Synthesis, characterization, and activity of cyclotriphosphazene-cyclene conjugates

Article abstract of DOI:10.1080/10426507.2012.741159

Two novel cyclotriphosphazene derivatives were synthesized from hexachloro cyclotriphosphzene. Their structures were characterized by ¹H, ³¹P, and ¹³C NMR spectroscopy as well as by IR spectroscopy and electrospray ionizat

Full text of DOI:10.1080/10426507.2012.741159

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Phosphorus, Sulfur, and Silicon and the
Related Elements
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Synthesis, Characterization, and
Activity of Cyclotriphosphazene-Cyclene
Conjugates
Jin-Hui Wang a , Cui-Cui Wang a , Di Zhang a , Cun-Jiang Liu a , Yong
Ye a b & Yufen Zhao a b c
a Department of Chemistry, Key Laboratory of Chemical Biology
and Organic Chemistry of Henan Province , Zhengzhou University ,
Zhengzhou , 450052 , China
b Department of Chemistry, Key Laboratory of Bioorganic Phosphorus
Chemistry and Chemical Biology (Ministry of Education) , Tsinghua
University , Beijing , 100084 , China
c Department of Chemistry, Key Laboratory for Chemical Biology of
Fujian Province , Xiamen University , Xiamen , 361005 , China
Accepted author version posted online: 02 Nov 2012.Published
online: 08 May 2013.
To cite this article: Jin-Hui Wang , Cui-Cui Wang , Di Zhang , Cun-Jiang Liu , Yong Ye &
Yufen Zhao (2013) Synthesis, Characterization, and Activity of Cyclotriphosphazene-Cyclene
Conjugates, Phosphorus, Sulfur, and Silicon and the Related Elements, 188:1-3, 54-58, DOI:
10.1080/10426507.2012.741159
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Phosphorus, Sulfur, and Silicon, 188:54–58, 2013
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2012.741159
SYNTHESIS, CHARACTERIZATION, AND ACTIVITY OF
CYCLOTRIPHOSPHAZENE-CYCLENE CONJUGATES
Jin-Hui Wang,1 Cui-Cui Wang,1 Di Zhang,1 Cun-Jiang Liu,1
Yong Ye,1,2 and Yufen Zhao1,2,3
1Department of Chemistry, Key Laboratory of Chemical Biology and Organic
Chemistry of Henan Province, Zhengzhou University, Zhengzhou 450052, China
2Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry
and Chemical Biology (Ministry of Education), Tsinghua University, Beijing
100084, China
3Department of Chemistry, Key Laboratory for Chemical Biology of Fujian
Province, Xiamen University, Xiamen 361005, China
GRAPHICAL ABSTRACT
Abstract Two novel cyclotriphosphazene derivatives were synthesized from hexachloro cy-
clotriphosphzene. Their structures were characterized by 1H, 31P, and 13C NMR spectroscopy
as well as by IR spectroscopy and electrospray ionization-mass spectrometry (ESI-MS). The
Zn complex of 4d was effective in hydrolytic DNA cleavage reactions.
Keywords Synthesis; cyclotriphosphazene; DNA; cleavage reactions
INTRODUCTION
DNA phosphodiester bonds are exceptionally resistant to hydrolysis at neutral pH and
25◦C: the half-life for the cleavage of dimethyl phosphate is estimated to be 130,000 years.1
Consequently, much effort has been devoted to the development of efficient catalysts for
this reaction.2–5 Chemical nucleases present some advantages over conventional enzymatic
nucleases in that they are smaller in size and thus can reach more sterically hindered regions
Received 5 August 2012; accepted 15 October 2012.
This work was financially supported by the NSFC (Nos. 20972143, 20972130) and the Program for New
Century Excellent Talents in University (NCET-11–0950).
Address correspondence to Yong Ye, Department of Chemistry, Key Laboratory of Chemical Biology
and Organic Chemistry of Henan Province, Zhengzhou University, Zhengzhou 450052, China. E-mail: yey-
ong03@tsinghua.org.cn
54

ACTIVITY OF CYCLOTRIPHOSPHAZENE-CYCLENE CONJUGATES
55
of a macromolecule. Therefore, designing new small molecules suitable for cleaving DNA
under hydrolytic conditions is of considerable importance.
Phosphazenes, particularly cyclophosphazenes, have received considerable interest,
not only because of their wide spectrum of chemical and physical properties, but also
due to their importance in synthetic chemistry.6–9 Different side-group structures affect the
chemical and physical properties of ring systems and high polymers based on a phosp-
hazene skeleton.10–17 Recently, we reported a series of polydentate cyclotriphosphazene
ligands,18,19 which showed good nuclease activity with hydrolytic cleavage mechanism. In
continuation of our efforts to obtain more efficient DNA cleavage reagent, we report here
the synthesis of two novel star-branched cyclotriphosphazene derivatives.
RESULTS AND DISCUSSION
The synthetic route to the new cyclotriphosphazene-cyclene conjugates is shown in
Scheme 1. Compound 2 was synthesized from the reaction of hexachloro cyclotriphosp-
hazene (1) with the potassium salt of 4-hydroxybenzaldehyde, followed by reduction with
sodium borohydride in THF/MeOH. Several reagents were available for converting hydroxy
groups of 2 to alkyl bromides. Bromination reagents based on hydrobromic acid/sulfuric
acid were found unsuitable and the reaction conditions were hard to control. Insoluble
intermediates were formed from the reaction with the hydroxy groups of compound 2.
The bromination reaction was achieved by using PBr3. Compound 3 was characterized by
1H NMR spectroscopy and electrospray ionization-mass spectrometry (ESI-MS). 1H NMR
spectroscopy showed that all six hydroxy groups were replaced by bromine atoms. In the
spectrum, two doublets for the aromatic ring protons appeared at 6.89 and 7.25 ppm, and a
single peak for the protons of the bromomethyl group was observed at 4.48 ppm.
Scheme 1
Compound 3 was treated with Boc-protected cyclene to generate compounds 4a,b.
Subsequent deprotection of the Boc group was then carried out by treatment of a CH2Cl2
solution of 4a,b with trifluoroacetic acid (TFA) at room temperature. Basification with
NaOH then yields the free cyclotriphosphazenes 4c,d.
1
The new cyclotriphosphazenes 4c,d were characterized by H, 31P, and 13C NMR
spectroscopy, IR spectroscopy and ESI-MS. ESI mass spectra for all compounds showed
the respective [M+H]+ ion. Their structures are similar and 4c was chosen as an example
for the discussion. In its 31P NMR spectrum a single peak is observed at 9 ppm. In the 1H

56
J.-H. WANG ET AL.
Figure 1 Effect of time on the cleavage activity of pUC19 DNA by the Zn complex of 4d (10 μM). pUC19 DNA:
0.5 ug/uL, 37◦C. Na2HPO4/HCl buffer (0.05 M Na2HPO4/HCl, 0.1 M NaCl), pH = 7.5. Lane 1: DNA control,
lanes 2–8: 1, 3, 5, 7, 12, 24, and 48 h.
NMR spectrum, two doublets at 6.9 ppm and 7.27 ppm were assigned to the protons of
the benzene ring. A singlet for methylene protons was observed at 3.8 ppm. A singlet at
3.52 ppm and two triplets at 3.11 and 2.58 ppm were assigned to the methylene protons of
the cyclone moiety.
The absorption band at 3039 cm−1 in the IR spectrum of 4c is assigned to the
stretching vibration of C–H. The stretching vibration of the methylene moiety appears at
2357 cm−1. The absorptions of the stretching vibration of the C=C bond appear at 1600
and 1499 cm−1. The absorption of the stretching vibration of C–O bond at 1155 cm−1 was
also observed.
The progress of the hydrolytic DNA cleavage by the Zn complex of 4d at pH 7.5 is
shown in Figure 1. Obviously the cleavage by the metal complex is time dependent. With
increasing reaction time the amount of form I of DNA diminished gradually, whereas the
amount of form II increased.
CONCLUSION
In conclusion, we designed and synthesized two novel cyclotriphosphazene deriva-
tives. The reactions are convenient and efficient. The target compounds were characterized
by 1H, 31P, and 13C NMR spectroscopy, IR spectroscopy and ESI-MS. The Zn complex of
4d was effective in the hydrolytic cleavage of DNA.
EXPERIMENTAL
Melting points were measured using a WC-1 microscopic apparatus and are cor-
rected. Infrared spectra were recorded with a Bruker VECTOR22 spectrophotometer in
KBr pellets. 1H and 13C NMR spectra were recorded using a Bruker DTX-400 spectrome-
ter. 31P NMR spectra are measured with a Bruker AMX 400 MHz spectrometer with 85%
H3PO4 as external reference.
Mass spectra were acquired in positive ion mode using a Bruker ESQUIRE 3000 ion
trap spectrometer equipped with a gas nebulizer probe, capable of analyzing ions up to m/z
6000. The high-resolution mass spectral data for compound 4c was recorded with a Waters
Q-Tof microTM spectrometer by ESI method. The sample was analyzed as a solution in
methanol. All chemicals and materials were of analytical grade and were obtained from
Chenzhuo Company. The solvents used were purified and dried by standard procedures.

ACTIVITY OF CYCLOTRIPHOSPHAZENE-CYCLENE CONJUGATES
57
Preparation of Compound 3
Into a 500-mL round-bottom flask was placed 3.495 g (4 mmol) of 2 with 200 mL of
THF under argon. Then, 3.8 mL (40 mmol) of phosphorus tribromide in 50 mL THF was
added dropwise at 0◦C and the mixture was stirred overnight. The solvent was removed.
The residue was dissolved in CHCl3 and washed with aq. NaHCO3. The organic layer
was dried over Na2SO4 and then the solvent was evaporated under reduced pressure.
After recrystallization from ethyl acetate white solid 4 was obtained (yield 81%). Mp:
144–146 ◦C; 1H NMR (400 MHz, CDCl3): δ = 6.89 (d, J = 8.2 Hz, 12H), 7.25 (d, J = 8.4
Hz, 12H), 4.48 (s, 12H); ESI-MS: m/z 1252 [M+H]+.
Synthesis of Compounds 4a,b
To a solution of compound 3 (0.254 g, 0.2 mmol) in 20 mL of CH3CN
was added 1 g Na2CO3. After the mixture was warmed to 80◦C, a solution of
BocCyclen (0.821 g, 1.7 mmol) in 10 mL of CH3CN was dropped in under N2,
followed by stirring for 48 h. Then, the reaction mixture was concentrated in
vacuo. Column chromatography of the residue (eluant: CHCl3/MeOH 50:1) pro-
vided 0.668 g (91.4%) of crude compound 4b; 1H NMR (400 MHz, CDCl3):
δ = 7.08 (d, J = 8 Hz, 12H, –C6H4), 6.93 (s, 12H, –C6H4), 3.74 (s, 12H, –CH2–pH), 3.55
(s, 24H, –CH2–cyclen), 3.39–3.26 (m, 48H, –CH2–cyclen), 2.61 (s, 24H, –CH2–cyclen),
1.44 (s, 162H, –CH3); 13C NMR (100 MHz, CDCl3): δ = 156.0, 155.6, 155.2, 149.9,
132.8, 131.4, 120.5, 79.5, 79.2, 55.1, 54.2, 53.4, 49.9, 47.5, 47.2, 28.7, 28.4; 31P NMR
(162 MHz, CDCl3): δ = 7.9; ESI-MS: [M+H]+ 3622.
By a similar method 4a was obtained in 90% yield. 1H NMR (400 MHz, CDCl3): δ
= 6.88 (d, J = 8 Hz, 12H, –C6H4), 6.73 (s, 12H, –C6H4), 3.64 (s, 12H, –CH2–aryl), 3.25
(s, 24H, –CH2–cyclen), 3.22–3.20 (m, 24H, –CH2–cyclen), 2.60 (s, 24H, –CH2–cyclen),
1.42 (s, 108H, -CH3); 13C NMR (100 MHz, CDCl3): δ = 156.0, 155.7, 155.4, 150.0, 132.8,
131.6, 121.5, 79.5, 70.9, 55.2, 54.2, 49.9, 47.3, 28.6, 28.4; 31P NMR (162 MHz, CDCl3):
δ = 8.6; ESI-MS: [M+H]+ 2742.
Synthesis of Compounds 4c,d
To a solution of 4b (0.668 g, 1.84 mmol) in dry CH2Cl2 (15 mL) at 0◦C, CF3COOH
(3 mL) was added dropwise under N2. The mixture was stirred for 7 h at room temperature.
After removal of the solvent, the residue was adjusted to pH 12 by aqueous solution of
NaOH and extracted with CHCl3 (3 × 30 mL). The organic phase was collected and dried
over Na2SO4. After removal of the solvent 0.313 g solid 4d was obtained. Yield: 93.8%; 1H
NMR (400 MHz, CDCl3): δ = 7.11 (d, J = 8.4 Hz, 12H, –C6H4), 6.99 (d, J = 8.4 Hz, 12H,
–C6H4), 3.59 (s, 12H, –CH2–aryl), 2.78 (d, J = 4.8 Hz, 24H, –CH2–cyclen), 2.64 (d, J =
4.8 Hz, 24H, –CH2–cyclen), 2.55 (d, J = 4 Hz, 48H, –CH2–cyclen); 13C NMR (100 MHz,
CDCl3): δ = 149.7, 134.8, 129.9, 120.8, 58.3, 50.9, 47.0, 46.2, 45.0; 31P NMR (162 MHz,
CDCl3): δ = 8.3.
Similarly compound 4c was obtained in 90% yield, mp: 68–70◦C. 1H NMR (400 MHz,
D2O): δ = 7.27 (d, J = 8.4 Hz, 12H), 6.90 (d, J = 8.4 Hz, 12H), 3.80 (s, 12H), 3.52 (s,
24H), 3.11 (t, J = 5.6 Hz, 24H), 2.58 (t, J = 5.6 Hz, 24H); 13C NMR (100 MHz, D2O):
δ = 149.3, 133.1, 131.8, 120.8, 57.7, 47.2, 43.6, 42.1; 31P NMR (162 MHz, CDCl3): δ =
9.0; IR (cm−1, KBr): 3039, 2911, 2843, 2357, 1895, 1660, 1599, 1552, 1499, 1450, 1356,

58
J.-H. WANG ET AL.
1266, 1198, 1155, 948, 887, 837, 747, 694; ESI-MS: [M+H]+: 1541.7. HR-MS (ESI): m/z
Calcd. for C78H120N21O6P3 [M+H]+: 1540.9012, found 1540.9010.
DNA Cleavage Experiments
The hydrolytic DNA cleavage activities of 4 were evaluated by monitoring the con-
version of supercoiled plasmid DNA (pUC19) (Form I) to nicked circular DNA (Form II)
or linear DNA (Form III) by agarose gel electrophoresis. In a typical experiment, reaction
solutions were prepared by mixing 2 μL of a Zn(II) complex solution (50 μM, pH 7.5)
with 2 μL of supercoiled DNA (50 μM), 2 μL of Na2HPO4/HCl buffer (100 mM), fol-
lowed by dilution with sterilized water to a final volume of 20 μL. All reaction mixtures
were incubated at 37◦C. Hydrolytic cleavage experiment was performed both at pH 7.5 in
Na2HPO4/HCl buffer. All reactions were quenched by addition of 2 μL of a quench buffer
containing EDTA, bromophenol blue, xylene cyanol, and sucrose. Cleavage products were
separated by electrophoresis on 1% agarose gels in 1 · TAE buffer for 1.5 h at 60 V and
stained with 0.5 μg/mL EB for 45 min. The gel was visualized by UV transillumination.
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