Phosphate diesters and DNA hydrolysis by dinuclear Zn(II) complexes featuring a disulfide bridge and H-bond donors

Article abstract of DOI:10.1016/j.tet.2010.01.050

The dinuclear ligand 1 based on the bis-(2-amino-pyridinyl-6-methyl)amine (BAPA) metal binding unit and featuring a two-atom disulfide bridge was synthesized and studied as hydrolytic catalysts for phosphate diesters. The Zn(II) complexes of BAPA are know

Full text of DOI:10.1016/j.tet.2010.01.050

Tetrahedron 66 (2010) 2189–2195
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Tetrahedron
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Phosphate diesters and DNA hydrolysis by dinuclear Zn(II) complexes featuring
a disulfide bridge and H-bond donors
Valentina Lombardo , Renato Bonomi , Claudia Sissi ,*, Fabrizio Mancin
a
b
a
b,
*
a
Dipartimento di Scienze Farmaceutiche, Universit a` di Padova, via Marzolo 5, I-35131 Padova, Italy
Dipartimento di Scienze Chimiche, Universit a` di Padova, via Marzolo 1, I-35131 Padova, Italy
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 October 2009
Received in revised form
The dinuclear ligand 1 based on the bis-(2-amino-pyridinyl-6-methyl)amine (BAPA) metal binding unit
and featuring a two-atom disulfide bridge was synthesized and studied as hydrolytic catalysts for
phosphate diesters. The Zn(II) complexes of BAPA are known to elicit the cooperation between the metal
ion and the hydrogen-bond donating amino groups to greatly increase the rate of cleavage of phosphate
2
December 2009
Accepted 11 January 2010
Available online 18 January 2010
2
diesters. The reactivity of the dinuclear complex 1$Zn(II) toward bis-p-nitrophenyl phosphate and
plasmid DNA was investigated and compared with that of reference complexes devoid of the disulfide
bridge or of the hydrogen-bond donating amino groups. The dimetallic Zn(II) complex produces re-
markable accelerations of the rate of cleavage of both the substrates accompanied by significant dif-
ferences. In the case of BNP, the presence of the disulfide bridge does not lead to the improvement of the
cooperative action of the two metal ions expected as the result of better preorganization. On the other
2
hand, in the case of DNA the complex 1$Zn(II) is much more reactive that the corresponding reference
devoid of the disulfide bridge. Hence, different requisites must be fulfilled by a good catalyst for the
cleavage of the two substrates. Moreover, binding studies with DNA indicated that the presence of two
metal ions in the complex or of the pyridine amino groups, but not of the disulfide bridge, results into an
enhanced affinity of the complexes toward this substrate.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
these systems, the metal ions can play several roles, mainly acting
as a Lewis acid: they provide the nucleophile for the reaction by
DNA phosphodiester bonds are hardly hydrolyzed under neutral
conditions and still represent a challenge for chemists. However,
facilitating the deprotonation of a coordinated water molecule,
activate the phosphate group toward nucleophilic attack, stabilize
the developing negative charge on the transition state, and, in
principle, may help the departure of the leaving group.
1
enzymes can reduce the half-life time of this reaction from 30 mil-
17
lion years to some milliseconds, bringing about a 1ꢀ10 -fold rate
acceleration.2 Such astonishing reactivity is the result of a perfect
organization of metal ions and organic groups in the active sites of
the enzymes.3 Research on artificial hydrolytic agents for DNA
cleavage has attracted a considerable effort over the last two decades
both for the intellectual challenge represented to design a catalyst
capable to rival enzymes’ reactivity but also for the important po-
tential applications of such systems, which span from detoxification
of pesticides and chemical weapons, to the realization of artificial
restriction enzymes for molecular biology and of anti-DNA drugs.1
Synthetic catalysts with good activity have been obtained by
using complexes of transition metal ions with high Lewis acidity
Still, the reactivity of enzymes is unmatched. First examples of
hydrolytic agents were monometallic complexes of transition metal
1a
ions with high Lewis acidity. In these systems, the role of the li-
gand is more or less to keep the hydroxide form of the complex in
solution. Several studies have been devoted to elucidate the effect
of the ligand structure in modulating and possibly enhancing the
5
reactivity of the metal center but it is quite clear that the desired
acceleration cannot be reached with simple mononuclear com-
6
plexes. The following step to increase the reactivity of artificial
hydrolytic agents has been the use of bimetallic complexes, as oc-
curs in several phosphatases and nucleases. In this case, the ligand
must keep the two metal centers at the right distance to obtain the
1
a,e
such as Ce(IV), Co(III), Cu(II), and Fe(III),
up to Komiyama’s
7
ARCUT system, based on the Ce(IV)/EDTA complex and pseudo-
complementary PNA strands, which recently allowed the first re-
maximum cooperation. Model studies performed with kinetically
inert Co(III) complexes have demonstrated that accelerations up to
4
12
7
ports of DNA manipulation with man-made restriction agents. In
10 -fold are achievable with this strategy. However, when com-
plexes of labile metal ions are used, complications arise from the
formation of
m-hydroxo bridges between the two metal centers,
*
Corresponding authors. Tel.: þ39 049 8275666; fax: þ39 049 8275239; e-mail
addresses: claudia.sissi@unipd.it (C. Sissi), fabrizio.mancin@unipd.it (F. Mancin).
which are poorly reactive and saturate the free binding sites on the
0
040-4020/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.01.050

2190
V. Lombardo et al. / Tetrahedron 66 (2010) 2189–2195
metal disfavoring the interaction with the substrate.8–10 This is
particularly true in the case of Zn(II), which is one of the metal ions
preferred by hydrolytic enzymes. Some Zn(II) features, such as low
toxicity, bioavailability, and absence of relevant redox chemistry,
make it a very attractive candidate for the development of artificial
The disulfide group, in fact, forms spontaneously when thiol de-
rivatives are exposed to air in basic conditions. This reactivity,
usually considered as a drawback in handling of thiols, reduces the
problem of preparing binucleating ligands to the simpler synthesis
of thiol containing mononuclear ligands (Chart 2).
1
c
agents. However, bimetallic Zn(II) complexes have so far shown
poor performances both with model substrates and DNA.
NH
2
H N
2
Another approach, again inspired by Nature, is the use of organic
groups appropriately located in the structure of the ligand, as co-fac-
N
N
N
N
N
N
N
N
11
N
S
S
N
N
S
S
N
tors to increase the reactivity of the metal complex. Such groups can
act as general bases, general acids, nucleophiles, or hydrogen-bond
donors similar to the aminoacid residues present in the active site of
nuclease enzymes. Several examples demonstrate that high activity
can be achieved by such strategy in the cleavage of model substrates
and RNA oligonucleotides, but reactivity on DNA is still not reported.
Recently, we12 and the groups of Williams and Mareque-Rivas
reported that Zn(II) complexes based on the bis-(2-amino-
pyridinyl-6-methyl)amine unit (BAPA, Chart 1), are very active in
promoting the cleavage of activated phosphate diesters, such as
bis-p-nitrophenyl phosphate (BNP, Chart 1) and hydroxypropyl-p-
nitrophenyl phosphate (HPNP, Chart 1). In the ultimate example
proposed by Williams and Mareque-Rivas, the bimetallic complex
A (Chart 1) produces million-fold accelerations of the cleavage of
NH
2
H N
2
N
N
N
1
2
NH
2
NH
2
S
S
13
NH2
H
N
5
2
N
N
N
N
N
N
N
N
N
N
N
N
NH2
H N
2
3
4
Chart 2. Ligands studied in this work.
13b
both HPNP and RNA dinucleotides at pH 7. The source of this high
reactivity is the ability of the complex to activate the substrate
toward the nucleophilic attack using both double metal activation
and four hydrogen bonds. Transposition of such high reactivity to
DNA would be highly desirable. With this in mind, we explored, and
here report, a new way to assemble binuclear Zn(II) complexes
containing the BAPA units and investigated their reactivity toward
model substrate BNP and plasmid DNA.
With this in mind, compound 1 (Chart 1) was designed as binu-
clear ligand based on the BAPA structure and containing the disulfide
group as two-atom bridging unit. Synthesis of 1 was performed
12d
(Scheme 1) from previously reported derivative I
through tosy-
lation of the hydroxyl group and substitution of the tosylate with
thioacetate. Removal of the protecting acetyl form II in basic con-
ditions provides 1. Ligand 2, devoid of the pyridine amino groups,
O N
NO
2
O
2
N
12d
N
H
2
O
P
O
O P O
O
was prepared in a similar way from previously described III.
Li-
N
N
O
O
gands 3 and 4, in which the disulfide bridging group is not present,
were prepared (Scheme 2) by reaction of 1,8-diaminooctane, re-
spectively, with compound V and picolyl chloride. Finally, the model
mononuclear ligand 5 was prepared (Scheme 1) by deprotection of II
in the presence of a large excess of propyl-1-thiol.
O
NH2
NH2
OH
BNP
HPNP
BAPA
H
H
H
H
H
H
H
N
N
H
N
N
N
N
N
2+ 2+
Zn
N
N
Zn
Zn2+
N
2+
N
Zn
O
A
N
N
N
N
N
N
a,b
N
N
N
N
N
N
N
B
Y
Y
Y
Y
OH
SAc
Chart 1. Activated phosphate esters and literature catalysts.
I, Y = NHAc
II, Y = H
III, Y = NHAc (51%)
IV, Y = H (2%)
2
2
. Results
c, d
.1. Ligand design and synthesis
e
Spatial organization of the bimetallic structure has been shown
to be crucial in determining the hydrolytic activity and the best
results have been reported with rigid structures that employ
N
N
bridging groups in the ligand structure to keep the metal ions in
N
a precise arrangement.1 Among these, one-atom bridging units
Y
Y
Y
Y
Y
Y
S
N
N
N
such as phenolate and alkoxide (i.e., complex A, Chart 1) are very
popular and found in many of the best performing RNA cleaving
S
N
S
S
N
systems.1
3,14
However, their efficiency in promoting the hydrolysis
of DNA and its models is apparently lower, at least when Zn(II) is
used as metal center. The studies of Meyer on di-zinc(II) complexes
based on the pyrazolate linking unit (Chart 1) indicate that the
greater intermetallic distance, allowed by this two-atom bridging
N
5
7
, Y = NH2 (12%)
, Y = H (82%)
1, Y = NH2 (43%)
2, Y = H (76%)
group, disfavors the formation
increased reactivity of the hydrolytic complex toward BNP.
On these bases, we found the disulfide group quite appealing as
m-hydroxo bridges leading to an
10
Scheme 1. Synthesis of ligands 1, 2, 5 and 7: (a) tosyl chloride, triethylamine, CH
4 h; (b) potassium thioacetate, acetone, reflux, 2 days; (c) HCl, EtOH/H O 1:1, rt, 16 h; (d)
propanethiol, NaOH, EtOH/H O 1:1, reflux, 16 h; (e) NaOH, EtOH/H O 1:1, reflux, 16 h.
2 2
Cl , rt,
2
2
15
a new bridging unit for the preparation of bimetallic complexes.
2
2

V. Lombardo et al. / Tetrahedron 66 (2010) 2189–2195
2191
NH2
0.12
Y
Y
Y
X
N
N
N
N
Y
N
a,b
4
x
N
N
0.08
Y
V, Y = NHAc, X = Br
VI, Y = H, X = Cl
H N
2
3
, Y = NH (37%)
2
0.04
4
, Y = H (82%)
Scheme 2. Synthesis of ligands 3 and 4: (a) For 3, K
NaOH, CTACl, water, rt, 20 h; (b) HCl, EtOH/H
2
CO
3
, CH
3
CN, reflux, 16 h; for 4:
2
O 1:1, rt, 16 h (only in the case of 3).
0.00
7
8
9
10
11
All the above ligands are sparingly soluble in water in their
neutral form and this prevented the investigation of the Zn(II)
binding through potentiometric titrations.
pH
20
Figure 2. pH dependence of the apparent second-order rate constants (k ) for the
reaction between BNP and Zn(II) complexes of ligands 3 (B), 1 (C), 5 (-), 4 (,) at
However, in the case of ligand 5, we could study the forma-
1
ꢂ4
ꢂ4
ꢂ2
ꢁ
tion of the Zn(II) complex through H NMR investigation (Fig. 1).
40 C ([ligand]¼0.5–2.0ꢀ10 M, [BNP]¼2.0ꢀ10 M, [buffer]¼5.0ꢀ10 M). The lines
12c
As usual for BAPA-based ligands,
formation of the Zn(II)
represent the best fit of the experimental data as described in the text.
complex causes significant changes on the resonances of the
ligand protons: pyridine H2 is downfield shifted while the res-
onances of H1 and H3 melt into a single signal. The methylene
protons H4 of the pyridinylmethyl moieties experience the most
important variation: their signal is downfield shifted and splits
into an AB system, reasonably, as a consequence of the loss of
conformational flexibility of this portion of the complex. Much
more significant are the changes of the signal relative to the
disulfide arm, which shows a significant modification of the
shape and, to a minor extent, of the chemical shift of the signals
relative to the methylene groups. Hence, also the disulphide
group binds to the Zn(II) ion and 5 acts, at least at neutral pH, as
a tetradentate ligand.
The reactivity of all the complexes increases with the pH up
to a maximum around pH 8–9 followed by a decrease, above pH
10. This behavior is diagnostic of the deprotonation of two acidic
functions of the complexes, the first deprotonation event leading
to a reactivity increase and the second to the opposite effect.
Fitting of the pH profiles with a kinetic model involving two
deprotonation equilibria (see Experimental Part) allowed the
a
determination of the pK values of the reacting species and of
the second-order rate constants for the reactive mono-
deprotonated metal complex. Comparison of the second-order
rate constants of Table 1 reveals the reactivity order of the
2 2 2
complexes: 3$Zn(II) z1$Zn(II) >5$Zn(II)>4$Zn(II) .
3
4
2
1
N
N
5
N
6
7
NH
NH
S
S
Table 1
Metal coordinated water molecule acidity constants (pK
a
) and second-order rate
8
9
constants (k ) for the reaction of BNP with the Zn(II) complexes of ligands 1–5 in
2
ꢁ
1
0
water at 40
Complex
C
pK1
pK2
k
ꢂ1 ꢂ1
s
a
a
2
, M
1
3
4
5
$Zn(II)
$Zn(II)
$Zn(II)
$Zn(II)
2
2
2
7.7
6.5
8.7
7.6
10.9
11.0
10.7
0.09
0.11
0.005
0.026
>11
ppm 7.50
7.00
6.50
4.00 3.50 3.00
2.50 2.00 1.50
1.00
Figure 1. 1H NMR spectra of 5 (upper trace) and 5$Zn(II) (lower trace) at 1.0 mM in
MeOD at 28 C.
2þ
However, if it is considered that the amount of Zn is double
in the case of bimetallic complex, the cooperativity between the
two metal centers in 1$Zn(II) appears to be quite limited. This
ꢁ
2
picture is confirmed by a kinetic experiment performed using
a fixed concentration of 1 ligand and increasing amounts of
metal ion (Fig. 3). The rate profile describes a smooth sigmoidal
curve reaching a plateau for a ligand to metal ion ratio around 2.
The reaction rate observed after the introduction of the second
metal ion is approximately 2.9 times larger than that observed
in the presence of 1 equiv of Zn(II), indicating again that the
effect of the two complexed subunits is more than additive but
the synergic action of the two metal ions is not very efficient.
A different profile (not shown) was however observed with li-
gand 3: in this case the kinetic data describe an almost linear
curve up to 2 equiv of added metal ion then going to plateau
reactivity.
2
.2. Reactivity toward BNP
Incubation of the Zn(II) complexes of 1, 3, 4, 5 with BNP (Chart
ꢁ
1
2
) in water at 40 C results into substrate hydrolysis, while
2
$Zn(II) was found to be completely unreactive. The kinetic
studies were performed by monitoring the increase of the
p-nitrophenoxide absorbance at 400 nm, using the initial rates
method. Apparent second-order rate constants were obtained at
each pH value by the linear fit of the pseudo-first order rate
constants versus complex concentration data. The pH de-
pendences of the apparent second-order rate constants are
reported in Figure 2.

2192
V. Lombardo et al. / Tetrahedron 66 (2010) 2189–2195
Control experiments performed in the presence of radical
5
4
3
2
1
0
scavengers DMSO and isopropanol revealed that these additives
have no effect on the extent of plasmid DNA cleavage by the Zn(II)
complexes, ruling out the involvement of diffusible radical species.
Moreover, the Ellmann test confirmed that the complexes con-
taining the disulfide unit are not cleaved, in the conditions used for
the DNA cleavage experiments, to form thiol containing species,
while the presence of diethyl disulfide does not affect the reactivity
of the complexes not containing the disulfide group.
Binding of the Zn(II) complexes to DNA was investigated by fol-
lowing the modification of the circular dichroism (CD) spectra of
linear calf thymus DNA after the additions of increasing concen-
trations of metal complex. In all the cases Figure 5, a decrease of the
dichroic signal, particularly of the 275 nm band, is observed, in-
dicating that the interaction of the complexes with the DNA leads to
a destabilization of the double-helix structure. Such effects are
expected in the case of a predominant electrostatic interaction be-
tween the negatively charged DNA backbone and the positively
charged complexes, which results into a partial charge neutraliza-
tion of DNA. Unfortunately, precipitation of DNA in the presence of
high metal complex concentrations and the overlap of the DNA di-
chroic bands with the ligand-induced dichroic bands hampered the
quantitative determination of the affinity constants of the different
complexes for DNA. However, if the amplitude of signal variation is
taken as an approximate indication of the binding strengths, the
0
.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0
Zn(II) equivalents
Figure 3. Kinetic profile for the BNP cleavage as a function of the Zn(II) to 1 ratio, in
HEPES buffer 0.05 M, pH 9.0. [1]¼2.0ꢀ10 M, [BNP]¼4.0ꢀ10 M, 40 C.
ꢂ5
ꢂ4
ꢁ
Finally, saturation kinetics were obtained measuring the re-
activity of the complexes 1$Zn(II) , 3$Zn(II) , and 5$Zn(II) in the
2 2
presence of increasing amounts of BNP at pH 9.0. This behavior is in
line with the accepted mechanism of the metal catalyzed hydrolysis
of phosphate diesters, which involves a pre-complexation step of
the substrate to the metal complex. Analysis of data with
a Michaelis–Menten like equation allowed to estimate the binding
constants of the substrate to the catalyst (K
and 8.4ꢀ10 M , respectively, 1$Zn(II)2, 3$Zn(II) , and 5$Zn(II).
2 2 2 2
DNA affinity order is: 1$Zn(II) z3$Zn(II) >2$Zn(II) >4$Zn(II)
(Fig. 5).
3
3
b
) of 2.7ꢀ10 , 4.3ꢀ10 ,
2
ꢂ1
2
8
6
4
2
000
000
000
000
0
8
6
000
000
2
.3. Cleavage of plasmid DNA
Incubation of plasmid DNA pBR 322 with the Zn(II) complex of
ligands 1–5 at pH 7 for 24 h at 37 C results in different degrees of
cleavage of DNA form I to form II (relaxed circular) depending on
the structure of the ligand (Fig. 4). Surprisingly enough, the order of
reactivity is quite different than that observed in the case of BNP
cleavage. With plasmid DNA, in fact, the reactivity order is
ꢁ
0
10 20 30 40 50 60
4000
[Complex], μM
2000
1
$Zn(II)
2
>5$Zn(II)z3$Zn(II)
2
,
while 4$Zn(II)
2
is substantially
unreactive. A peculiar behavior is observed for 2$Zn(II)2, which
produces a remarkable (about 20%) cleavage at low concentrations
0
(
10 mM) but becomes less reactive at higher concentrations.
0
5
10 15 20 25 30 35 40 45 50
4
3
2
1
0
0
0
0
0
[Complex], μM
Figure 5. DNA molar ellipticity ([
q
]) at 275 nm as a function of the Zn(II) complexes of
]) at 275 nm as
(B) and (,).
ligands 1 (C), 2 (-), 3 (B), and 4 (,). Inset: DNA molar ellipticity ([
q
a
function of the Zn(II) complexes of ligands
5
(C),
6
7
ꢁ
[
ctDNAbp]¼180
m
M. [HEPES]¼20 mM, pH 7, 25 C, 24 h, complex concentration is ex-
pressed as total Zn(II) concentration.
In order to obtain better insight into the reasons for the different
affinities of the ligands for DNA, monometallic Zn(II) complexes
$Zn(II) and 7$Zn(II) (Chart 3) were investigated and compared
with 5$Zn(II). When the comparison is restricted to mononuclear
complexes, affinity order for DNA emerging from circular dichroism
experiments is 5$Zn(II)z 6$Zn(II)>7$Zn(II).
6
N
N
0
10
20
30
40
50
N
N
N
N
[
Zn(II)], μM
NH2
NH2
S
S
Figure 4. Extent of pBR 322 plasmid DNA form II (nicked) produced after cleavage
with different concentrations Zn(II) complexes of ligands 1 (C), 2 (B), 3 (,), and 5
6
7
ꢁ
(
-). [DNAbp]¼12
m
M. [HEPES]¼20 mM, pH 7, 37 C, 24 h, complex concentration is
expressed as total Zn(II) concentration (the lines connecting the data point are for
a clearer inspection).
Chart 3. Reference ligands used in CD experiments.

V. Lombardo et al. / Tetrahedron 66 (2010) 2189–2195
2193
3
. Conclusions
In conclusion, the use of disulfide bridges appears as an in-
teresting strategy to the easy preparation and modification of
bimetallic complexes. In perspective, mixed binuclear ligands
featuring two different binding units or catalyst selection by the
dynamic combinatorial approach can be envisaged using this
structural element. Moreover, the complexes herein reported are
quite reactive toward BNP and DNA, where they rank among the
few Zn-based bimetallic complexes capable to promote DNA
When the literature on artificial hydrolytic catalysts for DNA and
model phosphate diesters is examined, one is surprised by the lack
of efficient bimetallic Zn(II)-based systems. This appears in striking
contrast with natural systems, which often use two Zn(II) ions in
9
the active sites of several nuclease enzymes. The studies of Bencini
and Meyer10 have quite clearly pointed out that the source of such
1
low efficiency lies in the formation of
m
-hydroxo bridges that are
cleavage. This confirms that the use of BAPA-based ligands is
poor nucleophiles and, on the other hand, block the available
binding sites on the metal ions preventing the binding of the
substrate.
The reactivity here reported for 1$Zn(II) and 3$Zn(II) toward
2 2
the model substrate BNP confirms such a scenario. The two com-
plexes are indeed highly reactive toward BNP, producing in the case
a promising strategy for the realization of efficient hydrolytic
catalysts for phosphate esters and also for DNA. However, the
results obtained indicate that the simple use of a two-atom bridge
is not sufficient to overcome the difficulty to find the right spatial
arrangements of the metal ions and achieve optimum cooperative
action: a further design effort is needed to achieve the optimum
geometry.
of 3$Zn(II)
reaction at pH 7 and 40 C, and at a complex concentration as low
as 50 M. However, both the bimetallic complexes are only 3–4 fold
2
a 40,000-fold rate acceleration over the background
ꢁ
16
m
4
4
. Experimental section
more reactive that the monometallic reference 5$Zn(II), indicating
that cooperative action of the two metal centers is limited. This was
.1. General
expected for complex 3$Zn(II)
produces very little pre-organization of the structure. It is more
surprising in the case 1$Zn(II) , which is even slightly less reactive
than 3$Zn(II)
2
, where the non-bridging spacer
17
Solvents were purified by standard methods. All commercially
2
available reagents and substrates were used as received. TLC
analyses were performed using Merck 60 F254 precoated silica gel
glass plates. Column chromatography was carried out on
Macherey-Nagel silica gel 60 (70–230 mesh). NMR spectra were
recorded using a Bruker AC250F spectrometer operating at
2
.
Some insight into the causes of such low reactivity come from
the analysis of the behavior of the other two dinuclear complexes
studied, 2$Zn(II)
reactivity of 2$Zn(II)
only much lower than that of the bridging group devoid 4$Zn(II)
2
and 4$Zn(II)
2
. Here the difference is dramatic: the
2
, which contains the disulfide bridge, is not
1
13
2
50 MHz for H and 62.9 MHz for C and a Bruker AV300 oper-
2
,
1
ating at 300 MHz for H. Chemical shifts are reported relative to
internal Me
but virtually absent. This indicates that the presence of the disulfide
bridge is potentially detrimental for the reactivity of the system,
probably because the arrangement of the two Zn(II) ions realized in
4
Si. Multiplicity is given as follows: s¼singlet,
d¼doublet, t¼triplet, q¼quartet, qn¼quintet, m¼multiplet,
br¼broad peak. ESI MS mass spectra were obtained with an Agilent
Technologies LC/MSD Trap SL mass spectrometer. UV–visible
spectra and kinetic traces were recorded on Perkin–Elmer Lambda
the complex still allows for the formation of unreactive
bridges. On these bases, the similar reactivity of complexes
$Zn(II) and 3$Zn(II) , accompanied by a similar affinity for the
m-hydroxo
1
2
2
16 and Lambda 45 spectrophotometers equipped with thermo-
substrate as indicated by the BNP binding constants measured,
could be attributed to the lack of or to a weak coordination of both
the Zn(II) ions to the sulfur atoms of the bridge that would make 1
and 3 structurally equivalent.
stated multiple cell holders. Zn(NO and Cu(NO were analyt-
3
)
2
3 2
)
ical grade products (Aldrich). Metal ion stock solutions were
titrated against EDTA following standard procedures. The buffer
components were used as supplied by the manufacturers: acetic
acid (Aldrich), 2-morpholinoethanesulfonic acid (MES, Fluka), 4-
Unfortunately, the solubility of the bimetallic complexes was too
low to allow the investigation of their structures. In the case of
monometallic 5$Zn(II), coordination of sulfur to Zn(II) is supported
by the NMR spectral changes, and the same occurs with similar
ligands featuring a thioether arm studied by Berreau and co-
(
4
2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Sigma),
-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPES,
Sigma), 2-[cyclohexylamino]ethanesulfonic acid (CHES, Sigma), 3-
N-cyclohexylamino]-1-propanesulfonic acid (CAPS, Sigma). The
[
workers.18 Also the absence of reactivity observed with 2$Zn(II)
supports the coordination of the Zn(II) ions to the sulfur atoms of
the bridge. However, in the case of 1$Zn(II) , steric hindrance be-
2
substrate bis-p-nitrophenyl phosphate sodium salt (BNP) is an
Aldrich product, used as received. Ligands 1–5 and 7 were
prepared as described in the following paragraphs, synthesis of
2
tween the pyridine amino groups, as emerges by inspection of
molecular models, could disfavor the interaction between the
metal ions and the disulfide bridge.
Differently from the behavior observed with the model sub-
strate BNP, reactivity toward plasmid DNA is quite interesting. In
fact, not only is the bimetallic complex 1$Zn(II) substantially more
reactive, at low concentrations, than the mononuclear counterpart
12d
ligand 6 was previously reported. Concentrations of ligands 1–7
prepared) stock solutions in methanol were determined by
spectroscopic titrations with Cu(NO monitoring the increase of
(
3 2
)
the Cu(II) absorption band (d–d transition) at 700 nm.
4.2. DNA cleavage experiments
but this time it is also much more reactive than complex 3$Zn(II)
and the same occurs if 2$Zn(II) and 4$Zn(II) are compared). With
2
(
2
2
DNA cleavage experiments were performed using pBR 322
DNA, hence, the presence of the disulfide group produces an im-
portant effect on the reactivity. Binding experiments give some
further hint: first, DNA affinity of bimetallic complexes is, as
expected, generally higher than that of monometallic one; second,
the presence, of the pyridine amines in the ligand substantially
increases DNA affinity while insertion of the disulfide group has no
(Fermentas) in 20 mM HEPES, pH 7.0. Reactions were performed
ꢁ
incubating DNA (12
m
M base pairs) at 37 C in the presence/ab-
sence of increasing amount of Zn(II) complexes for 24 h and
stopped by addition of EDTA and SDS to a final concentration of
0.06 M and 1%, respectively. Reaction products were resolved on
a 1% agarose gel in TAE buffer (40 mM TRIS base, 20 mM, acetic
acid, 1 mM EDTA) and visualized by ethidium bromide staining.
The relative amounts of different plasmid structures were quan-
tified using a Geliance 600 Imaging System (Perkin–Elmer)
interfaced to a PC workstation.
sensible effects. Hence, the greater reactivity of 1$Zn(II)
2
compared
with 3$Zn(II) is not due to higher affinity but must be attributed to
2
some effect of the disulphide bridge either on the geometry of the
complex when bound to DNA or on the metal properties.

2194
V. Lombardo et al. / Tetrahedron 66 (2010) 2189–2195
4
.3. CD experiments
(d, 2H, 8 Hz), 3.68 (s, 4H), 2.89 (t, 2H, 7 Hz), 2.55 (t, 2H, 7 Hz), 2.32
s, 3H), 2.22 (s, 6H), 1.86 (qn, 2H, 7 Hz).
(
CD measurements were performed using Jasco J-810 spec-
tropolarimeter equipped with a thermostated cell holder. CD
spectra were recorded in HEPES 20 mM pH 7.0 using a 1 cm path-
length quartz cells. Titrations were performed by addition of in-
4.4.2. Bis(3-amino-N-bis(6-amino-2-pyridylmethyl)-propyl) disul-
fide (1). Compound III (0.170 g, 0.40 mmol) was dissolved in a 1 M
NaOH solution in H O/EtOH 1:1 (75 mL) and refluxed for 16 h under
2
creasing complex concentrations to a calf thymus DNA solution
stirring. After this time, ethanol was evaporated and the resulting
aqueous solution was extracted with chloroform. The organic phase
ꢁ
1
80
mM (base pairs) in HEPES 20 mM pH 7.0 at 25 C. For each
measurement two scans were run in the 220–350 nm range and
recorded with 0.1 nm step resolution. Each measurement was re-
peated at least in triplicate in independent experiments. At the end
of each measurement, observed ellipticities were converted to
was dried with Na
were obtained as a orange oil (43%). H NMR (CD
7.39 (t, 4H, 8 Hz), 6.78 (d, 4H, 8 Hz), 6.41 (d, 4H, 8 Hz), 3.53 (s, 8H),
2 4
SO and the solvent evaporated. 0.103 g of 1
1
3
OD, 250 MHz), d:
13
2.58 (t, 4H, 7 Hz), 2.54 (t, 4H, 7 Hz), 1.81 (qn, 4H, 7 Hz). C NMR
2
ꢂ1
.
mean residue ellipticity [
q]¼degꢀcm ꢀdmol
(CDCl
3
, 62.9 MHz),
d
: 160.3, 158.4, 139.5, 112.9, 108.2, 61.1, 53.8, 37.3,
þ
27.7. APCI MS (m/z): 605.4 [MþH ]. Elemental analysis, calcd for
4
.4. Kinetic measurements
C
30
H
40
N
10
S
2
(604.84): C 59.57, H 6.67, N 23.16, S 10.60; found: C
59.68, H 6.70, N 23.20, S 10.55%.
The kinetic traces were recorded on Perkin–Elmer Lambda 16 and
Lambda 45 spectrophotometers equipped with a thermostated
multiple cell holders. Reaction temperature was maintained at
4.4.3. Propyl-(3-amino-N-bis(6-amino-2-pyridylmethyl)-propyl) di-
sulfide (5). Compound III (0.386 g, 0.90 mmol) was dissolved in
ethanol (20 mL) and concentrated HCl (1.8 mL) was added. The
mixture was stirred at room temperature for 12 h and the solvent
ꢁ
ꢁ
2
2
5.0ꢃ0.1 C or 40.0ꢃ0.1 C. The reactions were started by adding
ꢂ3
0
m
L of a 2.0ꢀ10 M solution of substrate (BNP) to a 2 mL solution
containing the appropriate buffer (0.05 M), Zn(NO
3 2
) , and ligands 1–
was evaporated to obtain the corresponding thiol as a brown solid
1
5
in the desired amount, and monitored by following the absorption
(0.355 g, 95%). H NMR (CD
3
OD, 250 MHz),
d
: 7.80 (t, 2H, 8 Hz), 6.87
of p-nitrophenoxide at 400 nm or p-nitrophenol at 340 nm. Re-
actions were followed up to about 5% of substrate hydrolysis. The
(d, 2H, 8 Hz), 6.80 (d, 2H, 8 Hz), 3.83 (s, 4H), 2.61 (t, 2H, 7 Hz), 2.51
(t, 2H, 7 Hz), 1.74 (qn, 2H, 7 Hz). The above thiol (0.265 g,
pseudo-first order rate constants (k
of the absorbance versus time data (the fit error was always less than
%) divided by the molar absorbivity of the p-nitrophenoxide and the
j
) were obtained from the slope
2
0.88 mmol) was dissolved in a 0.1 M NaOH solution in H O/EtOH
1:1 (40 mL) and 1-propanethiol (1.00 g, 13.1 mmol) was added
under stirring. The mixture was refluxed under nitrogen for 12 h,
and then 50 mL of chloroform were added. The organic solution
1
concentration of substrate. Each experiment was performed in
triplicate and the errors on the rate constants are always below 10%.
was separated, extracted with 10% NaHCO
with Na SO . After solvent evaporation, the crude product was
purified by flash chromatography (silica gel, eluent: CH Cl /MeOH
20:1). 0.039 g (12%) of 5 were obtained as a colorless oil. H NMR
(CDCl , 250 MHz), : 7.38 (t, 2H, 8 Hz), 6.83 (d, 2H, 8 Hz), 6.34 (d, 2H,
8 Hz), 3.62 (s, 4H), 2.68 (t, 2H, 7 Hz), 2.60 (t, 4H, 7 Hz), 1.87 (qn, 2H,
3
(2ꢀ100 mL), and dried
0
Apparent pH dependent second-order rate constants (k
obtained by linear regression fitting of the k
2
) were
2
4
j
versus metal complex
values and second-order rate con-
) for the monodeprotonated complexes were obtained by
non-linear regression analysis of the apparent second-order rate
2
2
1
n
concentration data. Kinetic K
stants (k
a
2
3
d
0
1
13
constants versus pH date according to the equation: k
2
¼k
2
$(K
a
/
7 Hz), 1.66 (m, 2H, 7 Hz), 0.95 (t, 3H, 7 Hz). C NMR (CDCl
62.9 MHz), : 158.1, 158.0, 112.9, 107.0, 60.4, 52.9, 41.2, 36.9, 26.9,
22.7, 13.3. ESI MS (m/z): 378.2 [MþH ]. Elemental analysis, calcd for
(377.57): C 57.26, H 7.21, N 18.55, S 16.98; found: C
57.35, H 7.17, N 18.42, S 17.08%.
3
,
þ
þ
2
[
H ]þ1þ[H ]/K
a
). BNP rate saturation experiments with Zn(II)
d
þ
complexes were fitted according to the Michealis–Menten equation:
rate¼kmax$[BNP]/(1/KBNPþ[BNP]), where kmax is the limiting reaction
rate in the experimental conditions and KBNP is the apparent binding
constant of the substrate to the catalyst.
30 40 10 2
C H N S
4
.4.4. N-Bis(2-pyridylmethyl)-1-acetylsulfanyl-3-propylamine
4
.4.1. N-Bis(6-acetylamido-2-pyridylmethyl)-1-acetylsulfanyl-3-pro-
(IV). N-Bis(2-pyridylmethyl)-3-hydroxy-propylamine (II) was
12d
pylamine (III). N-Bis(6-acetylamido-2-pyridylmethyl)-3-hydroxy-
prepared as previously reported.
15.7 mmol) was dissolved in CH Cl
Compound II (4.00 g,
(40 mL). Tosyl chloride
12d
propylamine (I), was prepared as previously reported. To a stirred
solution of compound I (0.675 g, 1.80 mmol) in dry pyridine (8 mL)
were added p-toluenesulfonyl chloride (0.673 g, 3.50 mmol) and
triethylamine (0.421 g, 4.16 mmol). The reaction mixture was stir-
2
2
(6.000 g, 31.4 mmol) and triethylamine (5.10 mL, 36.0 mmol) were
added and the resulting mixture was refluxed for 3 days. During
this time, the pH of the mixture was controlled with pH paper and
adjusted to 9–10 with triethylamine. The reaction mixture was
2
red under N at room temperature for 24 h. The pyridine was
evaporated, the crude product was dissolved in chloroform, and
allowed to cool, diluted with CH
(3ꢀ20 mL). The organic phase was dried with Na
rated. The crude product was purified by flash chromatography
(silica gel, eluent: CH Cl /MeOH 10:1). N-Bis(2-pyridylmethyl)-3-
chloro-propylamine (0.440 g (7%)) was obtained as a yellow oil. H
NMR (CDCl , 250 MHz), : 8.41 (d, 2H, 8 Hz), 7.55 (t, 2H, 8 Hz), 7.40
2
Cl
2
, and extracted with 5% NaHCO
3
washed with water (4ꢀ50 mL). The organic phase was dried over
2
SO and evapo-
4
2 4
Na SO and evaporated. 0.403 g (57%) of N-bis(6-acetylamido-2-
pyridylmethyl)-3-chloro-propylamine were obtained as a white
2
2
1
1
3
solid. H NMR (CDCl , 250 MHz), d: 8.05 (d, 2H, 8 Hz), 7.85 (br s, 2H),
7.68 (t, 2H, 8 Hz), 7.17 (d, 2H, 8 Hz), 3.70 (s, 4H), 3.58 (t, 2H, 7 Hz),
3
d
2
.70 (t, 2H, 7 Hz), 2.21 (s, 6H),1.99 (qn, 2H, 7 Hz). ESI MS (m/z): 390.8
(d, 2H, 8 Hz), 7.04 (t, 2H, 8 Hz), 3.72 (s, 4H), 3.46 (t, 2H, 7 Hz), 2.61 (t,
2H, 7 Hz), 1.87 (qn, 2H, 7 Hz). The above compound (0.472 g,
1.21 mmol) was dissolved in acetone (40 mL) and potassium thio-
acetate (0.420 g, 3.60 mmol) was added. The mixture was refluxed
under nitrogen for 60 h, the solvent was evaporated and the solid
þ
þ
[
1
16%, MþH ], 412.2 [100%, MþNa ]. The above compound (0.403 g,
.03 mmol) was dissolved in acetone (30 mL) and potassium thio-
acetate (0.240 g, 2.1 mmol) was added. The mixture was refluxed
under nitrogen for 60 h, the solvent was evaporated and the solid
residue dissolved in CH
extracted with water (5ꢀ20 mL) and dried with Na
solvent evaporation, the crude product was purified by flash chro-
matography (silica gel, eluent: CH Cl /MeOH/NH 20:1:0.01).
.386 g (90%) of III were obtained as a orange oil. H NMR (CDCl
50 MHz), : 8.08 (br s, 2H), 8.06 (d, 2H, 8 Hz), 7.67 (t, 2H, 8 Hz), 7.14
2
Cl
2
(20 mL). The organic solution was
residue dissolved in CH
extracted with water (5ꢀ20 mL) and dried with Na
vent evaporation, the crude product was purified by flash chro-
matography (silica gel, eluent: CH Cl /MeOH/NH 10:1:0.01).
Compound IV (0.110 g (29%)) was obtained as a brown oil. H NMR
(CDCl , 250 MHz), : 8.43 (d, 2H, 8 Hz), 7.59 (t, 2H, 8 Hz), 7.43 (d, 2H,
2
Cl
2
(20 mL). The organic solution was
2
SO . After
4
2
SO . After sol-
4
2
2
3
2
2
3
1
1
0
2
3
,
d
3
d

V. Lombardo et al. / Tetrahedron 66 (2010) 2189–2195
2195
8
7
Hz), 7.07 (t, 2H, 8 Hz), 3.72 (s, 4H), 2.78 (t, 2H, 7 Hz), 2.52 (t, 2H,
Hz), 2.19 (s, 3H), 1.72 (qn, 2H, 7 Hz). ESI MS (m/z): 315.0 [MþH ].
27.7 mmol) in NaOH 5 M (30 mL) were added 1,8-diaminooctane
(1.00 g, 6.93 mmol) and hexadecyltrimethylammonium chloride
þ
(
0.44 g, 0.14 mmol). The mixture was stirred for 20 h and the so-
lution was extracted with CH Cl
(3ꢀ20 mL). The organic yellow
solution was washed with brine (2ꢀ60 mL), water (2ꢀ60 mL), and
dried with Na SO . After solvent evaporation, the crude product
was purified by flash chromatography (silica gel, eluent: CH Cl
MeOH 10:0.5). Compound 4 (2.902 g (82%)) was obtained as a white
4.4.5. Propyl-(3-amino-N-bis(2-pyridylmethyl)-propyl)
disulfide
2
2
(7). Compound IV (0.055 g, 0.17 mmol) was dissolved in ethanol
(
4 mL) and 6 M HCl (2 mL) was added. The mixture was stirred at
2
4
room temperature for 3 h, and the solvent was evaporated to obtain
2
2
/
1
the desired thiol as a brown solid (0.063 g, 97%). H NMR (CD
3
OD,
: 8.88 (d, 2H, 8 Hz), 8.61 (t, 2H, 8 Hz), 8.20 (d, 2H, 8 Hz),
.05 (t, 2H, 8 Hz), 4.39 (s, 4H), 2.79 (t, 2H, 7 Hz), 2.46 (t, 2H, 7 Hz),
OD, 62.9 MHz), : 153.2, 147.6,
42.1, 127.9, 126.8, 56.0, 53.4, 30.1, 21.67. The above compound
0.048 g, 0.126 mmol) was dissolved into a 0.3 M NaOH solution of
O/EtOH 1:1 (30 mL), 1-propanethiol (0.66 g, 8.7 mmol) was
1
2
50 MHz),
d
solid. H NMR (CDCl
3
, 250 MHz), d: 8.33 (d, 4H, 8 Hz), 7.46 (t, 4H,
8
1
8 Hz), 7.38 (d, 4H, 8 Hz), 6.94 (t, 4H, 8 Hz), 3.65 (s, 8H), 2.36 (t, 4H,
.83 (qn, 2H, 7 Hz). 13C NMR (CD
d
7 Hz), 1.36 (m, 4H), 1.02 (m, 8H). C NMR (CDCl
13
3
3
, 62.9 MHz), d:
1
(
H
159.9, 148.7, 136.1, 122.6, 121.6, 60.3, 54.2, 29.2, 27.0, 26.8. ESI MS
þ
þ
(m/z): 509.4.0 [100%, MþH ], 531.2 [59%, MþNa ]. Elemental
analysis, calcd for C32 (508.70): C 75.55, H 7.93, N 16.52;
2
40 6
H N
added and the mixture was refluxed for 16 h. After this time 50 mL
of chloroform were added and the organic solution was extracted
found: C 75.76, H 7.70, N 16.58.
with 10% NaHCO
evaporation, the crude product was purified by flash chromatog-
raphy (silica gel, eluent: CH Cl /MeOH 20:1). 0.036 g of 7 (82%)
were obtained as a yellowish oil. H NMR (CDCl
3
(2ꢀ100 mL) and dried with Na
2
SO
4
. After solvent
Acknowledgements
2
2
1
The authors thank Marco Pedroni for preliminary experiments.
C.S. and V.L. were supported by University of Padova (grant #
CPDA078422/07).
3
, 250 MHz),
d: 8.52
(
d, 2H, 8 Hz), 7.67 (t, 2H, 8 Hz), 7.51 (d, 2H, 8 Hz), 7.15 (t, 2H, 8 Hz),
3
3
.82 (s, 4H), 2.64 (m, 6H), 1.92 (qn, 2H, 7 Hz), 1.65 (m, 2H), 0.96 (t,
H, 7 Hz). 13C NMR (CDCl
, 62.9 MHz), : 159.8, 149.2, 136.6, 123.1,
3
d
References and note
1
22.2, 60.6, 53.0, 41.3, 36.9, 27.0, 22.7, 13.3. ESI MS (m/z): 348.2
þ
[MþH ]. Elemental analysis, calcd for C18
H
25
N
3
S
2
(357.54): C 62.21,
1. Recent reviews: (a) Mancin, F.; Tecilla, P. In Metal Complex–DNA Interactions;
Hadjiliadis, N., Sletten, E., Eds.; Wiley: Chichester, UK, 2009; pp 369–394; (b)
Gahan, L. R.; Smith, S. J.; Neves, A.; Schenk, G. Eur. J. Inorg. Chem. 2009, 2745–
H 7.25, N 12.09, S 18.45; found: C 62.35, H 7.17, N 12.38, S 18.23%.
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4
.4.6. Bis(3-amino-N-bis(2-pyridylmethyl)-propyl) disulfide (2).
Compound IV (0.291 g, 0.92 mmol) was dissolved in a 1 M NaOH
solution in H O/EtOH 1:1 (40 mL) and refluxed under stirring for
6 h. After this time ethanol was evaporated and the resulting
aqueous solution was extracted with chloroform. The organic phase
was dried with Na SO and the solvent evaporated to obtain 0.199 g
of product (4) as a brown solid (76%). H NMR (CDCl
T.; Lonnberg, H. Org. Biomol. Chem. 2006, 4, 15–25; (e) Mancin, F.; Scrimin, P.;
Tecilla, P.; Tonellato, U. Chem. Commun. 2005, 2540–2548.
. Schroeder, G. K.; Lad, C.; Wyman, P.; Williams, N. H.; Wolfenden, R. Proc. Natl.
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. Katada, H.; Komiyama, M. Chem. Bio. Chem. 2009, 10, 1279–1288.
2
4
. (a) Mathews, R. A.; Rossiter, C. S.; Morrow, J. R.; Richard, J. P. Dalton Trans. 2007,
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1
3
, 250 MHz),
d:
8
8
.51 (d, 4H, 8 Hz), 7.66 (t, 4H, 8 Hz), 7.51 (d, 4H, 8 Hz), 7.15 (t, 4H,
13
Hz), 3.82 (s, 8H), 2.65 (t, 8H, 7 Hz), 1.89 (qn, 4H, 7 Hz). C NMR
1
995, 305–306.
6. Morrow, J. Comments Inorg. Chem. 2008, 29, 169–188.
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(
CDCl
3
, 62.9 MHz),
d
: 159.5, 148.8, 136.3, 122.8, 121.9, 60.3, 52.6,
þ
7
8
3
6.4, 26.6. ESI MS (m/z): 567.9 [MþNa ]. Elemental analysis, calcd
. (a) Chapman, W. H.; Breslow, R. J. Am. Chem. Soc. 1995, 117, 5462–5469; (b)
Mancin, F.; Rampazzo, E.; Tecilla, P.; Tonellato, U. Eur. J. Org. Chem. 2004, 281–288.
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6
H
36
N
6
S
2
(544.78): C 66.14, H 6.66, N 15.43, S 11.77; found: C
6.31, H 6.75, N 15.05, S 11.90%.
9. (a) Arca, M.; Bencini, A.; Berni, E.; Caltagirone, C.; Devillanova, F. A.; Isaia, F.;
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Inorg. Chem. 1997, 36, 2784–2790.
0 0
.4.7. N,N,N ,N -Tetra(6-amino-2-pyridylmethyl)-1,8-dioctylamine
4
(3). Compound V (1.500 g, 6.55 mmol), prepared as previously
12d
2 3
reported, and K CO (1.143 g, 8.27 mmol) were added to a solution
1
0. (a) Meyer, F. Eur. J. Inorg. Chem. 2006, 3789–3800; (b) Bauer-Siebenlist, B.;
Meyer, F.; Farkas, E.; Vidovic, D.; Dechert, S. Chem.dEur. J. 2005, 11, 4349–4360;
of 1,8-diaminooctane (0.210 g, 1.46 mmol) in acetonitrile (20 mL).
The mixture was stirred for 3 h under nitrogen at reflux, then in-
soluble salts were removed by filtration. After solvent evaporation,
the crude product was purified by flash chromatography (silica gel,
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Polizzi, S.; Mancin, F.; Tonellato, U.; Scrimin, P. J. Am. Chem. Soc. 2008, 130,
1
eluent: CH
2 2 3
Cl /MeOH/NH 10:1:0.01). 0.407 g (38%) of acetyl pro-
1
1
tected 3 were obtained as a yellow oil. H NMR (CDCl
8
3
The above compound (0.407 g, 0.55 mmol) was dissolved in a 5 M
NaOH in H O/EtOH 1:1 (45 mL). The reaction mixture was refluxed
for 7 h. After this time, ethanol was evaporated and the resulting
aqueous solution was extracted with chloroform. The organic phase
3
250 MHz),
.93 (s, 4H), 8.05 (d, 4H, 8 Hz), 7.63 (t, 4H, 8 Hz), 7.19 (d, 4H, 8 Hz),
.46 (s, 8H), 4.11 (t, 4H, 7 Hz), 2.18 (s, 12H), 1.45 (m, 4H), 1.15 (m, 8H).
d:
15744–15745; (c) Livieri, M.; Mancin, F.; Saielli, G.; Chin, J.; Tonellato, U. Chem.
dEur. J. 2007, 13, 2246–2256; (d) Livieri, M.; Mancin, F.; Tonellato, U.; Chin, J.
Chem. Commun. 2004, 2862–2863.
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Am. Chem. Soc. 2008, 130, 4232–4233; (b) Feng, G. Q.; Natale, D.; Prabaharan, R.;
Mareque-Rivas, J. C.; Williams, N. H. Angew. Chem., Int. Ed. 2006, 45, 7056–7059;
(c) Feng, G. Q.; Mareque-Rivas, J. C.; Williams, N. H. Chem. Commun. 2006, 1845–
1
2
1
847; (d) Feng, G. Q.; Mareque-Rivas, J. C.; de Rosales, R. T. M.; Williams, N. H. J.
Am. Chem. Soc. 2005, 127, 13470–13471.
4. (a) Morrow, J. R.; Amyes, T. L.; Richard, J. P. Acc. Chem. Res. 2008, 41, 539–548;
b) O’Donoghue, A.; Pyun, S. Y.; Yang, M. Y.; Morrow, J. R.; Richard, J. P. J. Am.
was dried with Na
2 4
SO and the solvent evaporated. Compound 3
1
(
0.302 g (97%)) was obtained as a orange oil (43%). H NMR (CD
3
OD,
d: 7.38 (t, 4H, 8 Hz), 6.82 (d, 4H, 8 Hz), 6.40 (d, 4H, 8 Hz),
1
2
50 MHz),
.53 (s, 8H), 2.45 (t, 4H, 7 Hz), 1.47 (m, 4H), 1.17 (m, 8H). C NMR
, 62.9 MHz), : 158.8, 158.2, 138.1, 112.6, 106.7, 60.5, 54.3, 29.4,
7.3, 26.9. Elemental analysis, calcd for C32 10 (568.76): C 67.58,
H 7.80, N 24.63; found: C 67.71, H 7.67, N 24.55.
(
13
3
Chem. Soc. 2006, 128, 1615–1621; (c) Iranzo, O.; Kovalevsky, A. Y.; Morrow, J. R.;
Richard, J. P. J. Am. Chem. Soc. 2003, 125, 1988–1993.
5. Gavrilova, A. L.; Bosnich, B. Chem. Rev. 2004, 104, 349–383 and references therein.
(
CDCl
3
d
1
1
2
44
H N
ꢁ
6. The rate estimated for the spontaneous hydrolysis of BNP at pH 7.0 and 40 C is
ꢂ10 ꢂ1
1.1ꢀ10
s
; see Chin, J.; Banaszczyk, M.; Jubian, V.; Zou, X. J. Am. Chem. Soc.
1
989, 111, 186–190.
7. Iranzo, O.; Elmer, T.; Richard, J. P.; Morrow, J. R. Inorg. Chem. 2003, 42, 7737–7746.
8. Garner, D. K.; Fitch, S. B.; McAlexander, L. H.; Bezold, L. M.; Arif, A. M.; Berreau,
L. M. J. Am. Chem. Soc. 2002, 124, 9970–9971.
1
1
0
0
4.4.8. N,N,N ,N -Tetra(2-pyridylmethyl)-1,8-dioctylamine (4). To
a
stirred solution of 2-chloromethyl pyridine hydrochloride (4.55 g,