The catalytic activity of ribose-containing polymers for the hydrolysis of phosphodiester and the cleavage of nucleic acid

Article abstract of DOI:10.1016/S0040-4039(02)01123-1

In order to investigate the catalytic activity for the hydrolysis of phosphodiester and the cleavage of nucleic acids, ribose-containing polymers 11, 12 and 13 were synthesized. While polymer 11 showed no catalytic activity, polymers 12 and 13 catalyzed the cleavage of nucleic acids and the hydrolysis of a phosphodiester substrate. The catalytic activity was attributed to the vic-cis diols of riboses on polymer chains, which formed hydrogen bonds with two phosphoryl oxygen atoms of phosphates so as to activate the phosphorous atoms to be attacked by nucleophiles (H2O).

Full text of DOI:10.1016/S0040-4039(02)01123-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 5597–5600
The catalytic activity of ribose-containing polymers for the
hydrolysis of phosphodiester and the cleavage of nucleic acid
Man Jung Han,a,* Kyung Soo Yoo,a Young Heui Kima and Ji Young Changb
aDepartment of Molecular Science and Technology, Ajou University, Suwon 442-749, Republic of Korea
bSchool of Materials Science and Engineering, and Hyperstructured Organic Materials Research Center,
Seoul National University, Seoul 151-744, Republic of Korea
Received 10 June 2002; accepted 12 June 2002
Abstract—In order to investigate the catalytic activity for the hydrolysis of phosphodiester and the cleavage of nucleic acids,
ribose-containing polymers 11, 12 and 13 were synthesized. While polymer 11 showed no catalytic activity, polymers 12 and 13
catalyzed the cleavage of nucleic acids and the hydrolysis of a phosphodiester substrate. The catalytic activity was attributable to
the vic-cis diols of riboses on polymer chains, which formed hydrogen bonds with two phosphoryl oxygen atoms of phosphates
so as to activate the phosphorous atoms to be attacked by nucleophiles (H2O). © 2002 Elsevier Science Ltd. All rights reserved.
In the biological system there are plenty of biopolymers
containing riboses. One of the typical biopolymers con-
taining riboses with vic-cis-diols is poly (ADP-ribose)
formed from NAD+ in chromatin. Since its discovery in
1966,1 the polymer has been suggested to be involved in
numerous biological reactions,2 although its functions
are not clear so far. Poly (ADP-ribose) forms during
apoptosis3 and DNA repair,4 where nuclease will be
required. Recently we reported that the alternating
copolymers of ribose derivatives and maleic anhydride
catalyzed cleavage of ss oligonucleotides.5,6 The poly-
mers, however, failed to cleave ds DNA of high molec-
ular weights, which may be in part due to the stiffness
of the polymer backbones, preventing them from bind-
ing DNA effectively. Herein, we described the synthesis
for the flexible vinyl polymers containing riboses and
the cleavage of a dinucleotide, ds DNA, and RNA as
well as the hydrolysis of a phosphodiester substrate.
Synthesis of monomer 5 was accomplished in four steps
from 3%-O-acetyl-1%,2%-O-isopropylidenene-5%,6%-diol-a-
D
-allofuranose (1), which was synthesized by deprotec-
tion of 5%,6%-acetonide groups of 1%,2%:5%,6%-di-
O-isopropylidene-a- -allofuranose according to the lit-
D
Scheme 1. Synthesis of monomer 5 and 10. Reagents and conditions: (a) NaIO4, silica gel, 25°C, 40 min; (b) NH3 in MeOH, 25°C,
24 h; (c) Pd/C, 4 atm H2, 25°C, 48 h; (d) acryloyl chloride, 25°C, 2 h; (e) PPh3, I2, Py., 25°C, 24 h; (f) NaN3, 110°C, 24 h; (g)
PPh3, H2O, 25°C, 24 h; (h) acryloyl chloride, 0°C, 11 h.
* Corresponding author. Tel.: +82-31-219-2519; fax: +82-31-214-8918; e-mail: mjhan@madang.ajou.ac.kr
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01123-1

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M. J. Han et al. / Tetrahedron Letters 43 (2002) 5597–5600
erature (Scheme 1).7 Oxidation of 1 with the aid of
NaIO4 in the suspension of silica gel in CH2Cl2 afforded
(97.5%) 2, which was confirmed by the appearance of
aldehyde carbonyl peak at 197.3 ppm in the 13C NMR
spectrum. Reaction of 2 with ammonia saturated in
MeOH at 25°C afforded (95.7%) 3, whose structure was
for acrylamido groups at 124.7 ppm (C
6
H2ꢀCH), 130.4
ppm (CH2ꢀC
6
H), and 164.9 ppm (C
spectrum. The elemental analysis results were satisfac-
tory for all the compounds.
6
ꢀO) in the 13C NMR
The polymerization of monomers 5 (2.47 mM) and 10
(1.15 mM) was carried out in water with the initiator
K2S2O8 (2 mol%) at 80°C to yield poly(5%-acrylamido-5%-
supported by the appearance of the peak for NHꢀC6 H at
139.8 ppm in the 13C NMR spectrum. Hydrogenation of
the imine moiety of 3 with the aid of Pd on charcoal in
MeOH (25°C, 24 h, 60 psi H2) yielded the corresponding
amine 4 (70.6%), identified by the chemical shift change
deoxy-1%,2%-O-isopropylidene-a-
D
-ribose) (11) (83.7%)
and poly(5%-acrylamido-5%-deoxy-1%-O-methyl-
D
-ribose)
(13) (68.1%), respectively. The polymers were isolated
of the peak for C6 from 139.8 to 49.1 ppm. Reaction of
by precipitation in acetone. The structures of polymers
4 with acryloyl chloride in anhydrous THF at 25°C gave
the corresponding monomer 5 (54.3%), supported by
the appearance of signals for the acrylic amide carbon
1
11 and 13 were identified by H NMR spectroscopy.
The acryl amide proton signals at 6.3–6.8 ppm com-
pletely disappeared and a broad peak at 2.0–2.6 ppm of
ethylene groups on the polymer chain showed up. Poly-
mer 11 was hydrolyzed by stirring for 24 h at 25°C in
1N HCl solution (Scheme 2). After neutralization with
a dilute NaOH solution, poly(5%-acrylamido-5%-deoxy-a-
(C
(C6
6
ONH) at 165.1 ppm and double bond carbon
H2ꢀCH) at 125.9 and 131.2 (CH2ꢀCH) ppm.
6
Monomer 10 was synthesized with starting from 1%-O-
methyl- -riobse (6) (Scheme 1), which was obtained by
methylation of a-
-ribose according to the literature.8
D
D
-ribose) (12) was isolated (64.6%) by dialysis through
D
a cellulose membrane of molecular weight cut off of
1000 and then by freeze-drying. After hydrolysis, ace-
tonide proton signals at l=1.34 and 1.59 ppm disap-
Reaction of 6 in a dioxane solution containing PPh3, I2
and pyridine yielded (60.9%) 5%-deoxy-5%-iodo-1%-O-
methyl- -ribose (7), which was reacted with sodium
D
1
peared in the H NMR spectrum of polymer 12. The
azide in DMF at 60°C to give (97.8%) azido moiety 8.
The structure of 8 was supported by the signal of the
polymerization data are summarized in Table 1.
carbon next to azide (C6 H2N3) at 51.1 ppm and the
appearance of the stretching band at 2119 cm−1 in the
IR spectrum. Reaction of 8 with PPh3 in THF/H2O
afforded (64.4%) the corresponding amine 9, identified
The extent of active center formation will depend on the
polymer chain length, i.e. molecular weight. To obtain
the polymers having different molecular weights with
narrow distribution, polymer 12 was successively dia-
lyzed through a series of cellulose membranes of molec-
ular weight cut off of 3500, 8000, 15 000, and 25 000.
The four filtrates obtained by dialyzing through
by the chemical shift change of the peak for C6 from 51.1
to 39.5 ppm. Monomer 10 was obtained (47.1%) by the
reaction of 9 with acryloyl chloride in THF and the
structure was confirmed by the appearance of new peaks
Scheme 2. Synthesis of polymers 11, 12 and 13. Reagents and conditions: (a) K2S2O8, H2O, 10 h, 80°C; (b) HCl, 18 h; (c) K2S2O8,
H2O, 15 h, 70°C.
Table 1. Polymerization data
Polym. no.
Monomer (mmol/L)
K2S2O8 (mmol/L)
Yield (%)
Mna
PDb
11
12
13
5 (2.47)
–
10 (1.15)
49.4
–
23.0
83.7
64.6
68.1
19 300
18 000
18 500
1.55
1.67
1.53
a Number-average molecular weights of the polymers were measured by GPC (Waters Co.) on Ultrahydrogel 250 column with poly(ethylene
glycol) standards (eluent: aqueous 0.1N NaNO3 solution).
b Polydispersity.

M. J. Han et al. / Tetrahedron Letters 43 (2002) 5597–5600
5599
the series of the membranes and the polymer solution
remained in the last tube were freeze-dried to give five
polymer portions having different number-average
molecular weights of 2500, 6400, 9200, 17 600, and
27 100, respectively. We have employed ethyl p-nitro-
phenyl phosphate (ENPP), as a phosphodiester substrate
model, to evaluate the catalytic activity of the polymers.
Hydrolysis rates were determined by the time dependent
release of p-nitrophenol (m400nm=10 268) in Tris-buffer
(pH 7.4, ionic strength=0.02, KCl) at 50°C. The hydroly-
sis of phosphate substrates and nucleic acids was often
catalyzed by metal ions with various ligands. To exclude
this possibility, we used the deionized water (resistivity
>18 MV cm−1) and crystallized Tris-buffer materials. The
initial hydrolysis rates of the substrate (ENPP) in the
presence of polymer 12 were measured6 and plotted
against the number-average molecular weights of the
polymers (Fig. 1). The initial hydrolysis rate was jumped
above Mn of 17 800 and therefore we used the polymer
with Mn of 17 800 as a catalyst for the hydrolysis of
natural nucleic acids.
The dinucleotide, 2%-deoxy-adenylyl (3%“5%)-2%-deoxy-
adenosine (dApdA), was incubated in the presence of 12
at pH 7.4 (Tris-buffer), v=0.02 (KCl), and 37°C for
24 h and the reaction mixture was investigated by HPLC
(Fig. 2). 92% of dApdA was hydrolyzed to give 2%-
deoxyadenosine (dA), 2%-deoxy-adenosine-3%-phosphate
(dAp), and 2%-deoxyadenosine-5%-phosphate (pdA). The
mole ratio of dA to dAp to pdA was found to be 5:4:1.
Figure 1. The initial rates as a function of number-average
molecular weights at pH 7.4 (Tris-buffer), 50°C, v=0.02 (KCl).
[substrate]=1.87×10−3 M, [polymer 12]†=5.26×10−6 M.
Supercoiled ds DNA (pBR 322 DNA Plasmid) and RNA
(BMV RNA) were incubated at 37°C in the presence of
polymers 11 and 12 at pH 7.4 (Tris-buffer) as well as in
a buffer solution alone for 6 h, and visualized by
EtBr-agarose gel electrophoresis. The cleavage of pBR
322 DNA Plasmid in the presence of polymer 12 (lane
1) is clearly shown in Fig. 3A. The circular supercoiled
DNA (form I) was converted to circular relaxed DNA
(form II) via single-strand cleavage. Fig. 3B shows that
BMV RNA was cleaved in the presence of polymer 12
to yield lower base pair segments (lane 1). Only partial
Figure 2. Liquid chromatogram of the hydrolysis products of
dinucleotide (dApdA). Conditions: C18 m-Bondapak reverse
phase column, UV detector u=254nm, eluent; 0.25 M pH 7.4
phosphate buffer, flow rate; 0.8 mL/min, [dApdA]=4×10−4 M,
[polymer]†=9.6×10−6 M.
Figure 3. Electrophoresis diagram of the reaction mixtures on the agarose gel: (A) supercoiled ds DNA (pBR 322) and (B) RNA
(BMV); lane 1: in the presence of polymer 12, lane 2: in the presence of polymer 11, and lane 3: in buffer solution.
[polymer]†=2.63×10−7 M, [nucleic acid]=50 mg/L.
† Concentration of repeating unit.

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M. J. Han et al. / Tetrahedron Letters 43 (2002) 5597–5600
cleavage was observed in a buffer solution alone (lane
3) and in the presence of polymer 11 (lane 2) under the
same conditions.
638–643; (b) Sugimura, T.; Fugimura, S.; Hasegawa, S.;
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In conclusion, we showed that the flexible vinyl poly-
mers containing vic-cis diols of riboses catalyzed the
cleavage of a dinucleotide and nucleic acids (DNA and
RNA) as well as the hydrolysis of phosphodiester sub-
strate. This catalytic activity was attributable to the
vic-cis diols of ribose on polymer chains, which formed
hydrogen bonds with two phosphoryl oxygen atoms of
phosphates so as to activate the phosphorous atoms to
be attacked by nucleophiles (H2O).9
4. (a) Malanga, M.; Althaus, F. R. J. Biol. Chem. 1994, 269,
17691–17696; (b) Satoh, M. S.; Poirier, G. G.; Lindahl, T.
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Acknowledgements
5. Han, M. J.; Yoo, K. S.; Cho, T. J.; Chang, J. Y.; Cha, Y.
J.; Nam, S. H. Chem. Commun. 1997, 163–164.
6. Han, M. J.; Yoo, K. S.; Kim, K. H.; Lee, G. H.; Chang,
J. Y. Macromolecules 1997, 30, 5408–5415.
This work was supported by a grant from the Ministry
of Education and Korean Science and Engineering
Foundation.
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