Polydentate cyclotriphosphazene ligands: Design,

synthesis and bioactivity

Le Wang a, Yong Ye a,b,*, Shang Bin Zhong a, Yu Fen Zhao a,b,*a Key Laboratory of Chemical Biology and Organic Chemistry, Department of Chemistry, Zhengzhou University, Zhengzhou 450052, China

b Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education),

Department of Chemistry, Tsinghua University, Beijing 100084, China

Received 3 July 2008

Abstract

Five multinuclear cyclotriphosphazene ligands were synthesized and tested for their cleavage activities to plasmid DNA. All of

these new compounds were confirmed by MS, 1H NMR, 31P NMR, 13C NMR and IR. Preliminary studies on the cleavage of pUC19

DNA in the presence of metal complexes were performed. The results revealed that these complexes could act as powerful catalysts

under physiological conditions. The complexes 3b + Cu can effectively cleave DNA to nicked form, giving hydrolysis rate constant

of 0.08/h under physiological conditions. An acid–base catalyzed DNA phosphate-diester hydrolysis mechanism was also

proposed.

# 2008 Yu Fen Zhao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Keywords: Cyclotriphosphazene; Polydentate ligand; DNA cleavage; Hydrolysis

Artificial nuclease is an important tool in molecular biology and genetic engineering, and it is also used as a kind of

nucleic acid structure and function probe [1–3]. With the high abilities in recognizing specific DNA sequence and

catalyzing the hydrolysis of phosphate diester bonds, chemical nucleases have rapidly become an invaluable research

tool in the fields of biology, bioorganic chemistry, therapy, and molecular biology [4]. Many complexes have been

designed and studied as artificial enzyme models. Moreover, multi-nuclear complexes have attracted move interest in

this field due to their potential cooperative effects between the metal centers [5].

Cyclotriphosphazene and conjugates with a backbone of alternating phosphorus and nitrogen atoms, have a similar

structure of the benzene ring. Due to the special structure, they can exhibit excellent properties in biochemistry. For

example, biological intermiscibility and degradation product for the non-toxic small molecules [6]. In addition, the

rich substitution chemistry at the phosphorus center has the possibility for introducing other bioactive group. Our

group had reported peptides, which have imidazole group, could act as powerful catalysts for the cleavage of plasmid

DNA under physiological conditions [7,8]. In order for excellent bioactivity, four other N-heterocyclics were

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Chinese Chemical Letters 20 (2009) 58–61

* Corresponding authors at: Key Laboratory of Chemical Biology and Organic Chemistry, Department of Chemistry, Zhengzhou University,

Zhengzhou 450052, China.

E-mail address: yeyong@zzu.edu.cn (Y. Ye).

1001-8417/$ – see front matter # 2008 Yu Fen Zhao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

doi:10.1016/j.cclet.2008.10.020

introduced into new compounds besides imidazole cyclic. In this paper, we design and synthesis five multinuclear

cyclotriphosphazene ligands. The reaction route is as follows (Scheme 1).

Compound 1 was synthesized according to lit. 9, however, we found the yield could increase to 91.5% when sodium

borohydride was added in batches and reaction time is 24 h (lit.: 79.0%) [9]. Compound 2 was firstly synthesized

according to lit. 10 with HBr + H2SO4 as bromination reagent in reflux temperature [10]. In fact, the reaction condition

is hard to control. After optimization design, bromination reaction was finished with PBr3 at 25 8C and got good yield

81%. The target compounds were synthesized by similar method and their 1H NMR, 31P NMR, 13C NMR, IR, and ESI-

MS spectra were recorded. 3c is chosen as an example to discussed. Compound 3c was synthesized firstly in THF and

sodium hydroxideas as base. However, as indicated by the 31P NMR spectra, the reaction has not finished enough. In

addition, there are three or four spots in TLC, which are very close. When triethylamine was selected as base in THF/

CH3OH and reaction time prolonged to 24 h in 50 8C, purity compound 3c was got easily in moderate yield (69.7%)

[11]. From the 31P NMR spectra, a single peak is observed around 8.226 ppm. In 1H NMR spectra, the double peaks

observed around 6.9 ppm and 6.8 ppm assign to the H atom on benzene ring, due to its interaction with each other.

Preliminary studies on the cleavage of pUC19 DNA in the presence of Cu(II) complexes have also been performed

in this paper. The interaction of metal complexes with pUC19 was studied by monitoring the conversion of circular

supercoiled DNA (Form I) to nicked (Form II) and linear (Form III) DNA. The amount of strand scission was assessed

by agarose gel electrophoresis. The results are shown in Fig. 1.

As Fig. 1 shown, Cu(NO3)2 and ligands 3a–3e almost have no bioactivity, and we only found Form I after 24 h at

37 8C. However, when 3a–3e complexes (Cu(II)) were added, we can observe the conversion of circular supercoiled

DNA (Form I) to nicked (Form II). Preliminary study results show that all the tested compounds could act as powerful

catalysts for the cleavage of plasmid DNA under physiological conditions, especially for 3b + Cu.

In order to investigate the cleavage reactivity of these complexes, the quantities of supercoiled, nick and linear DNA

were recorded varying with the time. 48 h later, there was almost no Form III and only Form II appeared, which means

L. Wang et al. / Chinese Chemical Letters 20 (2009) 58–61 59

Scheme 1. (a) PBr3/KHCO3, THF, 25 8C, 36 h, 81%; (b) RH/Et3N, THF/CH3OH, 50 8C.

Fig. 1. Cleavage reactivity of pUC19 DNA (5 mg/mL) catalyzed by Cu(II) complexes in Tris–HCl buffer (0.1 mol/L pH 7.0) at 37 8C for 24 h. Lane

1: DNA control, lane 2: Cu(NO3)2 (1.2 mmol/L), lanes 3–7: ligands 3a–3e (0.2 mmol/L), lanes 8–12: complexes 3a–3e + Cu (0.2 mmol/L).

that the complexes (3b + Cu) have good selectivity. Fig. 2(B) shows the mass fractions of DNA species present during

a reaction under these mild conditions. We could find a time course plot of Form I decrease and Form II formation

during cleavage by complexes 3b + Cu. As shown, the decrease of Form I fits well to a single-exponential decay curve

according to the single-exponential equation [12]:

Ft ¼ F1ð1� ekt1obsÞ þ F2ð1� ekt

2obsÞ þ F0

We used these data to perform simple kinetic analyses. The value for k1obs was obtained from the slope of a best-fit

line of the fraction cleaved versus time (Fig. 2(C)). From this curve fit, the hydrolysis rate constant of the complex

3b + Cu (0.2 mmol/L) was estimated to be 0.08/h (R2 = 0.99) for the decrease of Form I at 37 8C. Burstyn et al. [13]

had reported that the hydrolysis rate constant for pBR322 DNA was 0.0136/h in the present of 1 mmol complexes

[(en)Co(OH)(OH)2]+. Comparing catalyzed hydrolysis constant 0.0136 and itself hydrolysis constant 2 � 10�10

(24 8C, pH 7.4) [14], the constant 0.08 is better and our complexes have better catalyze activity.

We also investigated the affection of the buffer in different pH value (Fig. 3(A)). Fig. 3(B) shows the varying of

mass fractions (Form II) under the condition of different pH values. Mass fractions of DNA plasmid cleavage show a

bell-shaped curve with a maximum around pH 8. This implies a typical acid–base catalysis mechanism [15], with

optimal activity close to the pKa value of the catalyst [16]. A possible active species of the catalyst and a possible

mechanism for complexes catalyzed DNA hydrolysis are shown in Scheme 2.

In this letter, we investigated the cleavage reactivity of the 3b + Cu system and obtained its hydrolysis rate constant

under physiological conditions. An acid–base catalyzed DNA phosphate-diester hydrolysis mechanism was

preliminary proposed according to different pH value of buffer. Thus, the complex 3b + Cu may be used as simple

models in elucidating the precise role of metal ions in natural nuclease. Further research is in progress.

L. Wang et al. / Chinese Chemical Letters 20 (2009) 58–6160

Fig. 2. (A) Effect of time on the cleavage of pUC19 DNA (5 mg/mL) with complex 3b + Cu (0.2 mmol/L) in a buffer (pH 7.0, 0.1 mol/LTris–HCl)at

37 8C. Lane 1: DNA control, lanes 2–8: 1, 3, 5, 7, 12, 24 and 48 h. (B) Mass fractions of DNA species during the cleavage reaction with 3b + Cu

(0.2 mmol/L). (C) Kinetics plot for the decrease of Form I DNA under the above conditions.

Fig. 3. (A) Effect of different pH value on the cleavage of pUC19 DNA (5 mg/mL) with complex 3b + Cu (0.2 mmol/L) in a buffer (pH 7.0, 0.1 mol/

L Tris–HCl)at 37 8C. Lane 1: DNA control, lanes 2–8: pH: 6, 6.5, 7, 7.5, 8, 8.5, resp. (B) Effect of different pH value on pUC19 DNA cleavage by

3b + Cu under the above conditions.

Acknowledgments

We all thank the financial supports from the NNSFC (Nos. 20602032, 20732004 and 20572061).

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L. Wang et al. / Chinese Chemical Letters 20 (2009) 58–61 61

Scheme 2. Proposed mechanism for the hydrolysis of DNA by the 3b + Cu complex.