Metalated hybrid polymers as catalytic reagents for phosphate ester hydrolysis and plasmid modification

Article abstract of DOI:10.1016/j.bmcl.2003.12.082

Pendant pyrazolylcyclophosphazene containing hybrid cross-linked polymer (CPPL) has been utilized for binding Zn(II). The metalated polymer (CPPL-Zn) has been found to be very effective catalyst for the hydrolysis of a RNA model phosphodiester substrate [

Full text of DOI:10.1016/j.bmcl.2003.12.082

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1559–1562
Metalated hybrid polymers as catalytic reagents for phosphate
ester hydrolysis and plasmid modification
Vadapalli Chandrasekhar,* Pravas Deria, Venkatasubbaiah Krishnan,
Arunachalampillai Athimoolam, Sanjay Singh, C. Madhavaiah, S. G. Srivatsan
and Sandeep Verma*
Department of Chemistry, Indian Institute of Technology, Kanpur-208016, India
Received 21 October 2003; revised 22 December 2003; accepted 22 December 2003
Abstract—Pendant pyrazolylcyclophosphazene containing hybrid cross-linked polymer (CPPL) has been utilized for binding
Zn(II). The metalated polymer (CPPL–Zn) has been found to be very effective catalyst for the hydrolysis of a RNA model
phosphodiester substrate [2-(hydroxypropyl)-p-nitrophenyl phosphate, hNPP]. In addition, CPPL–Zn also cleaved supercoiled
plasmid DNA pBR322 thus providing a novel structural motif of inorganic–organic hybrid polymers as synthetic nucleases.
# 2004 Elsevier Ltd. All rights reserved.
Chlorocyclophosphazenes are excellent precursors for
the construction of multi-site coordination ligands due
to their robust framework and reactive periphery.1
Thus, replacement of the P–Cl bond in N3P3Cl6 by
appropriate substituents containing donor atoms can
afford a library of diverse ligand systems.2 We have
carried out extensive work on the pyrazolyl cyclophos-
phazenes N3P3(3,5-Me2Pz)6 and gem-N3P3Ph2(3,5-
Me2Pz)4.3 More recently we have incorporated the pyr-
azolylcyclotriphosphazene structural motif as a pendant
group in a cross-linked polymer, CPPL, and have
shown its utility for binding Cu(II).4 In this paper, we
report the preparation, characterization and catalytic
activity of CPPL–Zn.5 The effectiveness of CPPL–Zn as
a heterogeneous catalyst has been tested by choosing
hydrolysis of a RNA model substrate [2-(hydroxy-
propyl)-p-nitrophenyl phosphate, hNPP]. We have also
found that CPPL–Zn is effective towards cleaving
supercoiled plasmid DNA pBR322 in the absence of
other exogenous reagents. Though copper(II) has
slightly higher Zeff/r value6 to polarize the coordinated
water in lower pH, we have chosen zinc in view of its
coordination flexibility. This study represents one of the
foremost examples of a catalytic application for this
widely studied family of pyrazolylcyclophosphazene
polymer systems.
The synthetic strategy followed for the preparation of
the multi-site coordinating cross-linked polymer CPPL
and its zinc complex, CPPL–Zn, is outlined in Scheme
1. The amount of zinc incorporated in the polymeric
matrix was estimated by atomic absorption spectro-
scopy and was found to be 11.5 mg/g of the polymer.
The metal ion does not leach out of CPPL–Zn even
after prolonged incubation in the reaction buffer used
for the hydrolysis of the phosphate diester, suggesting
high stability of the metal–ligand system. This is in con-
.
trast to the model compound N3P3(3,5-Me2Pz)6 ZnCl2
which decomposes in aqueous methanol. The proposed
coordination environment around zinc(II) in CPPL–Zn
is based on an X-ray structure reported in the literature.7
Hydrolysis of the RNA model substrate, hNPP assis-
ted by CPPL–Zn was conveniently monitored by the
time-dependent release of p-nitrophenolate anion
(e400=1.65ꢀ104 Mꢁ1cmꢁ1)8 and was found to proceed
with a significant rate enhancement. Pseudo-first-order
rate constant (kobs) for the hydrolysis of hNPP
(1.52ꢀ10ꢁ3 minꢁ1) shows a 1000-fold rate enhancement
over the uncatalyzed hydrolysis.9 Importantly, the use
of unmetalated cross-linked polymer CPPL as a control
for the acceleration of phosphate ester hydrolysis under
conditions similar to those used for CPPL–Zn, revealed
a lack of rate enhancement, indicating a crucial role for
Keywords: Cyclophosphazene; Pendant polymer; Catalyst; Zinc;
Phosphate ester; Hydrolysis.
* Corresponding authors. Tel.: +91-5122-597259; fax: +91-5122-
597259; e-mail: vc@iitk.ac.in
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.082

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V. Chandrasekhar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1559–1562
coordinated zinc(II) ions in CPPL–Zn. This system
followed typical saturation kinetics as evident from the
Vi versus concentration of substrate plot (Fig. 1), thus
prompting us to perform a thorough kinetic analysis.
time-dependant 31P NMR analysis. Exclusive formation
of cyclic phosphate with increasing peak intensity at
18.6 ppm, as a function of time, clearly indicates acti-
vation of internal hydroxyl group perhaps by the metal-
bound hydroxide acting as a general base (Fig. 3).12
Michaelis–Menten kinetic parameters, Km and Vmax
,
were determined from the Lineweaver–Burk plot (1/Vi
versus 1/[S] plot, Fig. 2) and were found to be 1.53 mM
and 3.2ꢀ10ꢁ3 mMmin ꢁ1, respectively.
In order to demonstrate that the hydrolytic activity is
solely due to zinc contained in the cross-linked polymer
and not due to the leached out metal ions, the following
experiments were carried out. In the reaction arrest
experiment, CPPL–Zn was filtered from the reaction
mixture after a reaction time of 4 h and the hydrolysis of
hNPP was further monitored for 45 h. The hydrolysis
was completely arrested as soon as the polymer was
filtered, which strongly suggests that the hydrolytic
activity is solely due to the coordinated zinc ions in the
polymeric matrix (Fig. 4). Secondly, AAS analysis of
the fresh and used catalyst confirmed that the metal ion
has not leached out during the course of the catalytic
A profile of the initial velocity (Vi) versus pH displayed a
strong dependence of rate on pH variation. The bene-
ficial role of transition metal ions in assisting phosphate
ester hydrolysis is well documented.10 We believe that
the coordinated metal ion in our system activates the
water molecule and electrostatically mitigates the negative
charge on the phosphate moiety by its Lewis acidity.11
The mode of the phosphate ester hydrolysis assisted
by our polymeric matrix was also studied by the
Figure 2. Lineweaver–Burk plot for hNPP hydrolysis.
Scheme 1.
Figure 3. 31P NMR spectra for the hydrolysis of hNPP by CPPL–Zn.
Chemical shifts for hNPP and the cyclic phosphate product are ꢁ4.32
ppm and 18.6 ppm, respectively (ref 12d). The chemical shifts are with
respect to 85% H3PO4 used as external reference.
Figure 1. Saturation kinetics plot for hNPP hydrolysis.

V. Chandrasekhar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1559–1562
1561
Figure 5. Cleavage of supercoiled plasmid DNA pBR322 by CPPL–
Zn. Lane 1: DNA alone; Lane 2: DNA alone (24 h); Lanes 3-5:
DNA+CPPL–Zn (12, 16, 24 h respectively).
Figure 4. Reaction arrest experiment for hNPP hydrolysis.
Figure 6. Lysozyme cleavage experiment by using CPPL–Zn. Lane 1:
Molecular weight markers (Da) (17,000; 14,200; 6,500 from top to
bottom); Lane 2: Lysozyme alone; Lanes 3, 4: Lysozyme with polymer
at 24 and 48 h, respectively.
reaction as in both cases the zinc content remained
invariant. Thirdly, CPPL–Zn was separated from
the reaction mixture, washed, dried and reused for the
hydrolysis of hNPP under the same conditions.
The initial velocities obtained for the catalyzed reaction
with the reused catalyst are comparable with those
obtained for the fresh catalyst (Table 1) indicating that
CPPL–Zn retains its catalytic behavior through several
cycles of hydrolytic reaction.
In summary, we have described phosphate ester and
DNA cleaving properties of a metalated inorganic-
organic hybrid polymer. The catalysis is heterogeneous
in nature and the ease of separation of CPPL–Zn from
the reaction medium and its ready recyclability are few
attractive features of this system that can be further
optimized for diverse chemico-biological applications.
Attempts were made to explore the nuclease activity of
CPPL–Zn towards the cleavage of supercoiled plasmid
pBR322. The cleavage reaction was performed by sus-
pending the polymer in cacodylate buffer containing
pBR322.13 In a 24 h reaction, there was nearly ꢂ30%
conversion of supercoiled DNA form I to nicked form
II (Fig. 5). Here, it is important to mention that the
polymeric system worked under heterogeneous reaction
conditions and it did not require any external activating
agent to produce observed DNA cleavage in contrast to
numerous transition metal ion based complexes, which
require exogenous oxidants to induce strand scission.14
However, CPPL–Zn did not produce any detectable
cleavage of lysozyme even when incubated for pro-
longed time periods (Fig. 6).15 Heterogeneous nature of
DNA cleavage is a useful observation and this prop-
erty can be harnessed in devising possible applications
in selective degradation of nucleic acid contaminants in
various biological applications.
Acknowledgements
We thank DST and CSIR, India for financial support.
P.D., V.K., A.A., C.M. and S.G.S. thank CSIR for the
award of Senior Research Fellowships.
References and notes
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Table 1. Recycle experiment for hNPP hydrolysis with CPPL–Zna
Catalyst
Viꢀ10ꢁ4
(mMmin ꢁ1
)
Fresh CPPL–Zn
First recycle
Second recycle
Third recycle
0.67
0.52
0.58
0.67
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1.49 mmol) and 1, 4-divinylbenzene (80%, mixtures of
isomers, 0.6 g) were polymerized together using AIBN (33
mg, 0.2 mmol) as the initiator in 1,2-dichloroethane. The
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a All hydrolytic reactions were performed in duplicate in 0.01 M N-
ethyl morpholine buffer (pH 8.0, T=30 ꢃC), prepared in 50% aqu-
eous methanol. Weight of the polymer used was 3 mg/3 mL, corres-
ponding to 0.17 mMof Zn if the polymer were to be completely
soluble in the buffer. Concentration of the hNPP was 0.5 mM.

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V. Chandrasekhar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1559–1562
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duplicate in centrifuge tubes containing 3 mL of the sub-
strate prepared in 0.01M N-ethylmorpholine buffer (pH
8.0) in 50% aqueous methanol at 32 ꢃC. Methanol was
used in the reaction solvent to ensure optimum wetting of
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mL of the buffer, corresponding to 0.29 mMof zinc, if the
polymer was completely soluble in the buffer. Concen-
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of zinc if the polymer was to be completely soluble in
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35 ꢃC), containing pBR322 supercoiled plasmid DNA (10
ng/mL, New England Biolabs). 4 mL of methanol was used
for polymer wetting. Individual reactions were quenched
by adding gel loading buffer (5 mL) containing EDTA
(100 mM) at regural time intervals. Reactions were loaded
onto 0.7% agarose gel, containing ethidium bromide (1
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1
1
For Km, Vmax, concentrations of hNPP: 2.7–5.5 mM. For
kobs, concentration of hNPP was 5.5 mM.
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