, Cu-1, and Cu-2 at 8.0 mM in
methanol.
Fig. 2 Plots of kobs vs. [M-L] (M = Zn(II) ꢀ, Cu(II) ꢁ) for the cleavage of
HPNPP (4.0 ¥ 10-5 M) at [-OCH3]/[M(II)] = 0.5 and 25.0 0.1 ◦C. (A) L =
Catalytic cleavage of HPNPP
ss pH
ss pH
1,
= 10.0 0.1 for Zn-1 and
= 10.0 0.1 for Zn-2 and
= 8.9 0.1 for Cu-1. (B) L = 2,
ss pH
= 8.8 0.1 for Cu-2.
We have evaluated the catalytic activities of the metal complexes
of ligands 1–6 on the cleavage of 2-hydroxypropyl-p-nitrophenyl
phosphate (HPNPP) (Scheme 5), which is often used as model
compound of RNA.1,5,19 To avoid the effect of triflate counter
ions, the kinetic data for all zinc(II) and some copper(II) complexes
were corrected for their respective triflate inhibition constants (see
supporting information)
ss pH
Table 1 Kinetic data for the cleavage of HPNPP mediated by the
mononuclear complexes of ligands 1 and 2 at 25.0 0.1 ◦
C
Cat
103Kdis (M)
k2 (M-1 s-1)
103k3 (M-2 s-1)
-OMe
—
—
—
—
—
1.4 0.2
1.7 0.6
2.56 ¥ 10-3a
18.9a
—
Zn-[12]aneN3
Cu-[12]aneN3
Zn-1
1.8 0.4a
—
23.17b
3.8 0.2
21.5 1.2
2.9 0.2
0.21 0.02
0.18 0.11
8.5 2.5
—
Cu-1
Zn-2
Cu-2
—
a See reference 31. b The measured k2 see supporting information.
Scheme 5 Cleavage of HPNPP catalyzed by metal complexes of 1–6.
constant (Kdis) and conditional second rate constant (km) for the
reactive mononuclear complex.
i) Cleavage of HPNPP with mononuclear metal complexes. Fig.
2 shows the plots of the corrected observed pseudo-first order rate
constants for the cleavage of HPNPP as a function of [M:L] (M =
Zn(II), Cu(II), L = 1, 2) in the presence of 0.5 eq. of CH3O- to set
ss pH
kobs ={kmKdis ( 1 + 8[M2+ ]t Kdis −1)}/ 4
(2)
the
of the solution to self-buffered conditions.
For ligand 1, the plots of Cu-1 and Zn-1 show a slight upward
curvature, indicating a bimolecular process which is similar to that
of Zn-[12]aneN3 in the literature.29 By fitting the plots in the eqn
All of the kinetic data are listed in Table 1. It can be seen that
the second-order rate constant of Cu-1 (21.5 M-1 s-1) is the highest
among the mononuclear complexes in this study, which is 8.5 ¥
103-fold higher than that of methoxide (2.56 ¥ 10-3 M-1 s-1).31
For the mononuclear zinc(II) complexes, incorporating benzyl
and benzyl-triazole moieties reduced the activity. Compared to
benzyl substituent in Zn-1, the presence of the triazole group in
Zn-2 further reduced the activity but not significantly. For the
mononuclear copper(II) complexes, apparently the presence of the
benzyl group has almost no effect on the catalytic activity when
compared to that of the Cu-[12]aneN3 complex. However, the
obs
obs
(1), it affords k2 and k3 for Cu-1 and Zn-1, respectively.
kobs = k2obs[M-1] + k3obs[M-1]2
(1)
For ligand 2, the plots of both Zn-2 and Cu-2 are bowed, which
is not due to substrate-complex saturation binding process but
rather a monomer-dimer equilibrium behaviour, which requires
a square-root dependence.30 The data can be fit via a standard
NLLSQ treatment to eqn (2), which affords the dimer dissociation
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Table 2 Kinetic constants (maximum catalytic pH rate constant (kmax), dissociation constants (KM) and second order rate constants (k2 or kmax/KM) for
the cleavage of HPNPP mediated by different dinuclear catalysts at 25.0 0.1 ◦
C
ss pH
Catalysts
Ki (OTf)(mM)
103kmax (s-1)a
103KM (M)
k2 (M-1 s-1)
Synergistic effect
Zn2-3a
Zn2-3b
Zn2-3c
Zn2-3d
Zn2-3e
Zn2-4b
Zn2-4d
Zn2-4g
Zn2-5b
Zn2-6b
Cu2-3d
Cu2-4b
Cu2-4d
9.8 0.1
10.1 0.1
9.9 0.1
9.2 0.1
10.2 0.1
9.6 0.1
9.8 0.1
10.1 0.1
9.9 0.1
9.8 0.1
7.9 0.2
9.2 0.1
6.9 0.2
2.4 0.3
0.20 0.21
1.4 0.3
0.59 0.08
3.98 0.13
8.7 1.1
10.3 0.7
—
—
—
9.7 0.8
—
14.1 0.8
20.3 2.6
—
—
—
7.9 0.4
—
16.1 0.6
7.9 1.3
19.5 1.5
4.2 0.2
1.27 0.05
—
0.31 0.07
—
—
65 15
22b
700b
36b
34b
9b
2030 120
105 3.8
99.6 2.7
27.2 3.7
231 12
23.0 0.8
34 13
19.7 1.5
10.8 1.9
9.8 1.5
—
0.29 0.04
—
61c
6c
0.70 0.04
0.23 0.09
0.99 0.15
0.39 0.07
0.13 0.02
—
9c
5c
3c
—
10d
—
209
2
12.2 2.5
1.01 0.26
12.1 2.5
a kmax and KM determined by fits of the Michaelis–Menten equation. b Compared to that of Zn-2. c Compared to that of Zn-1. d Compared to that of Cu-1.
incorporation of the triazole moiety in Cu-2 dramatically reduced
the catalytic activity by 100-fold.
The different catalytic performances of M-1 and M-2 can
be attributed to their different coordination behavior in the
bimolecular process. It is known that [12]aneN3 coordinates to
metal ions through its three nitrogen donors in a facial mode,
the coordination geometries are four-coordinate tetrahedral for
zinc(II) ion and five-coordinate square-pyramidal for copper(II)
ion.32 Generally, copper(II) ion can strongly coordinate to nitrogen
donors. Thus the triazole moiety in 2 can coordinate to copper(II)
ion and leave one less coordinate site available for the activation of
Fig. 3 Plots of kobs vs. Zn -3 (3a ᭡, 3b ᭛, 3c ) for the cleavage of HPNPP
a substrate or a nucleophile. This has been supported by the UV-
Visible spectroscopic study (Fig. 1). The coordination of triazole
moieties to copper(II) ions has also been reported.33
᭹
2
ss pH
ss pH
(4.0 ¥ 10-5 M) at [-OCH3]/[Zn(II)] = 0.5,
= 9.8 0.1 for Zn2-3a (right
= 9.9 0.1 for Zn2-3c at 25.0
ss pH
axis),
= 10.1 0.1 for Zn2-3b and
0.1 ◦C.
ii) Cleavage of HPNPP with dinuclear metal complexes. The
catalytic cleavage of HPNPP in the presence of zinc(II) and
copper(II) complexes of ligand 3(a–e), 4(b, d, g), 5b and 6b
were carried out under self-buffered conditions as described
in the Experimental Section and Supporting Information. In
the preliminary work, the reaction conditions were optimized
by varying equivalents of base (NaOCH3), metal ion, and by
mixing time. It was found that the catalysts formed by the
literature procedure29 are the most active, i.e. the catalysts were
obtained in situ through sequential addition of stock solutions of
sodium methoxide, macrocyclic polyamine ligand, and Zn(OTf)2
(or Cu(OTf)2) to anhydrous methanol such that [CH3O-] : [di-
[12]aneN3] : [M(II)] = 1 : 1 : 2.29
(3) afforded kmax and KM to be (20.3 2.6) ¥10-3 s-1 and 0.31
0.07 mM.
kobs = kmax [Sub]/(KM + [Sub])
(3)
For the meta-substituted di-[12]aneN3 system Zn2-3b, it was
corr
found that kobs
increased sharply beyond 0.2 mM of Zn2-
3b and showed linearity at higher concentration ranges of the
measurement. It was assumed that the dinuclear complex for Zn2-
3b was not fully formed at lower concentrations. The gradient
of the linear part of the plot gave a second rate constant of
(2.03 0.12) ¥ 103 M-1 s-1. The plot of kobs versus [Zn2-3c] is
corr
almost linear, and the second order rate constant for the catalytic
processes (k2), evaluated from the gradients of the linear plot, is
105.1 3.8 M-1 s-1.
The plots of kobs versus [catalyst] for the dinuclear zinc(II)
complexes of 3d and 3e (Figure 4S) showed linear dependence and
saturation dependence, respectively. The analyzed kinetic data are
listed in Table 2. It is obvious that incorporating a substituent at
the 5- and 2-position of the meta-substituted di-[12]aneN3 system
reduced the catalytic activity.
When it came to the copper complexes of the above di-
[12]aneN3 ligands, the catalytic properties dramatically decreased.
For example, the catalytic activity of Cu2-3d was much lower and
showed a saturation kinetics with kmax of (1.27 0.05) ¥ 10-3 s-1
(see Figure 9S). Even after the correction of triflate inhibition
a) The cleavage of HPNPP with dinuclear metal complexes
containing triazole moieties. Ligands 3a–3c contain two triazole-
attached [12]aneN3 units but with different spatial arrangements.
We observed a strong inhibition from triflate counter ions on
catalytic rate constants for the dinuclear zinc(II) complexes (see
Ki data in Table 2). After correcting for the triflate inhibition, the
plots of kobs versus [catalyst] are shown in Fig. 3, which indicates
that the arrangement of the two [12]aneN3 units plays an important
role in the catalysis.
It is apparent that ortho substituted Zn2-3a provided much
corr
poorer catalytic activity, the plot of kobs versus [Zn2-3a] shows a
saturation curve. Fitting the data with the Michaelis–Menten eqn
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which is obviously not strong when compared with those of the
corresponding zinc(II) complex, the calculated k2 is lower than that
of Cu-1.
All of the kinetic data are listed in Table 2. It can be seen that
Zn2-3b in this series showed the highest catalytic activity with the
second-order rate constant of 2.03 ¥ 103 M-1 s-1 after correcting for
triflate inhibition. Compared with the activity of the mononuclear
complex Zn-2, Zn2-3b showed synergistic effects up to 700-fold
between the two metal ions.
Zn2-5b and Zn2-6b reduced the synergistic effects to be only 5 and
3-fold, respectively, when compared to that of Zn-1.
c) Mechanism of the catalytic cleavage of HPNPP. Kinetic
data for the mono-nuclear complexes M-1 and M-2 indicated a
bimolecular process in the studied concentration range. For ligand
1, the bimolecular process was dependent on both the neutral
(CH3OH):M-1 and basic (CH3O-):M-1 forms with the former
acting to bind the HPNPP and the latter delivering a M(II)-bound
methoxide that acts as a base to deprotonate the 2-hydroxypropyl
group, similar to that of Zn-[12]aneN3.29 Both species worked
synergistically, resulting in the upward kinetic plot of kobs versus
[M-1]. The activity of Zn-1 (k2 = 3.8 M-1 s-1) is lower than that of
Zn-[12]aneN3 (k2 = 18.9 M-1 s-1),29 which can be attributed to the
steric effect of the benzyl moiety. Less of a decrease on the catalytic
activity for Cu-1 may be attributed to the strong coordination
constants between [12]aneN3 and the copper(II) ion. For ligand 2,
the bimolecular process afforded the bowed kinetic curve due to
the formation of an inactive dimer in which the coordination of the
triazole moiety to the metal ion reduced the free coordination sites
for the activation of the phosphates substrate or the nucleophile.
Compared with the zinc(II) complex, the catalytic activity of Cu-2
decreased greatly because of the strong coordination of the triazole
moiety to the copper(II) ion.
For the cleavage of HPNPP by a dinuclear Zn(II) complex
of PP-Di-[12]aneN3 ligand, Brown et al. proposed the process
involving at least two binding events, followed by one or more
chemical steps that released the observed p-nitrophenol product.
The binding steps were suggested to consist of phosphate diester
association with one metal ion followed by a rearrangement where
it became doubly activated via binding to the second metal ion. The
chemical step was proposed to involve internal deprotonation of
the 2-hydroxyl group followed by cyclization with expulsion of the
phenoxy leaving group. The catalytic process of the dinuclear metal
complexes of ligands 3(b,d,e)–6 should be consistent with the same
mechanism (see Scheme 6). However, depending on the structure
of the ligands and the nature of the metal ions, the synergistic
effects between two the [12]aneN3 units and the rate-limiting steps
should differ.
b) The cleavage of HPNPP with dinuclear complexes of 4–6.
Further, we monitored the catalytic activity of the dinuclear metal
complexes lacking the triazole moieties, i.e. di-[12]aneN3 ligands
4–6, which contain two [12]aneN3 units linked directly by meta-
xylyl backbones but with different steric effects.
The plots of kobs versus [catalyst] of ligands 4b, 5b and 6b for the
cleavage of HPNPP are shown in Fig. 4, and the fitting results of
the plots are listed in Table 2. The kinetic data clearly indicates
that N-methylation of the [12]aneN3 units reduced the catalytic
activity, which can be attributed to the steric effects. For ligand 4b,
both the dinuclear zinc(II) and copper(II) complexes show linear
dependence on the catalyst concentrations (the plot for Cu2-4b
shown as Figure 11S). The plots of Zn2-5b and Zn2-6b, which
respectively contain two methyl and four methyl groups on the
[12]aneN3 units, show saturation behavior, the data were analyzed
by standard Michaelis–Menten kinetics (eqn (3)).
Fig. 4 Plots of kobs vs. Zn -L (L = 4b, 5b , 6b ꢀ) for the cleavage of
᭹
2
ss pH
HPNPP (4.0 ¥ 10-5 M) at [-OCH3]/[Zn(II)] = 0.5,
= 10.0 0.1 for
ss pH
ss pH
= 9.8
Zn2-4b (left axis),
= 9.9 0.1 for Zn2-5b (right axis) and
0.1 for Zn2-6b (right axis), 25.0 0.1 ◦C.
For the dinuclear zinc(II) complexes of ligands 4d and 4g, which
contain 5-substituted nitro-, and carboxyl- moieties, their catalytic
activity (Figure 6S) is reduced when compared to that of Zn2-4b.
The same is true for the dinuclear copper(II) complex Cu2-4d,
which also shows saturation kinetics with kmax of (1.22 0.25) ¥
10-2 s-1 (see Figure 10S).
The Zn2-4b complex shows the highest catalytic activity and
synergistic effect among the zinc(II) complexes in this series.
The second-order rate constant is (231 12) M-1 s-1, which is
approximately 61-fold that of Zn-1, and 13-fold higher than that
of Zn-[12]aneN3. With a second-order rate constant of (209
2) M-1 s-1, Cu2-4b also showed the highest catalytic activity and
synergistic effect (10-fold higher than that of Cu-1) among the
copper(II) complexes in this work. At 1.0 mM of Zn2-4b and
Cu2-4b, the cleavage of HPNPP was accelerated by 2.6 ¥ 108-
fold and 3.0 ¥ 109-fold, respectively, when compared to that of the
background reactions. N-Methylation of the [12]aneN3 units in
Scheme 6 Pathway for the cleavage of HPNPP promoted by dinuclear
metal complexes.
From the kinetic data listed in Table 2, most of the dinuclear
metal complexes studied show synergistic effects but they varied
from 3- to 700-fold enhancements. After correcting for the
inhibition by triflate, the plots of kobs versus the concentration
of Zn2-3b, Zn2-3d, Zn2-4b, and Cu2-4b are linear, this may indicate
that the reactions are limited by a prior binding process and that
the actual chemical step of HPNPP cyclization is even faster. In
terms of the k2 values listed in Table 2, the activities of Zn2-
3b, Zn2-3d, Zn2-4b, and Cu2-4b are 7.9 ¥ 105, 3.9 ¥ 104, 9.0 ¥
104, and 8.1 ¥ 104-fold higher than that of methoxide. When
compared to that of Zn2-PP-Di-[12]aneN3, the activities of the
above dinuclear complexes are 2–3 orders lower, which can be
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attributed to the weaker substrate binding ability of the catalysts
caused by some of the following factors: the rigidity of the benzene
backbone, the large distance between the two 12[ane]N3 units, and
the coordination of triazole moieties to metal(II) ions.
are being constructed in our laboratory, their applications on the
catalytic cleavage of the phosphate diester linkage in RNA and
DNA are currently under investigation.
When the substrate binding ability becomes even weaker due to
the above aspects, the limiting step of the catalytic reaction will
move to the chemical step and the saturation kinetics will appear.
This should be the case for the reactions catalyzed by the dinuclear
metal complexes Zn2-3e, Zn2-4d, Zn2-4g, Zn2-5b, and Zn2-6b listed
in Table 2. It can be seen that steric effects from 2/5-positioned
substituents (3e, 4d, 4g), or N-methylation of [12]aneN3 units (5b,
6b) all significantly impaired the catalytic efficiency and resulted
in the Michaelis–Menten saturation kinetics for the cleavage of
HPNPP. The rate-limiting step can be assigned to the chemical
step of cyclization.
Experimental
1. Physical measurements
1H- and 13C-NMR spectra were recorded on a Bruker Avance
400 MHz spectrometer and chemical shifts in ppm are reported
relative to internal tetramethylsilane (TMS) or residual solvent
peaks. IR spectra were recorded on a Nicolet 380 spectrometer in
the range of 4000–400 cm-1, samples were prepared as KBr pellets.
ESI-MS and HRMS spectra were recorded on Quattro Mocro
and Bruker Daltonics Bio TOF mass spectrometer, respectively.
UV-visible spectra were obtained on a Varian Cary-300 UV-
visible spectrophotometer. Elemental analyses were obtained on
an Elementar Vario EL analyzer.
Conclusions
In the present study, we have designed and synthesized a series of
mono- and di-[12]aneN3 ligands 1–6 through nitrogen alkylation
and copper(I)-mediated alkyne-azide click reactions. All of the new
ligands have been fully characterized by 1H-NMR, 13C-NMR, IR,
and MS.
2. Chemicals
Methanol (water < 50 ppm) was purchased from Acros Organics
and used as supplied. Sodium methoxide, propargyl bromide,
Cu(CF3SO3)2, and Zn(CF3SO3)2 were purchased from Alfa Aesar
and used without further purification. The sodium salt of 2-
hydroxylpropyl-p-nitrophenyl phosphate (HPNPP) was prepared
according to the literature procedure.31 Compound 1,34 7,35 9,36
10a–f,37–44 and 1326 were synthesized according to the literature
procedures and characterized by 1H-NMR. Other chemicals and
reagents were obtained commercially and used without further
purification.
The catalytic activities of their zinc(II) and copper(II) complexes
on the cleavage of RNA model phosphate HPNPP have been
evaluated, which varied greatly with metal ions and the structures
of the ligands. The incorporation of the triazole moieties in the
ligands reduced the catalytic activities due to their coordination
with metal ions, especially for copper(II) complexes. It was also
found that the inhibition of counter ion triflate on the activity
is much strong for the dinuclear metal complexes containing
triazole moieties. For the ligands lacking triazole moieties, their
metal complexes showed good intermolecular (for mononuclear
complexes) or intramolecular (for dinuclear complexes) synergistic
effects between two metal ions. Specifically, dinuclear complexes
Zn2-3b, Zn2-3d, Zn2-4b and Cu2-4b exhibit second-order rate
constants of 2.03 ¥ 103, 99.6, 231, and 209 M-1 s-1, respectively,
3. Synthesis
(1) Preparation of compound 8. Boc2-[12]aneN3 7 (1.86 g,
5 mmol), propargyl bromide (0.59 g, 5 mmol), and triethylamine
(0.71 g, 7 mmol) were dissolved in 50 mL of dry THF and stirred
under N2. The reaction mixture was heated to reflux under N2 for
72 h, and then was evaporated under reduced pressure to afford
the crude product. The pure product was separated on a silica gel
column (petroleum ether/acetone = 2 : 1, Rf 0.7) to give a white
which are 7.9 ¥ 105, 3.9 ¥ 104, 9.0 ¥ 104, and 8.1 ¥ 104-fold
MeO-
larger than that of methoxide (k2
= 2.56 ¥ 10-3 M-1 s-1),31
they afforded the synergistic effects of 700, 42, 61, and 10-fold
when compared with the corresponding mononuclear complexes.
Relative to the background reactions, the cleavage of HPNPP
catalyzed by 1.0 mM of Zn2-4b, and Cu2-4b can be accelerated by
2.6 ¥ 108 and 3.0 ¥ 109-fold, respectively.
The above work clearly demonstrates that substituents on the
coordinating backbone, linkers between two [12]aneN3 units, and
the N-methylation on [12]aneN3 units all play important roles
in the catalytic activity. The meta-positioned di-[12]aneN3 system
showed good activity and evident synergistic effects. The presence
of 5/2-positioned substituents on the aryl group and the methyl
groups on [12]aneN3 units resulted in poor activities due to the
steric effects.
It should be noted that there is difference in the cleavage
mechanism between HPNPP and RNA. For HPNPP, the for-
mation of phosphane is generally the rate-limiting step due to
the presence of a good leaving group (4-nitrophenolate). With
RNA, the breakdown of the phosphane intermediate is rate-
limiting due to the poor leaving property of the alkoxyl group.
More [12]aneN3-derived compounds and their metal complexes
1
solid. Yield: 1.88 g, 92%. H NMR (400 MHz, CDCl3, ppm): d
3.38 (s, 2H), 3.33–3.25 (m, 8H), 2.54–2.51 (t, J = 12.0 Hz, 4H),
2.16 (s, 1H), 1.88–1.77 (m, 6H), 1.45 (s, 18H). 13C NMR (101
MHz, CDCl3, ppm): d 156.47, 79.41, 78.06, 73.13, 50.11, 46.20,
44.36, 39.95, 28.69, 26.98, 26.07. HR-MS (m/z) found (calcd) for
C22H39N3NaO4 (M + Na)+: 432.2860 (432.2838).
(2) Preparation of compound 11(a, b, c, d, e). General method:
Compound 10 (1.0 mmol) and NaN3 (0.33 g, 3.0 mmol) were
stirred in THF/H2O (v/v = 5 : 1) for 2 h. The mixture was extracted
with ethyl acetate; the organic layers were combined, washed
three times with brine, and subsequently dried with anhydrous
Na2SO4. After the solvent was removed under reduced pressure,
the obtained pale yellow liquid was further dried under vacuum.
11a):21 Yield: 0.17 g, 91%. 1H NMR (400 MHz, CDCl3, ppm):
d 7.39 (m, 4H), 4.44 (s, 4H).13C NMR (101 MHz, CDCl3, ppm): d
133.9, 130.2, 129.0, 52.3. ESI-MS (m/z) found (calcd) for C8H8N6
(M): [M + 1]+, 190.17 (189.20).
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11b):21 Yield: 0.165 g, 88%. 1H NMR (400 MHz, CDCl3, ppm):
d, 7.42–7.38 (t, J = 8.0 Hz, 1H), 7.30 (s, 1H), 7.28 (s, 2H), 4.35
(s, 4H).13C NMR (101 MHz, CDCl3, ppm): d 134.9, 128.1, 126.8,
126.6, 52.2. ESI-MS (m/z) found (calcd) for C8H8N6 (M): [M +
1]+, 190.29 (189.20).
acetate. The organic layers were washed once with brine, dried
over Na2SO4, and evaporated under reduced pressure. The crude
products were purified by flash chromatography on silica gel with
CH2Cl2/MeOH to yield the Boc-protected compounds. Subse-
quently, the respective Boc-protected compound was dissolved in
CH3OH (40 mL). Acetyl chloride (15 mL) was added dropwisely
into the solution and stirred at room temperature for 2 h. After the
solvent was removed under reduced pressure, the resultant white
precipitates were collected, washed with ether, and dried under
vacuum.
11c):21 Yield: 0.18 g, 96%. 1H NMR (400 MHz, CDCl3, ppm):
d 7.35 (s, 4H), 4.36 (s, 4H). 13C NMR (101 MHz, CDCl3, ppm) d
133.9, 130.2, 129.0, 52.3. ESI-MS (m/z) found (calcd) for C8H8N6
(M): [M + 1]+, 190.11 (189.20).
1
11d): Yield: 0.22 g, 96%. H NMR (400 MHz, CDCl3, ppm):
d 8.16 (s, 2H), 7.65 (s, 1H), 4.55 (s, 4H). 13C NMR (101 MHz,
CDCl3, ppm): d 148.6, 138.5, 132.9, 122.3, 53.5. ESI-MS (m/z)
found (calcd) for C8H7N7O2 (M): [M + 1]+, 234.10 (234.19).
11e): Yield: 0.29 g, 98%. 1H NMR (400 MHz, CDCl3, ppm): d
6.95 (s, 2H), 4.50 (s, 4H), 3.84 (s, 3H). 13C NMR (101 MHz, CDCl3,
ppm): d 156.7, 134.7, 112.8, 112.2, 53.3, 52.6. ESI-MS (m/z) found
(calcd) for C8H9BrN6O (M): [M + 1]+, 298.14 (298.12).
2·4HCl): 0.46 g, Yield: 94%. 1H NMR (400 MHz, D2O, ppm): d
7.98 (s, 1H), 7.31–7.23 (m, 5H), 5.51 (s, 2H), 3.97 (s, 2H), 3.21–3.13
(m, 8H), 3.87 (s, 4H), 2.16–2.13 (t, J = 11.2 Hz, 2H), 1.97–1.94
(t, J = 12 Hz, 4H). 13C NMR (101 MHz, D2O, ppm): d 134.50,
129.13, 128.84, 128.13, 126.53, 54.09, 48.77, 47.45, 43.08, 41.75,
19.83, 18.93. IR (KBr, cm-1): 3423 (s), 2951 (s), 2754 (s), 2635 (s),
1589 (s), 1456 (s), 1364 (w), 1231 (w), 1127 (w), 1055 (m), 1006
(w), 913 (w), 855 (w), 725 (m). Elemental analysis calcd (%) for
C19H34Cl4N6: C, 46.73; H, 7.02; N, 17.21. Found (%): C, 46.33; H,
7.10; N, 17.12. HR-MS (m/z) found (calcd.) for C19H31N6 (M): [M
+ H]+, 343.2604 (343.2610).
3a·8HCl): 0.41 g, Yield: 92%. 1H NMR (400 MHz, D2O, ppm):
d 8.19 (s, 2H), 7.34 (m, 4H), 5.62 (s, 4H), 4.31 (s, 4H), 3.31–3.21
(m, 24H), 2.24–2.14 (m, 12H).13C NMR (101 MHz, D2O, ppm):
d 137.72, 135.02, 128.77, 127.17, 53.60, 48.77, 47.99, 42.30, 41.17,
19.88, 18.32. IR (KBr, cm-1): 3441 (s), 2951 (s), 2642 (s), 2407 (s),
1590 (s), 1517 (w), 1482 (s), 1458 (s), 1440 (s), 1365 (m), 1213 (s),
1132 (m), 1055 (m), 1005 (s), 917 (m), 855 (w), 794 (w), 746 (m),
522 (m). Elemental analysis calcd (%) for C32H62Cl8N12: C, 42.77;
H, 6.95; N, 18.71. Found (%): C, 42.64; H, 7.13; N, 18.61. HR-
MS (m/z) found (calcd.) for C32H54N12 (M): [M + H]+, 607.4674
(607.4673).
3b·8HCl): 0.42 g, Yield: 93%. 1H NMR (400 MHz, D2O, ppm):
d 8.17 (s, 2H), 7.43–7.39 (t, J = 16 Hz, 1H), 7.31–7.29 (d, J = 6.4
Hz 3H), 5.62 (s, 4H), 4.28 (s, 4H), 3.33–3.28 (m, 16H), 3.17 (s, 8H),
2.25–2.23 (t, J = 5.6 Hz, 4H), 2.15–2.12 (t, J = 6.0 Hz, 8H). 13C
NMR (101 MHz, D2O, ppm): d 136.90, 135.37, 129.89, 128.46,
127.63, 53.73, 48.54, 47.55, 42.10, 41.08, 19.97, 18.01. IR (KBr,
cm-1): 3422 (s), 2959 (s), 2757 (s), 2641 (s), 1616 (m), 1588 (m),
1479 (m), 1457 (m), 1434 (m), 1364 (w), 1233 (w), 1128 (w), 1057
(m), 1006 (w), 913 (w), 747 (m). Elemental analysis calcd (%) for
C32H62Cl8N12: C, 42.77; H, 6.95; N, 18.71. Found (%): C, 42.66; H,
7.21; N, 18.68. HR-MS (m/z) found (calcd.) for C32H54N12 (M):
[M + H]+, 607.4668 (607.4673).
(3) Preparation of compound 12(b, d, f). General method:
compound 10 (1 mmol) and tricyclic orthodiamide 13 (0.36 g,
3 mmol) in 60 mL freshly distilled CHCl3 was refluxed for 2 h.
After cooling to room temperature, the white precipitates were
filtered, washed with ether (2 ¥ 10 mL), and then dried to afford
the corresponding compounds.
1
12b): Yield: 0.57 g, 92%. H NMR (400 MHz, D2O, ppm): d
7.97 (s, 2H), 7.32 (t, J = 7.4 Hz, 1H), 7.16–7.18 (d, J = 7.4 Hz, 2H),
7.04 (s, 1H), 3.74 (s, 4H), 3.59–3.66 (t, J = 13.0 Hz, 4H), 3.17–3.37
(m, 12H), 2.89–2.93 (d, J = 13.5 Hz, 4H), 2.33–2.40 (t, J = 12.8
Hz, 4H), 2.01–2.12 (m, 8H), 1.20–1.24 (d, J = 14.4 Hz, 4H), 13C
NMR (400 MHz, D2O, ppm): d 156.42, 135.23, 133.22, 129.99,
128.30, 55.33, 54.34, 53.34, 42.26, 22.46, 19.25. IR (KBr, cm-1):
2943 (m), 2839 (m), 1679 (s), 1503 (w), 1459 (m), 1426 (m), 1371
(m), 1332 (m), 1216 (m), 1116 (m), 1070 (m), 1044 (m), 736 (m),
676 (m), 532 (m), 459 (m), 436 (m). ESI-MS (m/z) found (calcd)
for C28H46Br2N6 (M): [M - Br]+, 545.4(545.3), 547.3(547.3).
1
12d): Yield: 0.64 g, 96%. H NMR (400 MHz, D2O, ppm): d
7.92 (s, 2H), 7.89 (s, 2H), 7.32 (s, 1H), 3.75 (s, 4H), 3.57–3.50 (t,
J = 28 Hz, 4H), 3.28–3.07 (m, 12H), 2.83–2.80 (d, J = 12 Hz, 4H),
2.30–2.37 (t, J = 26.4 Hz, 4H), 2.06–1.85 (m, 8H), 1.17–1.13 (d,
J = 14.6 Hz, 4H). 13C NMR (101 MHz, D2O, ppm): d 156.30,
147.80, 139.02, 137.27, 124.24, 55.21, 54.52, 53.05, 42.18, 22.69,
19.15. ESI-MS (m/z) found (calcd) for C28H45Br2N7O2 (M): [M -
Br]+, 591.33(591.61).
1
12f): Yield: 0.62 g, 90%. H NMR (400 MHz, D2O, ppm): d
8.03 (s, 2H), 7.77 (s, 2H), 7.33 (s, 1H), 3.86 (s, 4H), 3.75 (s, 3H),
3.66–3.62 (d, J = 12.3 Hz, 4H), 3.39–3.33 (d, J = 12 Hz, 4H), 3.26–
3.18 (m, 8H), 2.94–2.91 (d, J = 12 Hz, 4H), 2.50–2.44 (t, J = 16
Hz, 4H), 2.17–2.10 (m, 4H), 2.00–1.88 (m, 4H), 1.29–1.25 (d, J =
16 Hz, 4H). 13C NMR (101 MHz, D2O, ppm): d 168.80, 156.34,
137.77, 137.71, 130.57, 129.63, 55.26, 54.30, 52.92, 47.34, 42.32,
23.19, 19.32. ESI-MS (m/z) found (calcd) for C30H48Br2N6O2 (M):
[M - Br]+, 604.00(604.64).
3c·8HCl): 0.41 g, Yield: 91%. 1H NMR (400 MHz, D2O, ppm):
d 8.10 (s, 2H), 7.60 (s, 2H), 7.32 (s, 2H), 5.69 (s, 4H), 4.37 (s,
4H), 3.28–3.23 (m, 24H), 2.15 (m, 12H).13C NMR (101 MHz,
D2O, ppm): d 136.46, 132.50, 130.76, 130.03, 127.61, 51.39, 48.48,
47.28, 41.89, 40.90, 19.94, 17.83. IR (KBr, cm-1): 3442 (s), 2960
(s), 2760 (s), 2635 (s), 2413 (s), 1616 (s), 1483 (s), 1458 (s), 1214 (s),
1053 (m), 1005 (s), 919 (m), 747 (m), 523 (m). Elemental analysis
calcd (%) for C32H62Cl8N12: C, 42.77; H, 6.95; N, 18.71. Found
(%): C, 42.71; H, 6.99; N, 18.65. HR-MS (m/z) found (calcd.) for
C32H54N12 (M): [M + H]+, 607.4674 (607.4673).
3d·8HCl): 0.39 g, Yield: 82%. 1H NMR (400 MHz, D2O, ppm):
d 8.19 (s, 2H), 8.07 (s, 2H), 7.07 (s, 1H), 5.76 (s, 4H), 4.18 (s, 4H),
3.32–3.27 (m, 17H), 3.07 (m, 7H), 2.27–2.24 (t, J = 12 Hz, 4H),
2.11–2.08 (t, J = 12 Hz, 8H). 13C NMR (101 MHz, D2O, ppm): d
148.55, 137.37, 133.96, 126.66, 122.97, 122.55, 57.36, 52.80, 49.53,
(4) Preparation of compound 2 and 3(a, b, c, d, e). General
method: the respective azide compound (1.0 mmol for 2, 0.5 mmol
for 3) and compound 8 (0.41 g, 1 mmol) were added into
THF/H2O (v/v = 2 : 1), CuSO4·5H2O (0.013 g, 0.05 mmol) and
sodium vitamin C (0.019 g, 0.1 mmol) were also added into
the solution as catalyst. The mixture was stirred overnight at
room temperature, saturated with NaCl, and extracted with ethyl
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42.13, 19.88, 19.44, 16.73. IR (KBr, cm-1): 3431 (s), 2956 (m), 2928
(m), 2759 (s), 2105 (m), 1619 (m), 1590 (m), 1537 (s), 1456 (s), 1351
(s), 1232 (w), 1122 (w), 1055 (m), 1007 (w), 916 (w), 787 (w), 738
(m). Elemental analysis calcd (%) for C32H61Cl8N12O2: C, 40.73;
H, 6.52; N, 19.30. Found (%): C, 40.68; H, 6.40; N, 19.10. HR-MS
(m/z) found (calcd.) for C32H53N12O2 (M): [M + H]+, 652.4532
(652.4523).
2960 (s), 2758 (br, s), 1716 (s), 1593 (s), 1458 (s), 1369 (m), 1308
(s), 1227 (s), 1145 (m), 1085 (m), 1071 (m), 1023 (m), 884 (w),
818 (w), 746 (w), 702 (w), 567 (m), 513 (m). Elemental analysis
calcd (%) for C27H53Cl5N6O2: C, 48.33; H, 7.96; N, 12.52. Found
(%): C, 48.17; H, 8.12; N, 12.31. HR-MS (m/z): found (calcd.) for
C27H49N6O2 (M): [M + H] 489.3923 (489.3917).
3e·8HCl): 0.43 g, Yield: 85%. 1H NMR (400 MHz, D2O, ppm):
d 8.19 (s, 2H), 6.98 (s, 2H), 5.70 (s, 4H), 4.31 (s, 4H), 3.77 (s, 3H),
3.34–3.29 (m, 17H), 3.21 (s, 7H), 2.26–2.23 (t, J = 12 Hz, 4H),
2.16–2.13 (t, J = 12 Hz, 8H). 13C NMR (101 MHz, D2O, ppm): d
158.82, 138.27, 135.76, 127.37, 117.61, 115.89, 55.77, 54.64, 48.61,
47.67, 42.91, 41.63, 19.93, 18.80. IR (KBr, cm-1): 3407 (m), 3224
(m), 3084 (w), 2910 (w), 2881 (w), 1531 (s), 1465 (m), 1419 (s), 1392
(s), 1342 (s), 1197 (m), 1150 (m), 1096 (m), 1063 (m), 1038 (m), 928
(w), 868 (m), 784 (m), 746 (m), 695 (w), 651 (m). Elemental analysis
calcd (%) for C33H63BrCl8N12O: C, 39.34; H, 6.30; N, 16.68. Found
(%): C, 39.45; H, 6.46; N, 16.28. HR-MS (m/z) found (calcd.) for
C33H55BrN12O (M): [M + H]+, 715.3914 (715.3883).
(6) Preparation of compound 5b. Under an argon atmosphere,
a solution of 12b (1.0 g, 1.6 mmol) and NaBH4 (0.59 g, 10 eq) in
50 mL of ethanol was stirred in an ice bath overnight. The reaction
mixture was quenched with 6 M HCl, and then neutralized to
alkaline (pH ≥ 12) with 50% aqueous NaOH. The mixture was
extracted with CH2Cl2 (30 mL ¥ 4), and the organic layers were
combined together, dried over anhydrous Na2SO4. The solvent was
then removed in vacuo to afford the free ligand as a pale yellow
viscous oil. Yield: 0.49 g, 65%. 1H NMR (400 MHz, CDCl3, ppm):
d 7.17–7.20 (m, 4H), 3.38 (s, 4H), 2.70 (m, 4H), 2.55 (m, 4H),
2.43–2.45 (m, 8H), 2.34–2.36 (m, 8H), 2.08 (s, 6H), 1.58–1.61 (m,
12H). 13C NMR(101 MHz, CDCl3, ppm): d 139.43, 129.63, 128.32,
127.30, 58.31, 57.78, 54.23, 54.07, 50.09, 49.26, 48.39, 40.87, 24.61,
24.21, 18.44. IR (KBr, cm-1): 3321 (m), 2928 (s), 2801 (s), 1712 (s),
1668 (s), 1452 (s), 1431 (s), 1398 (s), 1363 (s), 1221 (m), 1204 (m),
1176 (m), 1148 (m), 1068 (m), 791 (w), 741 (m), 700 (w), 530 (w).
Elemental analysis calcd (%) for C28H52N6: C, 71.14; H, 11.09; N,
17.78. Found (%): C, 71.08; H, 10.93; N, 17.62. HR-MS (m/z)
found (calcd.) for C28H52N6 (M): [M + 1]+, 473.4348 (473.4332).
(5) Preparation of compound 4(b, d, g). General method for
preparing compounds 4(b, d): compounds 12(b or d) (0.5 mmol)
in 20 mL of 3 M HCl was refluxed for 12 h, and then neutralized
with NaOH solution of water (3.0 M) until alkaline (pH ≥ 12).
The mixture was then extracted with CH2Cl2 (30 mL ¥ 4). After
the combined organic layers were dried over anhydrous Na2SO4,
the solvent was removed by rotary evaporation under reduced
pressure to afford the free ligand as a pale yellow viscous oil.
Method for compound 4g: compound 12g (0.5 mmol) in 20 mL
of 3 M HCl was refluxed for 12 h, and then the solvent was removed
in rotary under reduced pressure to afford the ligand as a white
solid, which was washed twice with ethanol.
(7) Preparation of compound 6b. Under an argon atmosphere,
a mixture of 12b (1.0 g, 1.6 mmol), formic acid (10 mL), and
formaldehyde solution (10 mL, 37–40%) was refluxed overnight.
After the removal of solvent in vacuo, the residue was quenched
with 6 M HCl, and then neutralized to alkaline (pH ≥ 12) with
50% aqueous NaOH. The solution was extracted with CH2Cl2
(30 mL ¥ 4), the organic layers were combined, dried over
anhydrous Na2SO4, and the solvent was then removed under
vacuum to obtain the free ligand as pale yellow viscous oil. Yield:
1
4b): Yield: 0.16 g, 71%. H NMR (400 MHz, CDCl3, ppm): d
7.28–7.30 (m, 3H), 7.23 (s, 1H), 3.50 (s, 4H), 2.85 (m, 8H), 2.71
(m, 8H), 2.53 (m, 8H), 1.69–1.74 (m, 12H).13C NMR (101 MHz,
CDCl3, ppm): 138.93, 129.85, 127.94, 127.46, 57.35, 52.51, 49.48,
46.85, 26.45, 26.02. IR (KBr, cm-1): 3317 (m), 2928 (s), 2801 (s),
1667 (s), 1452 (s), 1431 (s), 1398 (m), 1374 (m), 1265 (m), 1246 (m),
1204 (m), 1176 (m), 1148 (m), 1095 (m), 1068 (m), 800 (w), 734(m),
700 (w). Elemental analysis calcd (%) for C26H48N6: C, 70.22; H,
10.88; N, 18.90. Found (%): C, 69.98; H, 10.97; N, 18.65. HR-
MS (m/z) found (calcd.) for C26H48N6 (M): [M + H]+, 445.3952
(445.3941).
1
0.48 g, 60%. H NMR (400 MHz, CDCl3, ppm): d 7.16–7.25
(m, 4H), 3.46 (s, 4H), 2.42–2.47 (m, 24H), 2.20 (s, 12H), 1.59–
1.61 (m, 12H). 13C NMR (101 MHz, CDCl3, ppm): d 139.95,
129.52, 127.79, 127.26, 62.37, 59.13, 57.96, 55.83, 52.25, 49.27,
45.49, 42.99, 42.23, 25.61, 25.27, 22.08. FT-IR (KBr, cm-1): 2934
(s), 2926 (s), 2834 (s), 2788 (s), 1677 (m), 1607 (w), 1589 (w), 1458
(s), 1366 (m), 1344 (m), 1241 (m), 1120 (m), 1102 (m), 1081 (m),
1060 (m), 963 (m), 861 (w), 803 (w), 741 (m), 698 (w), 568 (w).
Elemental analysis calcd (%) for C30H56N6: C, 71.95; H, 11.27; N,
16.78. Found (%): C, 71.75; H, 11.64; N, 16.42. HR-MS (m/z)
found (calcd.) for C30H56N6 (M): [M + 1]+, 501.4651 (501.4645).
1
4d): Yield: 0.21 g, 84%. H NMR (400 MHz, CDCl3, ppm): d
8.25 (s, 2H), 7.61 (s, 1H), 3.57 (s, 4H), 2.89–2.86 (t, J = 10.0 Hz,
8H), 2.70–2.67 (t, J = 10.2 Hz, 8H), 2.56–2.53 (t, J = 10.9 Hz, 8H),
1.73–1.68 (m, 12H). 13C NMR (101 MHz, CDCl3, ppm): d 148.64,
141.98, 135.19, 122.10, 56.95, 52.84, 50.13, 47.17, 26.58, 26.19. IR
(KBr, cm-1): 3421 (s), 2951 (s), 2753 (br, s), 1653 (s), 1588 (s), 1537
(s), 1459 (s), 1353 (s), 1177 (w), 1108 (m), 1069 (m), 1006 (m),
887 (m), 784 (m), 746 (s), 694 (m), 556 (br, m). Elemental analysis
calcd (%) for C26H47N7O2: C, 63.77; H, 9.67; N, 20.02. Found
(%): C, 63.57; H, 9.86; N, 19.69. HR-MS (m/z) found (calcd.) for
C26H48N7O2 (M): [M + H]+, 490.3884 (490.3869).
4. General methods for kinetic measurements
+
The CH3OH2 concentrations were determined using a combina-
tion glass electrode (Mettler Toledo FE20) calibrated with Fisher
Certified standard aqueous buffers (pH 4.00, 7.00 and 10.00) as
ss pH
described in the literature.45 The
values in methanol were
4g·5HCl): 0.31 g, Yield: 91%. 1H NMR (400 MHz, D2O, ppm):
d 8.21 (s, 2H), 7.89 (s, 1H), 4.42 (s, 4H), 3.34–3.32 (m, 24H),
2.25–2.178 (m, 12H). 13C NMR (101 MHz, D2O, ppm): d 167.26,
137.96, 133.07, 132.11, 131.54, 62.90, 57.13, 47.51, 44.63, 42.42,
41.25, 40.98, 20.26, 19.05, 18.17, 13.47. IR (KBr, cm-1): 3420 (s),
determined by subtracting a correction constant of -2.24 from
the readings obtained from the electrode, while the autoprotolysis
constant was taken to be 10-16.77 M2. The values for the kinetic
experiments were simply measured from the solution of the
complexes after reactions.
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The rates of catalyzed cleavage of HPNPP (0.04 mM) were
monitored spectrophotometrically using a UV-vis spectropho-
tometer thermostatted at 25.0
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determined from the rate of appearance of p-nitrophenol at
322 nm. All kinetic experiments were performed with cata-
lyst formed in situ through the sequential addition of stock
solutions (typically 20 mM) of sodium methoxide, ligand
and Zn(OTf)2 or Cu(OTf)2 to anhydrous methanol such that
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Acknowledgements
The authors gratefully acknowledge the financial assistance from
the Fundamental Research Funds for the Central Universities
(2009SC-1), Beijing Municipal Commission of Education; Spe-
cialized Research Fund for the Doctoral Program of Higher
Education (273911), Program for New Century Excellent Tal-
ents at Universities (15770703), the Ministry of Education of
China; and the Nature Science Foundation of China (202028).
The authors also thank Prof. R. S. Brown, Prof. H. K. Fun,
Prof. B. Coppola, and Dr Rui-Bing Wang for their helpful
discussions.
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used; if the electrode is calibrated in water and the ‘pH’ of the neat
ss pH
buffered methanol solution then measured, the term
is used; and
if the electrode is calibrated in the same solvent and the ‘pH’ reading is
ws pH
made, then the term
of methanol is 10-16.77, neutral
is used. Since the autoprotolysis constant
ss pH
is 8.4.
6796 | Org. Biomol. Chem., 2011, 9, 6788–6796
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The Royal Society of Chemistry 2011
©