Metal ion induced allosteric transition in the catalytic activity of an artificial phosphodiesterase

Article abstract of DOI:10.1039/b716196d

An artificial phosphodiesterase (1) bearing two types of metal binding sites, a catalytic site and a regulatory bipyridine site showed a unique allosteric transition in the catalytic activity against the metal concentration. The rate constants for the hydrolysis reaction of 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) and RNA dimer (ApA) with and without an effector metal ion were evaluated; the k_obs value of HPNP hydrolysis for 1?(Zn ²⁺)₃ (2.0 × 10^-4 s^-1) is 3.3 times larger than that for 1?(Zn²⁺)₂. In the case of 1 and Cu²⁺, a 19.4 times larger k_obs value was obtained for 1?(Cu²⁺)₃ (1.2 × 10^-3 s ^-1) against 1?(Cu²⁺)₂. The increase in the catalytic activity is ascribed to the allosteric conformational transition of 1 induced by the coordination of effector metal ion to the Bpy moiety. A detailed investigation revealed that a conformational change of 1 induced by the third M²⁺ complexation enhances the rate of hydrolysis rather than a change in the substrate affinity. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b716196d

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Metal ion induced allosteric transition in the catalytic activity of an artificial
phosphodiesterase†
Shinji Takebayashi,a Seiji Shinkai,*a Masato Ikedab and Masayuki Takeuchi*c
Received 19th October 2007, Accepted 26th November 2007
First published as an Advance Article on the web 12th December 2007
DOI: 10.1039/b716196d
An artificial phosphodiesterase (1) bearing two types of metal binding sites, a catalytic site and a
regulatory bipyridine site showed a unique allosteric transition in the catalytic activity against the metal
concentration. The rate constants for the hydrolysis reaction of 2-hydroxypropyl-p-nitrophenyl
phosphate (HPNP) and RNA dimer (ApA) with and without an effector metal ion were evaluated; the
kobs value of HPNP hydrolysis for 1·(Zn2+)3 (2.0 × 10−4 s−1) is 3.3 times larger than that for 1·(Zn2+)2. In
the case of 1 and Cu2+, a 19.4 times larger kobs value was obtained for 1·(Cu2+)3 (1.2 × 10−3 s−1) against
1·(Cu2+)2. The increase in the catalytic activity is ascribed to the allosteric conformational transition of
1 induced by the coordination of effector metal ion to the Bpy moiety. A detailed investigation revealed
that a conformational change of 1 induced by the third M2+ complexation enhances the rate of
hydrolysis rather than a change in the substrate affinity.
molecular recognition systems have been studied well, whereas
studies on artificial allosteric catalytic systems are still very
Introduction
rare.3,4 Furthermore, as far as we know, there are few reports
of the same metal ion playing both roles of a catalytic metal ion
and an effector metal ion, giving rise to an allosteric transition
phenomenon.3–5 Such novel systems would show a unique catalytic
activity response depending on the metal ion concentration.
There is considerable interest in creating artificial phosphodi-
esterases because of their potential application to gene therapy.
For this purpose, numerous catalysts containing essential metal
ions have been explored.6 Recently, we designed compound 1 as
an allosteric artificial phosphodiesterase, which has two different
types of metal ion binding sites, 2,2ꢀ-bipyridine (Bpy) as a
regulatory site and 2,2ꢀ-dipicolylamine (DPA) as catalytic sites
(Fig. 1).7
The DPA moieties have higher Zn2+ or Cu2+ affinities than the
Bpy moiety;8 as a result, the first two metal ions are bound to two
DPA moieties in 1 to produce 1·(M2+)2 (M2+ = Zn2+ or Cu2+). The
regulatory Bpy moiety adopts mostly a transoid conformation
because of the repulsion between lone-pairs of nitrogen atoms,9
where the amine distance between DPA is estimated to be 0.84 nm
by the molecular modeling (see Fig. 2: Insight II, Discover 3). The
The design of artificial allosteric systems is of great significance for
regulating the catalytic activities and complexation properties of
artificial receptors.1 We have been interested in the exploitation of
homotropic allosteric systems, which show the nonlinear amplifi-
cation of binding events and chemical signals.2 This phenomenon
is useful to control the activities of an artificial receptor in an
OFF–ON switching manner at a threshold condition.1,2 Allosteric
aDepartment of Chemistry and Biochemistry, Graduate School of Engi-
neering, Kyushu University, Fukuoka, 819-0395, Japan. E-mail: seijitcm@
mbox.nc.kyushu-u.ac.jp; Fax: (+81) 92 802 2818; Tel: (+81) 92 802 2820
bDepartment of Synthetic Chemistry and Biological Chemistry, Graduate
School of Engineering, Kyoto University, Kyoto, 615-8510, Japan
cMacromolecules Group, Organic Nanomaterials Center, National Institute
for Materials Science, Tsukuba, 305-0047, Japan. E-mail: TAKEUCHI.
Masayuki@nims.go.jp; Fax: (+81) 29 859 2101; Tel: (+81) 29 859 2110
† Electronic supplementary information (ESI) available: Fig. S1: 1H NMR
spectra (9.5–6.5 ppm) for 1 + Zn(ClO4)2. Fig. S2: 1H NMR spectra (6.0–
3.0 ppm) for 1 + Zn(ClO4)2. Fig. S3: ESI MS spectra for [Zn2+]/[1] =
2.0 and 8.0 and [Cu2+]/[1] = 2.0 and 3.0 in 33% ethanol–water (HEPES,
25 mM). Fig. S4: Lineweaver–Burk plots for HPNP cleavage. Fig. S5:
UV-Vis spectral changes of 1·(Zn2+)3 upon addition of EDTA-2Na in 33%
ethanol–water (HEPES, 25 mM). See DOI: 10.1039/b716196d
Fig. 1 Chemical structures of compounds 1–4 and substrates HPNP and ApA.
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Scheme 2 Synthesis of 2. Reagents and conditions: (i) BDH, benzyl
peroxide, dry CCl4, reflux, 4 h; (ii) 2,2ꢀ-dipicolylamine, KI, K2CO3, dry
CH3CN, 35 ◦C, 5 h.
nation of 2,2ꢀ-bipyridine-4,4ꢀ-dicarbaldehyde with 2,2ꢀ-dipicolyl-
amine. We selected sodium triacetoxyborohydride [NaBH(OAc)3]
as a reducing agent (Scheme 1).10 The methyl groups of
3,3ꢀ-dimethylbiphenyl were brominated with 1,3-dibromo-5,5ꢀ-
dimethylhydantoin (BDH) in carbon tetrachloride in the pres-
ence of benzoyl peroxide to give 3,3ꢀ-dibromomethylbiphenyl
(5) (Scheme 2).11 Compound 4 was reacted with lithium di-
isopropylaminde, and the anion thus formed was trapped with
trimethylsilyl chloride (TMSCl) to generate 4-trimethylsilyl-4ꢀ-
methyl-2,2ꢀ-bipyridine (6) (Scheme 3).12 The TMS group of 6
was removed using dry fluoride anion sources (CsF in DMF)
in the presence of BrF2CCF2Br to produce the 4-bromomethyl-4ꢀ-
methyl-2,2ꢀ-bipyridine (7). Compounds 2 and 3 were synthesized
from the corresponding bromomethyl precursors 5 and 7 by the
nucleophilic substitution with 2,2ꢀ-dipicolylamine.
Fig. 2 The molecular models of 1·(Zn2+)2 and 1·(Zn2+)3.
metal ion coordination to Bpy results in a conformational change
from transoid to cisoid by enforcing an alignment of the DPA
sites at a distance of 0.48 nm (catalytically more active); that is an
allosteric transition. Compounds 2–4 used for control experiments
cannot show such an allosteric transition phenomenon.
We demonstrated preliminary results on an allosteric transition
of 1 in the recent communication,7 wherein 1 can alter its catalytic
activity upon addition of metal ions (Zn2+ or Cu2+) in the
hydrolysis of 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP).
This preliminary finding stimulated us to further investigate in
detail the influence of the metal binding on the catalytic hydrolysis
reaction of HPNP. In this publication, we report the investigations
of the metal-induced allosteric transition in the catalytic activity
of 1 and further demonstrated the allosteric catalysis for RNA
dinucleotide, ApA, as a substrate.
In situ sequential formation of trinuclear metal complex of 1
The complexation behaviour of 1 with Zn2+ and Cu2+ (as perchlo-
rate salts) in ethanol–water (HEPES, 16 mM) = 1/2 (v/v) solution
at pH 7.7 and 25 ◦C was monitored by photometric titration, ESI
MS and 1H NMR spectroscopies.
Results and discussion
Synthesis of compounds 1–3
Binding processes of Zn2+ to 1 (0.40 mM) were accompanied by
three steps of spectral changes with isosbestic points observed at
293.2 nm (0 to 1 equiv. Zn2+), 296.8 nm (1 to 2 equiv. Zn2+) and
297.4 nm (more than 2 equiv. Zn2+) (Fig. 3(a)). The absorbance
of DPA moieties changed almost linearly upon addition of 0 to
1 equiv. Zn2+ and 1 to 2 equiv. Zn2+. This result clearly showed that
two Zn2+ ions are bound firstly to the DPA moieties quantitatively
to produce 1·(Zn2+)2 under these conditions. At [Zn2+] higher
than 2 equiv., a typical shift from 284.6 to 312.0 nm in the
absorption band of the Bpy moiety was observed; Bpy·Zn2+ is
formed at this stage (Fig. 4). A plot of the absorbance at 312.0 nm
against Zn2+ concentration showed a saturation behaviour and the
association constant for the formation of 1·(Zn2+)3 from 1·(Zn2+)2
was evaluated to be 9.1 × 102 M−1. Similar results were obtained
The synthetic pathways for compounds 1–3 are shown in
Schemes 1, 2, 3. Compound 1 was synthesized by reductive ami-
Scheme 1 Synthesis of 1. Reagents and conditions: (i) 2,2ꢀ-dipicolylamine,
NaBH(OAc)3, ClCH2CH2Cl, r.t., 18 h.
Scheme 3 Synthesis of 3. Reagents and conditions: (i) LDA/THF, TMSCl, dry THF, 0 ◦C, 30 min; (ii) CsF, BrF2CCF2Br, dry DMF, r.t., 2 h; (iii)
2,2ꢀ-dipicolylamine, K2CO3, KI, dry CH3CN, 35 ◦C, 2 h.
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Fig. 3 (a) UV-Vis spectral changes of 1 (0.4 mM) upon addition of Zn(ClO4)2 (broken lines; 0–2 equiv., solid lines; 2–11 equiv.) in 33% ethanol–water
(HEPES, 25 mM) at pH 7.7 and 25 ◦C, (b) UV-Vis spectral changes of 1 (1.0 mM) upon addition of Cu(ClO4)2 (broken lines; 0–2 equiv., solid lines;
2–6 equiv.) in 33% ethanol–water (HEPES, 25 mM) at pH 7.7 and 25 ◦C.
Fig. 4 Schematic representation for the sequential complexation of metal ions to 1.
for the 1 (1.0 mM) and Cu2+ system, where 2 equiv. of Cu2+ were
bound to the DPA moieties in 1 quantitatively (Fig. 3(b)). The
association constants for the formation of 1·(Cu2+)3 and 12·(Cu2+)5
from 1·(Cu2+)2 were evaluated to be 6.3 × 104 M−1 and 1.0 ×
108 M−2, respectively (Fig. 4).
406 nm. The kinetic studies at varying pH were performed
and pseudo-first-order-rate constants (kobs) were evaluated for
1·(Zn2+)3. The pH-rate profile for the transesterification of HPNP
catalyzed by 1·(Zn2+)3 is bell-shaped in the pH region 6.0–8.0 with
a maximum at pH 7.7 (Fig. 5). A bell-shaped pH rate profile
is often observed for metal-promoted HPNP cleavage. This bell-
type curve indicates that cooperativity between the metal centers in
DPA would be due to the occurrence of general-acid/general-base
1H NMR titration experiments (see supplementary
information†) also supported this sequential binding scheme
shown in Fig. 4. The in situ formation of 1·(M2+)3 and 12·(M2+)5
(for Cu2+) from 1·(M2+)2 was further supported by the ESI MS
measurement. The ESI MS spectra for [Zn2+]/[1] = 2.0, 8.0 in
33% ethanol–water (HEPES, 25 mM) showed strong peaks at
m/z = 1007.1 and 1271.0, which are assignable to the species of
[1·(Zn2+)2·(ClO4−)3]+ (calc. for [1·(Zn2+)2·(ClO4−)3]+ = 1007.0) and
[1·(Zn2+)3·(ClO4−)5]+ (calc. for [1·(Zn2+)2·(ClO4−)3]+ = 1270.8),
respectively (see supplementary data,† Fig. S3(a) and (b)). The
ESI MS spectrum for [Cu2+]/[1] = 2.0, 3.0 in 33% ethanol–water
(HEPES, 25 mM) showed strong peaks at m/z = 1003.1 and
1266.0, which are assignable to the species of [1·(Cu2+)2·(ClO4−)3]+
(calc. for [1·(Cu2+)2·(ClO4−)3]+ = 1003.0) and [1·(Cu2+)3·(ClO4−)5]+
(calc. for [1·(Cu2+)2·(ClO4−)3]+
=
1265.8), respectively (see
supplementary data,† Fig. S3(c) and (d)).
Hydrolysis of HPNP by Zn(II) and Cu(II) complexes of 1
We firstly employed HPNP as a substrate. Kinetic experiments
were conducted on the basis of the release rate of p-nitrophenolate
from HPNP by monitoring the increase in the absorbance at
Fig. 5 Effect of pH on the observed rate constants measured for the
cleavage of HPNP by 1·(Zn2+)3. Reagents and conditions: [1] = 0.40 mM,
[Zn(ClO4)2] = 2.0 mM, [HPNP] = 0.80 mM.
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catalysis. The increase in the rate with increasing pH is explained by
generation of metal-promoted hydroxide, which acts as a general
base in HPNP cleavage. The decrease in the rate at higher pH
might reflect the competitive binding of hydroxide to the catalyst
which prevents binding of the substrate.
Then, the kinetic studies at varying Zn2+ to Cu2+ concentrations
were performed and pseudo-first-order rate constants (kobs) were
evaluated for 1 and 2 (Fig. 6). Very interestingly, in the case of
1, there was a further significant increase in the catalytic activity
upon addition of more than 2 equiv. metal ion, whereas such
enhancement was not observed for 2 without the regulatory site
(Fig. 6). In Fig. 6(a), the rate constant for 1 with 8 equiv. Zn2+
is 3.3 times larger than for 1 with 2 equiv. Zn2+ (1·(Zn2+)2). The
saturation behaviour observed for 1 in Fig. 6(a) is complementary
to the results of photometric titration. In the case of 1 and Cu2+,
19.4 times larger kobs value was obtained for 1 with 3 equiv. Cu2+
against 1 with 2 equiv. Cu2+(1(Cu2+)2) (Fig. 6(b)). For 1 with
3 equiv. Cu2+, the ratio of [1·(Cu2+)3]/[12·(Cu2+)5]/[1·(Cu2+)2] is
estimated to be 81/11/8 by the association constants between Cu2+
and the Bpy moiety in 1.13 These results clearly show that 1·(M2+)3
and/or 12·(M2+)5 have higher catalytic activity than 1·(M2+)2.
In order to evaluate the contribution of the Bpy·Zn2+ complex
to the hydrolysis of HPNP, kobs values with control compounds 3
and 4 were measured. It was found that kobs value of 6.6 × 10−5 s−1
obtained from a mixture of 2·(Zn2+)2 and 4·Zn2+ is almost same as
that obtained from 2·(Zn2+)2 (6.2 × 10−5 s−1). In addition, the kobs
value of 3.5 × 10−5 s−1 for 3·(Zn2+)2 is 5.8 times smaller than that
for 1·(Zn2+)3. These results show that inter- or intra-molecular
reaction catalyzed by Bpy·Zn2+ and DPA·Zn2+ complexes is not
effective for the hydrolysis of HPNP (Table 1(a)–(d)). Similar
results were obtained from Cu2+ (Table 1(e)–(h)). The kobs value of
1.2 × 10−3 s−1 for 1·(Cu2+)3 + 12·(Cu2+)5 is 40 times larger than that
for other control complexes. The increase in the catalytic activity
is ascribed, therefore, to the allosteric conformational transition
of 1 induced by the coordination of effector metal ion to the Bpy
moiety as shown in Fig. 4.
Fig. 6 (a) Plots of pseudo-first-order rate constants (kobs) for the
hydrolysis of HPNP (0.80 mM) at various Zn2+ concentrations in 33%
ethanol–water (HEPES, 25 mM): (a) [1] = 0.40 mM (ꢀ), [2] = 0.40 mM
(᭹) pH 7.7 at 25 ◦C. (b) Plots of pseudo-first-order rate constants (kobs) for
the hydrolysis of HPNP (1.0 mM) at various Cu2+ concentrations in 33%
ethanol–water (HEPES, 25 mM): (a) [1] = 1.0 mM (ꢀ), [2] = 1.0 mM (᭹)
Michaelis–Menten kinetic parameters were evaluated from
saturation kinetic experiments to obtain a further insight into
the rate enhancement observed for 1. Saturation kinetic curves
were obtained from Zn2+ and Cu2+ catalysts. A Lineweaver–Burk
plot was applied to calculate the Michaelis–Menten constant (Km)
and the catalytic constant (kcat) (see supplementary information†).
The results are summarized in Table 1. A significant increase
in the value of kcat (4.1 times for Zn2+ and 55 times for Cu2+)
was obtained from the conditions (b) and (d) compared with (a)
and (c) in Table 2. A conformational change of 1 induced by the
pH 7.7 at 25 ◦C. Inset is an enlarged view for 2 and Cu2+
.
third M2+ complexation, that is, allosteric transition, enhanced the
rate of hydrolysis but not by a change in the substrate affinity. This
should be a consequence of the preferable preorganization of two
DPA·M2+ complex units toward the hydrolysis; the conformational
change of this catalyst would facilitate the attack of intramolecular
hydroxyl ion sitting on M2+ to the substrate.
Table 1 Pseudo-first-order rate constants (kobs) for the hydrolysis of HPNP Zn(II) and Cu(II) complexes of 1–4a
103kobs/s−1
(a) 1 (0.40 mM) with Zn(ClO4)2 (2.0 mM)b
(b) 2 (0.40 mM) with Zn(ClO4)2 (2.0 mM)b
(c) 2 (0.40 mM) + 4 (0.40 mM) with Zn(ClO4)2 (2.0 mM)b
(d) 3 (0.40 mM) with Zn(ClO4)2 (2.0 mM)b
(e) 1 (1.0 mM) with Cu(ClO4)2 (3.0 mM)c
0.20
0.062
0.066
0.035
1.2
0.030
0.028
0.032
(f) 2 (1.0 mM) with Cu(ClO4)2 (2.0 mM)c
(g) 2 (1.0 mM) + 4 (1.0 mM) with Cu(ClO4)2 (3.0 mM)c
(h) 3 (1.0 mM) with Cu(ClO4)2 (2.0 mM)c
a In 33% ethanol–water (HEPES, 25 mM), pH 7.7 at 25 ◦C. b [HPNP] = 0.8 mM. c [HPNP] = 1.0 mM
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sufficiently fast for monitoring at 25 ◦C. We confirmed that the
metal binding process are almost the same as those observed at
25 ◦C. The kobs value of 3.6 × 10−4 s−1 for 1·(Zn2+)3 was 33 times
larger than that for 1·(Zn2+)2 (kobs = 1.1 × 10−5 s−1). In the case of
Cu2+ system, the hydrolysis reaction with 1·(Cu2+)2 was too slow
to follow. On the contrary, kobs for 1·(Cu2+)3 was evaluated to be
1.4 × 10−5 s−1, which is consistent with the data demonstrated by
other groups.6 These results clearly show that 1·(M2+)n can also
alter their catalytic activities in the hydrolysis reaction of ApA.
Table 2 Michaelis–Menten kinetic parameters estimated by Lineweaver–
Burk plots for HPNP cleavage; in ethanol–water (HEPES, 25 mM), pH 7.7
at 25 ◦
C
103kcat/s−1
103Km/M
(a) 1 (0.40 mM) with Zn(ClO4)2 (0.8 mM)
(b) 1 (0.40 mM) with Zn(ClO4)2 (2.0 mM)
(c) 1 (1.0 mM) with Cu(ClO4)2 (2.0 mM)
(d) 1 (1.0 mM) with Cu(ClO4)2 (3.0 mM)
0.51
2.10
0.26
14.3
1.93
3.13
3.56
12.7
Upon addition and removal of the third metal ion bound to
BPy, we confirmed the conversion between 1·(M2+)3 and 1·(M2+)2
by UV-vis spectroscopic method (supplementary information†);
this indicates that the ON–OFF experiments using this system
can be demonstrated. At first, HPNP was added to a solution of
1·(M2+)2. Absorbance at 406 nm assignable to p-nitrophenolate
slowly increased. After 3 min, the addition of metal ion to the
reaction mixtures caused a sharp increase in the formation of p-
nitrophenolate. Next, the removal of metal ion by the addition
of EDTA resulted in the decrease in the rate of hydrolysis again
(Fig. 7). The results further support the view that the third metal
ion acts as an allosteric effector for this hydrolysis reaction.
Conclusions
In conclusion, we have demonstrated that compound 1 bearing
two types of Zn2+ or Cu2+ binding sites exhibits an allosteric
response in the catalytic activities toward the hydrolysis of
phosphodiester substrates such as HPNP and ApA. We anticipate,
therefore, that such a supramolecular catalytic system would
further produce intelligent artificial systems responding to various
types of chemical stimuli.
Experimental
1H NMR, absorption spectra, MALDI TOF MS, ESI MS spectra
were measured with Bruker DMX 600, Shimadzu UV-2500,
Perseptive Voyager RP MALDI TOF spectrometer, Perseptive
Mariner and JASCO J-720 WI, respectively.
For the cleavage of ApA the reaction was followed by HPLC
by withdrawing 10 ll of the mixture from 100 ll of the reaction
solution and 100 ll of mobile phase condition A (see below).
Separation condition: columnC30-UG-5(4.6nm, 250 nm); mobile
phase (condition A = 10 mM NaH2PO4, 10 mM Sodium octane-
sulfonate, 5 mM EDTA-2Na); eluent gradient with acetonitrile (A:
100 - 80% (0–8.0 min), 80–20% (8.0–8.1 min), 30% (8.1–25.0 min.),
30–100% (25.0–25.1 min), and 100% (25.1–30 min)); flow rate
1.0 ml min−1; temperature 40 ◦C; detection wavelength 254 nm.
Hydrolysis of ApA by Zn(II) and Cu(II) complexes
With such an outstanding catalyst in hands we turned to
more appealing substrates such as RNA dinucleotides; namely
ApA. The cleavage process was followed by HPLC, monitoring
the disappearance of the substrate and the formation 3ꢀ-AMP and
adenosine. The kobs values were calculated from changes in these
species (Table 3). We set the reaction temperature at 35 ◦C for Zn2+
complex and 45 ◦C for Cu2+ complex because the reaction was not
Table 3 Pseudo-first-order rate constants (kobs) for the hydrolysis of ApA
by Zn(II) and Cu(II) complexes of 1a
104kobs/s−1
Syntheses
b
1 with 2 equiv. Zn2+
1 with 8 equiv. Zn2+
1 with 2 equiv. Cu2+
1 with 3 equiv. Cu2+
0.11
3.6
—
b
c
c
4,4ꢀ-Dimethyl-2,2ꢀ-bipyridine 414 and HPNP15 were synthesized
according to the previously reported methods. 1H NMR, absorp-
tion spectra, MALDI TOF MS, ESI MS spectra were measured
with Bruker DMX 600, Shimadzu UV-2500, Perseptive Voyager
RP MALDI TOF spectrometer, Perseptive Mariner and JASCO
J-720 WI, respectively.
d
1.4
a In 37% ethanol–water (HEPES, 25 mM), pH 7.7, [ApA] = 0.10 mM.
b [1] = 1.0 mM, [Zn(ClO4)2] = 2.0, 8.0 mM, at 35 ◦C. c [1] = 1.0 mM,
[Cu(ClO4)2] = 2.0, 3.0 mM, at 45 ◦C. d The rate was too slow to evaluate.
Fig. 7 Control of hydrolysis for HPNP upon addition and removal of allosteric metal ion: (a) [1] = 0.40 mM, [Zn(ClO4)2] = 2.0 mM, [HPNP] = 1.0 mM
(0 min), addition of Zn(ClO4)2 (3.0 mM) after 3 min, and addition of EDTA (3.0 mM) after 6 min in ethanol–water (HEPES, 25 mM), pH 7.7 at 25 ◦C.
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4,4ꢀ-Bis(dipicolylaminomethyl)-2,2ꢀ-bipyridine (1)
was added saturated aqueous NaHCO3 (50 ml), and the product
was extracted with ethyl acetate (3 × 30 ml). The organic extract
was washed with brine (3 × 30 ml). The organic extract was dried
over Na2CO3, filtered, and concentrated to give crude 5 (408 mg,
28% calculated by 1H NMR spectrum). The residue was subjected
to the next step without further purification.
4,4ꢀ-Diformyl-2,2ꢀ-bipyridine (200 mg, 0.94 mmol) and 2,2ꢀ-
dipicolylamine (0.34 ml, 1.9 mmol) were dissolved in 30 ml of
dichloroethane. To this solution sodium triacetoxyborohydride
(540 mg, 2.5 mmol) was added and the resultant mixture was
stirred at room temperature for 18 h. After addition of 5%
aqueous NH3, the insoluble material was filtered off. The filtrate
was concentrated in vacuo. The residual aqueous solution was
extracted by chloroform and dried over anhydrous sodium sulfate.
The solution was evaporated to dryness, the oil residue being
chromatographed (silica gel, chloroform–methanol = 50 : 1 with
four drops of 28% aqueous NH3) to give yellow solid. The resultant
yellow solid was washed by ether to give 1 as a white powder
(253 mg, 46%); mp 135.9–137.9 ◦C. 1H NMR (600 MHz, CDCl3,
27 ◦C, d/ppm, J/Hz): 3.81 (s, 4H), 3.85 (s, 8H), 7.15 (t, J = 6.0,
4H), 7.47 (d, J = 4.8, 2H), 7.59 (d, J = 7.7, 4H), 7.67 (t, J = 7.5,
4H), 8.38 (s, 2H), 8.53 (d, J = 4.5, 4H), 8.63 (d, J = 4.9, 2H);
MALDI TOF MS (dithranol matrix): calc. (found) for [1 + H]+:
579.71 (579.71). Calc. for C36H34N8·0.5CH3OH: C, 73.71; H, 6.10;
N, 18.84. Found: C, 73.50; H, 5.93; N, 18.77%.
4-Bromomethyl-4ꢀ-methyl-2,2ꢀ-bipyridine (7)
1
To a solution of crude 6 (312 mg, 0.58 mmol calculated by H
NMR spectrum) with dry DMF (10 ml) were added 1,2-dibromo-
1,1,2,2-tetrafluoroethane (0.14 ml, 1.16 mmol) and CsF (0.2 g,
1.3 mmol) with stirring under N2 at room temperature for 2 hours.
The resultant precipitate was filtered off. To the filtrate was added
water (50 ml), and the product was extracted with ethyl acetate (3 ×
30 ml). The organic extract was washed with brine and water (3 ×
30 ml, respectively). The organic extract was dried over Na2CO3,
filtered, and concentrated to give crude 6 (269 mg, 35% calculated
by 1H NMR spectrum). The residue was subjected to the next step
without further purification.
4-Dipicolylaminomethyl-4ꢀ-methyl-2,2ꢀ-bipyridine (3)
3,3ꢀ-Dibromomethylbiphenyl (5)
1
To a solution of crude 7 (269 mg, 0.20 mmol calculated by H-
To a solution of 3,3ꢀ-dimethylbiphenyl (0.5 ml, 2.54 mmol) with
dry CCl4 (30 ml) were added BDH (940 mg, 3.30 mmol) and BPO
(108 mg, 0.48 mmol). The mixture was allowed to reflux under N2
for 4 h. The resultant precipitate was filtered off and the filtrate was
evaporated under reduced pressure. The residue was recrystallized
from hexane and chloroform to afford the prod◦uct as a white solid
(200 mg, 22%). 1H NMR (250 MHz, CDCl3, 27 C, d/ppm, J/Hz):
4.56 (s, 4H), 7.46 (m, 6H), 7.61 (s, 2H).
NMR spectrum) with dry DMF (3 ml) and dry acetonitrile (7 ml)
were added K2CO3 (83 mg, 0.60 mmol), KI (10 mg, 0.06 mmol)
and dipicolylamine (0.04 ml, 0.22 mmol) with stirring under N2 at
◦
35 C for 2 hours. The resultant precipitate was filtered off. The
filtrate was washed with brine (3 × 30 ml), and the organic extract
was dried over Na2CO3, filtered, and concentrated. The residue
was purified by column chromatography (silica gel, chloroform–
methanol = 30 : 1 (v/v) saturated 28% aqueous ammonia) to give
1
yellow oily ◦compound 3 (100 mg, 100%). H NMR (600 MHz,
CDCl3, 27 C, d/ppm, J/Hz) 2.43 (s, 3H), 3.81 (s, 2H), 3.85 (s,
4H), 7.15 (m, 3H), 7.46 (d, J = 4.8, 1H), 7.59 (d, J = 7.8, 2H),
7.66 (t, J = 7.6, 2H), 8.20 (s, 1H), 8.38 (s, 1H), 8.53 (m, 3H), 8.63
(s, 1H); MALDI TOF MS (CHCA matrix): calc. (found) for [3 +
H]+: 382.20 (382.24). Calc. for C24H23N5·0.25CHCl3: C, 70.81; H,
5.70; N, 17.03. Found: C, 70.93; H, 5.88; N, 16.81%.
3,3ꢀ-Bis(dipicolylaminomethyl)biphenyl (2)
To
a 5 (195 mg,
solution of 3,3ꢀ-dibromomethylbiphenyl
0.57 mmol) with dry acetonitrile (10 ml) were added K2CO3
(400 mg, 2.89 mmol), KI (38 mg, 0.23 mmol) and dipicolylamine
(0.20 ml, 1.1 mmol). The mixture was allowed to stir at 35 ◦C under
N2 for 5 h. The resultant precipitate was filtered off and the filtrate
was evaporated under reduced pressure. The residue was dissolved
in chloroform and washed with aqueous Na2CO3 (2 × 20 ml). The
organic extract was dried over anhydrous Na2SO4. After removal
of solvent under reduced pressure, the residue was purified by
column chromatography (silica gel, chloroform–methanol = 25 :
1 (v/v) saturated 28% aqueous ammonia) to give yellow ◦oil
compound 2 (158 mg, 48%). 1H NMR (600 MHz, CDCl3, 27 C,
d/ppm, J/Hz) 3.77 (s, 4H), 3.86 (s, 8H), 7.14 (t, J = 5.9, 4H),
7.42 (m, 6H), 7.63 (m, 10H), 8.52 (d, J = 4.7, 4H); MALDI TOF
MS (dithranol matrix): calc. (found) for [2 + H]+: 577.73 (577.40).
Calc. for C38H36N6·1.0CHCl3: C, 67.29; H, 5.36; N, 12.07. Found:
C, 67.35; H, 5.48; N, 12.00%.
Acknowledgements
This study was supported partially by a Grant-in-Aid for Scientific
Research B (17350071) (to M. T.) of the Ministry of Education,
Culture, Science, Sports, and Technology (Japan). We thank Prof.
Itaru Hamachi, Prof. Akio Ojida and Dr Masa-aki Inoue for
helpful discussions. M. T. and S. T. thank Prof. Fumito Tani and
Mr Jun Hagiwara for ESI MS measurements. S. T. thanks JSPS for
the Research Fellowship for Young Scientists for financial support.
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