Phosphodiester modification by zinc metalated adenine polymer with carboxyl pendants

Article abstract of DOI:10.1016/j.bmcl.2006.07.077

This report describes a novel carboxyl pendant containing adenylated polymeric template, its metalation with Zn (II), and manifestation of catalytic activity for the hydrolysis of model phosphodiester, bis(p-nitrophenyl) phosphate (bNPP), and plasmid clea

Full text of DOI:10.1016/j.bmcl.2006.07.077

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5364–5367
Phosphodiester modification by zinc metalated adenine
polymer with carboxyl pendants
a
b,
c
a,
*
*
Yogita Gupta, G. N. Mathur, Masood Parvez and Sandeep Verma
a
Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur 208016, India
Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, India
Department of Chemistry, The University of Calgary, 2500 University Drive NW, Calgary, Canada T2N1N4
b
c
Received 20 April 2006; revised 19 July 2006; accepted 22 July 2006
Abstract—This report describes a novel carboxyl pendant containing adenylated polymeric template, its metalation with Zn (II), and
manifestation of catalytic activity for the hydrolysis of model phosphodiester, bis(p-nitrophenyl) phosphate (bNPP), and plasmid
cleavage. Observation of a bell-shaped pH-Kobs profile suggested influence of pH variation over hydrolysis rate. This metalated
polymer also afforded facile relaxation of pBR322 supercoiled DNA, with an interesting reusability feature intricately associated
with heterogeneous catalysis.
ꢀ
2006 Elsevier Ltd. All rights reserved.
Redox inertness and hard–soft properties of zinc play an
important role in its coordination to various donor envi-
ronments in proteins thus making it available for biolog-
N-(6-purinyl)-caproic acid (1) was prepared according
to a reported method and further derivatized to give a
polymerizable monomer, 9-(4-vinylbenzyl)purine-6-yl
1
8
ical activities. Zinc coordinates to side chains of Cys,
His, Asp, and Glu, with water playing a crucial role in
amino caproic acid (2). Site of alkylation was confirmed
by spectroscopic evaluation and crystal structure studies
2
9
catalytic zinc sites. Several zinc enzymes and deoxyri-
bozymes have been described for their nuclease activity
(Figs. 1 and 2). Acidic polymer (AP1) was synthesized
1
0
using standard free-radical initiation chemistry. AP1
8
3
pertaining to hydrolysis of DNA and RNA. Interaction
4
was metalated with zinc chloride to yield AP1-Zn.
(Scheme 1).
of zinc with nucleobases has been investigated and
there have been reports of structurally characterized
5
zinc–adenine complexes. Several examples of phos-
6
Compound 1 exhibited purine–purine dimerization with
the help of carboxylate group hydrogen bonding with
phate ester hydrolysis and DNA cleavage by zinc com-
7
plexes have also been reported.
In the present paper, we report synthesis, characteriza-
tion, and catalytic activity of AP1-Zn homopolymer.
The effectiveness of AP1-Zn as a heterogeneous catalyst
has been extensively studied by choosing routinely used
phosphodiester substrate, bis(p-nitrophenyl)phosphate
(
by time-dependent release of p-nitrophenolate anion
bNPP). AP1-Zn assisted hydrolysis of bNPP monitored
4
À1
À1
(
e400 = 1.65 · 10 M cm ). Attempts were also done
to explore nuclease activity of the homopolymer toward
super-coiled plasmid, pBR322 relaxation.
Keywords: Plasmid cleavage; Catalysis; Adenine; Polymer; Zinc.
*
Corresponding authors. Tel.: +91 512 259 7643; fax: +91 512 259
436; e-mail: sverma@iitk.ac.in
7
Figure 1. ORTEP diagram of 1 and 2.
0
960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.077

Y. Gupta et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5364–5367
5365
2
1
1
0
5
0
5
8
8.5
9
9.5
pH
Figure 3. pH curve for bNPP hydrolysis catalyzed by AP1-Zn.
À8 À1
À6 À1
s
9
(
.1 · 10
s
(pH 8.28, 50 ꢁC) and 2.24 · 10
1
3
pH 9.2, 35 ꢁC).
Figure 2. POVERAY diagram showing intermolecular hydrogen
bonding in solid state structure of 1 and 2. Color code: Green:
Carbon; Blue: Nitrogen; Red: Oxygen; Pink: Hydrogen.
Such profiles have been reported for other synthetic
phosphodiesterases following hydrolytic pathway for
14
phosphate ester cleavage. It is established that transi-
tion metal ions lower pK of metal-bound water leading
a
to the generation of hydoxyl ion, which can act either as
a general base to activate solvent water or as a nucleo-
phile to attack at the phosphorus center. We assume
that our metal ion coordinated polymeric template acti-
vates water molecules and electrostatically mitigates the
developing negative charge on the phosphate moiety to
exhibit catalytic activity.
We also determined kinetic parameters for this catalyst
under Michaelis–Menten conditions. Michaelis constant
(
(
K ), maximal velocity (Vmax), and turnover number
m
kcat), from corresponding Lineweaver–Burk plot, were
À4
À1
found to be 1.27 mM, 1.25 · 10 mM min , and
4
the unmetalated homopolymers failed to assist phos-
phate ester hydrolysis in a control reaction confirming
the importance of metal center in catalysis.
À5
À1
.8 · 10 min , respectively (Fig. 4). Interestingly,
Scheme 1. Molecular structure of AP1 polymer.
Coordinated zinc ions are responsible for the catalytic
activity of AP1-Zn. We performed reaction arrest exper-
iment to confirm the fact that adventitiously leached out
metal ions are not responsible for hydrolytic assistance.
the Hoogsteen face of another modified purine, and this
dimer afforded extended interaction in the solid state via
base-pairing mediated by N3–H9 interaction. In case of
2
, alkylation at N9 resulted in the loss of dimeric struc-
ture, but a continuous hydrogen bonded supramolecular
lattice was observed due to hydrogen bonding of car-
boxylate group with the Hoogsteen face.
0.2
Commonly employed activated phosphodiester sub-
strate (bNPP) was chosen to evaluate hydrolytic poten-
0.15
1
1
tial of AP1-Zn. Pseudo-first-order rate constants for
bNPP hydrolysis catalyzed by AP1-Zn were determined
for various pH values between 8 and 9.5. A bell-shaped
curve with a maximum at pH 8.7 giving a clear indica-
tion that at this optimal pH, rate of bNPP hydrolysis
was fastest (Fig. 3). The observed rate constant, kobs
for the release of p-nitrophenolate anion was
0
.1
0
.05
À5
À1
5
0
1
enhancement over the uncatalyzed reaction.
9.0 · 10 min , which corresponded to ꢀ10 -fold
-
1
-0.5
0
0.5
1
1
2
-1
1
/[S](mM )
In comparison, two selected values for bNPP phospho-
diester hydrolysis reported from the literature are:
Figure 4. Lineweaver–Burk plot for bNPP hydrolysis catalyzed by
AP1-Zn.

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Y. Gupta et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5364–5367
dinated zinc ions in AP1-Zn. Interestingly, this cleavage
0
0
0
0
.037
.027
.017
.007
reaction was completely inhibited in the presence of
EDTA, which probably complexes zinc ions thus inter-
fering with AP1-Zn catalytic activity (Fig. 6, lane 9).
Similar effect of EDTA was observed for bNPP hydroly-
sis where its addition aborted hydrolytic activity pre-
sumably by zinc ion complexation (data not shown),
thus verifying crucial role of zinc in hydrolysis of natural
as well as non-natural substrates.
-0.003
0
10 20 30 40 50 60 70 80
Time (h)
Results presented above suggest that our water-stable
metalated homopolymer, AP1-Zn, affords facile hydro-
lysis of phosphodiester hydrolysis substrates when com-
pared to its unmetalated counterpart. Insolubility of
AP1-Zn in reaction medium facilitates its separation
subsequent to hydrolytic reaction. Recovered polymer
retains its catalytic activity making recyclability of
AP1-Zn as an extremely attractive feature for future
investigations. Synthetic nuclease activity of AP1-Zn
against pBR322 supercoiled DNA provides an indica-
tion that such reusable artificial models may become
important tools for various biochemical applications.
Figure 5. Reaction arrest experiment for bNPP hydrolysis catalyzed by
AP1-Zn filtration.
In this experiment, AP1-Zn was filtered off from the
reaction mixture after a reaction time of 7 h and
hydrolysis of bNPP was further monitored up to 72 h
at various time points. It was noticed that the hydrolysis
reaction aborted soon after the removal of AP1-Zn and
a lack of change in absorbance confirmed that
hydrolysis occurred due to assistance rendered by
AP1-Zn and not due to leached out zinc ions (Fig. 5).
AP1-Zn homopolymer was insoluble in aqueous and
organic solvents and hence it could be easily recovered
and recycled for further use. Recovered polymer was
washed with aqueous methanol, acetone and air-dried
prior to reuse. AAS analysis of the recycled AP1-Zn
polymer confirmed that zinc has not leached out during
the course of hydrolytic reaction (data not shown). Fur-
thermore, kinetic data for fresh and reused polymer
were comparable suggesting that AP1-Zn retains its cat-
alytic potential through several cycles of hydrolytic reac-
tion (supplementary data, Table 2).
Acknowledgment
Financial support from DMSRDE, Kanpur, to one of
us (SV) is gratefully acknowledged.
Supplementary data
Crystallographic data (excluding structure factors) for
structures 1 and 2 have been deposited with the
Cambridge Crystallographic Data Center. CCDC
number for 1 is 247273; for 2 is 247274. Copies of this
information can be obtained free of charge on applica-
tion to CCDC, 12 Union Road, Cambridge CB21EZ,
UK. [Fax: +44-1223/336-033; E-mail: deposit@ ccdc.
cam.ac.uk]. Supplementary data associated with this
article can be found, in the online version, at
doi:10.1016/j.bmcl.2006.07.077.
When compared with the catalysts reported in the liter-
ature, AP1-Zn combines superior catalytic activity
together with reusability. This property makes Zn-im-
pregnated polymeric matrices more suitable as artificial
oligozinc phosphate ester hydrolases compared to
reported models.
AP1-Zn was further evaluated for hydrolytic cleavage of
a natural substrate, supercoiled plasmid DNA pBR322.
A time-course experiment was performed under hetero-
geneous conditions in cacodylate buffer containing
pBR322. Complete conversion of supercoiled form I
to nicked form II was observed (Fig. 6, Lane 7), while
unmetalated polymer AP1 did not induce plasmid relax-
ation (Fig. 6, lane 2), confirming a crucial role of coor-
References and notes
1
5
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Commun. 1970, 1668.
Figure 6. Nicking of pBR322 supercoiled DNA catalyzed by AP1-Zn.
Each reaction contains 100 lg AP1-Zn. Lane 1: pBR322 alone; lane 2:
pBR322 with unmetalated polymer, AP1 (60 h); lanes 3–8:
pBR322 + AP1-Zn (12, 24, 36, 48, 60, 72 h, respectively); lane 9:
pBR322 + AP1-Zn + EDTA (60 h).
6. (a) Chandrasekhar, V.; Krishnan, V.; Azhakar, R.;
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1
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11. Phosphate ester hydrolysis. All hydrolytic reactions were
performed in duplicate in centrifuge tubes thermostated at
30 ꢁC for bNPP hydrolysis. The assay mixture contained
3 mL of substrate solution of appropriate concentration
prepared in 0.01 M N-ethylmorpholine (NEM) aqueous
buffer in 90% aqueous methanol (pH 8.6). The reference
cell contained substrate without metal conjugates, to
(
d) Sissi, C.; Rossi, P.; Felluga, F.; Formaggio, F.; Palumbo,
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correct for background hydrolysis. AP1-Zn weight was
and Vmax, concentration of bNPP was
À1
2
1 mg mL . For K
m
8
. Hausch, F.; J a¨ schke, A. Tetrahedron 2001, 57, 1261, 9-(4-
vinylbenzyl)-purin-6-yl-amino caproic acid (2). N-(6-Puri-
nyl)-caproic acid (1) (1.0 g, 4.0 mmol), 4-vinylbenzyl
chloride (0.5 mL, 3.6 mmol), and sodium hydride (0.21 g,
1.0–5.0 mM. For Kobs, concentration of bNPP was
12 mM. Control experiments of unmetalated AP1 alone,
with phosphate ester substrates, did not accelerate hydro-
lysis, suggesting a magnificent purpose of coordinated Zn
(II) ions in AP1-Zn. Initial velocities were determined as a
function of time-dependent release of p-nitrophenolate
anion at 400 nm (e400 = 1.65 · 104 M cm ), with a
Shimadzu UV-160 spectrophotometer. Michaelis–Menten
parameters were determined using corresponding Linewe-
aver–Burk plots. All hydrolytic reactions were performed
at least over five half-lives of each substrate.
8
3
.8 mmol) were stirred in anhydrous DMF (20 mL) at
0 ꢁC. After 12 h of stirring, solvent was removed under
À1
À1
vacuum to dryness. Residue was dissolved in water,
filtered, and pure product precipitated by addition of
1
f
N HCl, 0.95 g (2.5 mmol, 65%). R (silica gel) = 0.6
1
(CH Cl /MeOH/CF COOH 9:0.5:0.5). Mp 170 ꢁC.
NMR (400 MHz, CDCl
2
H
2
2
3
3
, 25 ꢁC, TMS): d = 1.45–1.53(m,
H); 1.69–1.79 (m, 4H); 2.35 (t, 2H, J = 7.3Hz); 3.6–3.63
12. (a) Vance, D. H.; Czarnik, A. W. J. Am. Chem. Soc. 1993,
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173.
(
m, 2H); 5.24(d, 1H, J(H,H) = 11 Hz); 5.3 (s, 2H); 5.7 (d,
1
1
H, J(H,H) = 18 Hz); 6.7 (dd, 1H, J(H,H) = 18 and
1 Hz); 7.2 (d, 2H, J(H,H) = 8 Hz); 7.36 (d, 2H,
J(H,H) = 8 Hz); 7.7 (s, 1H); 8.4 (s, 1H). FABMS
M+1) = 366. Zinc metalated AP1 (AP1-Zn). Potassium
salt of AP1 (1.2 g) dissolved in MeOH (70 mL) and ZnCl2
0.75 g) was added to this solution. The reaction mixture
(
(
was stirred for 12 h at room temperature. A white
precipitate formed which was filtered, washed with meth-
anol (4· 25 mL), acetone (2· 20 mL), and ether (2·
2
0 mL). Resulting metalated complex (1 g) was dried
under vacuum. The metalated polymer, AP1-Zn was
cream in color and insoluble in all common solvents and
thus acted as heterogeneous catalyst in the hydrolytic
reaction. The amount of Zn incorporated in the polymeric
matrix was determined by AAS analysis and was found to
be 16.7 mg/g of the polymer. The resulted zinc complex
was used for phosphate ester hydrolysis and DNA
cleavage reactions..
15. pBR322 cleavage experiment. All cleavage reactions were
performed by incubating 100 lg AP1-Zn (corresponding
to 0.127 mM of zinc concentration, if the polymer was to
be completely soluble in buffer) in sodium cacodylate
buffer (10 mM, pH 7.5), at 35 ꢁC, unless otherwise
mentioned. Total reaction volume was 20 lL and weight
of the DNA was 8 ng/lL of buffer. Two microliters of
methanol was used for polymer wetting. All reactions were
quenched with 5 lL of loading buffer containing 100 mM
EDTA, 50% glycerol in Tris–HCl, pH 8.0, at regular time
intervals. The samples were loaded onto 0.7% of agarose
gel containing ethidium bromide (1 lg/mL) and were
electrophoresed for 1 h at constant current, 70 mA.
Finally gels were imaged on PC-interfaced Bio-Rad Gel
Documentation system 2000.
1
9
.
H NMR spectra recorded using a JEOL 400 MHz
spectrometer in CDCl3 (Aldrich). FABMS recorded at
RSIC, Lucknow, and AAS values recorded at FEAT
Laboratory, IIT-Kanpur. Single crystal X-ray studies:
Light green colored crystals of 1 were grown from
methanol and acetone (1:1) solvent mixture by slow
evaporation method and mounted on an Enraf Nonius