Efficient syntheses of artificial nucleases containing mono-, di- and tri-[12]aneN3 ligating units through click chemistry

Article abstract of DOI:10.1016/j.inoche.2010.06.009

Mono-, di-, and tri-nucleating [12]aneN₃ ligands 3-5 have been efficiently prepared through the copper-mediated click reactions between propargyl modified [12]aneN₃ 2 and mono-, bis-, and tri-azido compounds. Their corresponding zinc

Full text of DOI:10.1016/j.inoche.2010.06.009

Inorganic Chemistry Communications 13 (2010) 1054–1056
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Inorganic Chemistry Communications
journal homepage: www.elsevier.com/locate/inoche
Efficient syntheses of artificial nucleases containing mono-, di- and tri-[12]aneN3
ligating units through click chemistry
Ju Zan a, Hao Yan a, Zhi-fo Guo a, Zhong-Lin Lu a,b,
⁎
a
The College of Chemistry, Beijing Normal University, Beijing, 100875, PR China
b
State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Mono-, di-, and tri-nucleating [12]aneN3 ligands 3–5 have been efficiently prepared through the copper-
mediated click reactions between propargyl modified [12]aneN3 2 and mono-, bis-, and tri-azido compounds.
Their corresponding zinc(II) and copper(II) complexes showed excellent catalytic activity and strong
synergetic effects in the cleavage of RNA model phosphate diester.
Received 16 April 2010
Accepted 2 June 2010
Available online 9 June 2010
© 2010 Elsevier B.V. All rights reserved.
Keywords:
Artificial nucleases
Click chemistry
[12]aneN3
Methanolysis
Phosphate diester
The development of artificial nucleases capable of mimicking the
action of nature enzymes is a long-standing goal in bioorganic
chemistry [1–6]. These studies will offer many applications, such as in
understanding the chemistry of the natural nucleases, providing
valuable tools in the manipulation of DNA, and developing the drugs
for gene diagnosis and treatments. According to the active structures
of the natural enzymes, much attention has been paid to the design
and preparation of synthetic metallonucleases where metal ions
complexed to suitable organic ligands perform their catalytic function
in the cleavage of phosphodiester bonds [7–12]. Ligands capable of
coordinating two or more metal ions play important roles in the
design of artificial nucleases due to the following advantages of
multinuclear complexes: 1) by double Lewis acid activation of a
phosphate; 2) through bifunctional catalysis in which the metal ions
activate the phosphate and supply a metal-coordinated hydroxide
acted as nucleophile or base; 3) as an electrostatic reservoir of positive
charge to interact with the anionic phosphate to stabilize the
transition state for the phosphoryl transfer reaction; 4) by assisting
the departure of the phosphate's leaving group through coordination.
However, syntheses of these ligands are often expensive and time-
consuming. To develop an efficient method in the preparation of
multi-nucleating ligands is always desirable.
and alkynes, which provides 1,4-disubstituted 1,2,3-triazole ring in
high yield without the need for further purification, without
generating side reactions and proceeding under friendly conditions,
in aqueous media at room temperature. On the other hand, [12]aneN3
unit is a very useful building block in the design and construction of
artificial nucleases due to its facial tridentate coordinating ability. The
systems containing one or more [12]aneN3 units have shown
prominent results in the detailed mechanism studies and excellent
catalytic performance [16–20]. Recently we found that the dinuclear
zinc(II) complex of 1,3-bis-[12]aneN3-propane accelerates the cleav-
age of model phosphate diesters by a remarkable factor of 1012-fold,
when compared to the background reactions [21,22].
With the above consideration, we report here on the syntheses and
characterization of artificial nucleases containing mono-, di- and tri-
nucleating [12]aneN3 ligating units through click reactions between
propargyl modified [12]aneN3 and various azides.
Our syntheses started from tri-tert-butyl-3-hydroxy-1,5,9-triaza-
cyclododecane-1,5,9-tricarboxylate 1, which was prepared according
to the literature reported by Lönnberg [16]. The synthetic route of the
propargyl modified [12]aneN3 compound, tri-tert-butyl-3-(prop-2-
ynyloxy)-1,5,9-triazacyclododecane-1,5,9-tricarboxylate 2 is outlined
in Scheme 1. The reaction of 1 with propargyl bromide in freshly
distilled anhydrous DMF smoothly afforded 2 in 96% yield in the
presence of sodium hydride at room temperature [23].
As a modular synthetic approach, click chemistry has been
explosively applied in all areas of modern chemistry from drug
discovery to material science in recent years [13–15]. This method
refers to a Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition of azides
The mono-, bis-, and tri-azido compounds used for the following
click reactions were prepared from the corresponding bromides
following literature methods [24–26]. Among them, ethyl azide was
not isolated but directly used in the click reaction.
The click reactions between 2 and the mono-, di- and tri-azide
were processed smoothly and efficiently in DMF–H2O–THF mixture
⁎
Corresponding author. The College of Chemistry, Beijing Normal University, Beijing,
100875, PR China. Fax: +86 10 58801804.
E-mail address: luzl@bnu.edu.cn (Z.-L. Lu).
1387-7003/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.inoche.2010.06.009

J. Zan et al. / Inorganic Chemistry Communications 13 (2010) 1054–1056
1055
Scheme 1. Preparation of the propargyl modified [12]aneN3.
Scheme 2. Reagents and conditions: i) VcNa, CuSO4·5H2O, DMF–H2O–THF, RT, 3–4 h; ii) MeOH, HCl(aq.), reflux, 6 h; iii) NaOH (10 M), 0.5 h; iv) Zn(OTf)2 or Cu(OTf)2.
(volume ratio 4:3:3) in the presence of hydrated copper(II) sulfate
and sodium ascorbate at room temperature under N2 (Scheme 2) [27].
The reactions were monitored by TLC and stopped when the starting
material was found disappearing. After the treatment with saturated
NH4Cl aqueous solution, the resulted Boc-protected triazole com-
pounds were isolated through flash chromatography on silica gel with
the yields of 80–86%, they were not completely dried but directly used
in the next step. The removal of Boc protecting groups was processed
in refluxing methanol in the presence of concentrated HCl for 6 h. The
resulted hydrochloride salts were then neutralized with sodium
hydroxide (10 M) to afford the free ligands 3–5.
methanolysis of phosphate diester, 2-hydroxypropyl-p-nitrophenyl
phosphate (HPNPP), which is often used as model compound of RNA
(Scheme 3), were tested [29]. The results are summarized in Table 1.
It can be seen that relative to the methoxide promoted background
reaction, the mono-nuclear, di-nuclear, and tri-nuclear zinc(II)
complexes at 1.0×10−4 mol dm−3 could accelerate the reactions by
6.5×105, 4.4×107, and 1.5×109 folds, respectively. kobs of the di-
nuclear and tri-nuclear zinc(II) complexes are 56 and 210-fold larger,
respectively, than that of the mononuclear complex Zn:3. For the
corresponding copper complexes, excellent catalytic activities were
also observed. At the concentration of 1.0×10−4 mol dm−3, the
mono-nuclear, di-nuclear, and tri-nuclear copper(II) complexes could
accelerate the reactions by 4.9×105, 4.7×107, and 7.3×108 folds,
respectively. The di-nuclear and tri-nuclear complexes also showed
good synergistic effects, kobs of the di-nuclear and tri-nuclear copper
The three new ligands were characterized with 1H NMR, 13C NMR,
and high resolution mass spectra [28]. It can be seen that the protons on
the triazole moieties generally appear at 7.50–7.60 ppm in singlet, the
protons from CH−O−CH2 moieties between triazole and [12]aneN3
units appear at 3.66 (multiplet) and 4.70 (singlet) ppm, respectively.
The protons from methylene groups of [12]aneN3 generally appear
around 1.60, 2.50, and 2.80 ppm as multiplet, which are consistent with
those reported in the literature [16].
The corresponding mono-, di-, and tri-nuclear zinc(II) and copper
(II) complexes were prepared in situ following the literature method,
i.e. by mixing ligand, base, and metal salts in appropriate ratio [21,22].
The catalytic activities of zinc(II) and copper complexes on the
Table 1
Kinetic data for the cleavage of HPNPP (0.04 mM) by zinc(II) and copper(II) complexes
(0.1 mM) with ligands 3, 4, and 5 at 25.0 0.1 °C.
b
Catalyst
kobs s−1
sspHa
kMobesO−s−1
Acceleration
Relative kc
Zn:3
3.1×10−4
1.74×10−2
6.51×10−2
6.2×10−5
5.5×10−3
1.2×10−2
10.04
9.96
9.35
9.40
9.40
8.70
4.77×10−10
3.96×10−10
9.73×10−11
1.26×10−10
1.17×10−10
1.65×10−11
6.5×105
4.4×107
1.5×109
4.9×105
4.7×107
7.3×108
Zn2:4
Zn3:5
Cu:3
Cu2:4
Cu3:5
56
210
89
194
The reaction sspH for the self-buffered metal complex solutions [30].
Data for the methoxide promoted background reaction. Second-order rate
a
b
constants for the cleavage of HPNPP by methoxide are 2.56×10−3 M−1 s−1 [31].
c
kobs(M2:4)/kobs(M:3) or kobs(M3:5)/kobs(M:3).
Scheme 3. Methanolysis of phosphate HPNPP.

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J. Zan et al. / Inorganic Chemistry Communications 13 (2010) 1054–1056
over Na2SO4, filtered, and concentrated in vacuo. The crude material was purified by
flash chromatography on silica gel (EtOAc/Pet. Spirit 1:8) to give 2 as a colorless oil
(0.26 g, 96% yield): 1 H NMR (400 MHz, CDCl3) δ 4.24 (d, J=2.0, 2H), 4.03–3.93 (m,
1H), 3.60–3.33 (m, 8H), 3.25–3.07 (m, 4H), 2.39 (t, J = 2.1, 1H), 2.10–1.93 (m, 2H),
1.86–1.71 (m, 2H), 1.45 (s, 18H), 1.44 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 156.44,
156.16, 80.14, 79.80, 77.36, 75.09, 74.49, 57.45, 49.52, 46.85, 45.15, 29.46, 28.60,
28.55. HR-MS cacld. for C27H48N3O7 (M+H)+: 526.3492. Found: 526.3487.
[24] L. Beaufort, L. Delaude, A.F. Noels, Tetrahedron 63 (2007) 7003.
[25] Farhanullah, T. Kang, E.-J. Yoon, E.-C. Choi, S. Kim, J. Lee, Eur. J. Med. Chem. 44
(2009) 239.
(II) complexes are 89 and 194-fold larger than that of the
mononuclear copper(II) complex.
In conclusion, we have successfully applied the copper-mediated
click reactions for the preparation of mono-, di-, and tri-nucleating
[12]aneN3 ligands in higher yields. The synthetic route is efficient and
versatile. Compound 2 is a useful building block in the construction of
the functional bio-molecules containing [12]aneN3, such as modified
sugar and peptides. The preliminary work has shown that the zinc(II)
and copper complexes of those novel [12]aneN3 ligands are very
efficient in the cleavage of RNA model compound HPNPP. Further
kinetic works in methanol and aqueous solution are being undertaken
in our group.
[26] J. Shen, R. Woodward, J.P. Kedenburg, X. Liu, M. Chen, L. Fang, D. Sun, P.G. Wang, J.
Med. Chem. 51 (2008) 7417.
[27] Caution: due to their potentially explosive character, all reactions involving
mono-, bis-, and tri-azido compounds were carried out with the appropriate
protection under a well-ventilated hood. General procedure for the preparation of
ligands 3–5: i) preparation of triazole compounds through click reactions: to a
solution of 2 (1.8 mmol 0.945 g) in 10 ml of DMF–H2O–THF mixture (volume ratio
4:3:3) were added sodium ascorbate (10 mol%, 35, 5 mg 0.18 mmol), CuSO4·5-
H2O (5 mol%, 14.4 mg 0.09 mmol), and the respective azides to make sure that the
ratio of alkyne and azido groups is 1:1. The mixture was stirred at room
temperature for 4 h, then poured into 10 ml of saturated NH4Cl aqueous solution.
THF was removed under reduced pressure, and the aqueous solution was
extracted with DCM (5×15 ml). The combined organic phases were washed with
water (3×50 ml), dried over Na2SO4, and concentrated in vacuo. The crude
material was purified by flash chromatography on silica gel (EtOAc/Pet. Spirit 5:1)
to give Boc-protected compounds of 3–5 as a white solid with yield of 80–86%. All
these compounds were used without identification for the next step. ii) Removal
of Boc protecting groups through hydrolysis: The above prepared compounds
were dissolved in MeOH (20 ml), and concentrated HCl (1.0 ml) was added to the
above solution. The mixture was stirring under reflux for 5 h. After cooling the
reaction mixture to 0 °C, the hydrochlorides salts of 3–5 were precipitated as
white solid. iii) Preparation of free ligands by neutralization: to the solution of the
above hydrochlorides salts in H2O (20 ml) was added 10 ml of 10 M NaOH. The
mixture was stirred for 0.5 h at room temperature, then extracted with DCM
(7×15 ml). The combined organic phase was dried over Na2SO4 and solid NaOH,
filtered, and concentrated under reduce pressure to get the free ligands 3–5 as
yellow oil.
Acknowledgements
The authors wish to thank the financial assistance from the Beijing
Municipal Commission of Education, the Program for New Century
Excellent Talents in Universities, the Specialized Research Fund for the
Doctoral Program of Higher Education, the Scientific Research
Foundation for the Returned Overseas Chinese Scholars (213006),
and the Ministry of Education of China.
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13
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13
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13
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25 0.1 °C. The stock solutions of the complexes at 1×10−3 mol dm−3 were
prepared in pure methanol by sequential addition of aliquots of stock solutions of
sodium methoxide, ligand and Zn(CF3SO3)2 such that the relative amounts were
0.5:1.0:1.0, 1.0:1.0:2.0, and 1.5:1.0:3.0 for mononuclear, dinuclear, and trinuclear
complexes, respectively.
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1,5,9-triazacyclododecane-1,5,9-tricarboxylate (0.51 mmol 0.25 g) in 4 ml dry DMF
was added propargyl bromide (0.62 mmol 0.095 g) and NaH (1.3 mmol 0.060 g).
The mixture was stirred for 3 h at room temperature. Then 2 ml MeOH was added
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,
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