Polymeric enzyme mimics: catalytic activity of ribose-containing polymers for a phosphate substrate.

Article abstract of DOI:10.1039/b301166f

The polymers containing ribose rings: poly(5'-acrylamido-5'-deoxy-1',2'-O-isopropylidene-alpha-D-ribose) (11), poly(5'-acrylamido-5'-deoxy-alpha-D-ribose) (12) and poly(5'-acrylamido-5'-deoxy-1'-O-methyl-D-ribose) (13) were prepared as enzyme mimics. Polymers 12 and 13 with free vic-cis-diol groups catalyzed the hydrolysis of phosphodiester (ethyl p-nitrophenyl phosphate and N-methylpyridinium 4-tert-butylcatechol cyclic phosphate) and phosphomonoester substrates with a rate acceleration of 10 approximately equal to 10(3) compared with the uncatalyzed reaction. They also catalyzed the reverse reactions, i.e., the esterification of phosphomonoester to phosphodiester and the phosphorylation of alcohols with phosphate ions. 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 phosphorus atoms to be attacked by nucleophiles. The catalytic activity was negligible for polymer 11 where vic-cis-diol groups were blocked with isopropylidene groups. 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 phosphorus atoms to be attacked by nucleophiles.

Full text of DOI:10.1039/b301166f

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Polymeric enzyme mimics: catalytic activity of ribose-containing
polymers for a phosphate substrate
Man Jung Han,*a Kyung Soo Yoo,a Young Heui Kima and Ji Young Changb
a Department of Molecular Science and Technology, Ajou University, Suwon 442-749, Korea.
E-mail: mjhan@ajou.ac.kr; Fax: (82) 31 219 1592
b School of Materials Science and Engineering and Hyperstructured Organic Materials
Research Center, Seoul National University, Seoul 151-744, Korea
Received 3rd February 2003, Accepted 8th May 2003
First published as an Advance Article on the web 27th May 2003
The polymers containing ribose rings: poly(5Ј-acrylamido-5Ј-deoxy-1Ј,2Ј-O-isopropylidene-α--ribose) (11),
poly(5Ј-acrylamido-5Ј-deoxy-α--ribose) (12) and poly(5Ј-acrylamido-5Ј-deoxy-1Ј-O-methyl--ribose) (13)
were prepared as enzyme mimics. Polymers 12 and 13 with free vic-cis-diol groups catalyzed the hydrolysis of
phosphodiester (ethyl p-nitrophenyl phosphate and N-methylpyridinium 4-tert-butylcatechol cyclic phosphate)
and phosphomonoester substrates with a rate acceleration of 10 ∼ 103 compared with the uncatalyzed reaction.
They also catalyzed the reverse reactions, i.e., the esterification of phosphomonoester to phosphodiester and the
phosphorylation of alcohols with phosphate ions. 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 phosphorus atoms to be attacked by nucleophiles. The catalytic activity was negligible for polymer 11 where
vic-cis-diol groups were blocked with isopropylidene groups. 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 phosphorus atoms to be attacked by nucleophiles.
estingly, the polymer showed nearly the same catalytic activities
Introduction
(nuclease, ligase, phosphatase, and phosphorylase activities) for
There has been great interest in the synthesis of enzyme-like
the phosphate reactions as the cyclodextrinyl enzyme model.
polymers.1–9 The discovery of catalytic nucleic acids such as
The polymer is believed to form a secondary structure com-
parable to the cavity of β-cyclodextrin where a substrate is
captured inside. The polymer also catalyzed the cleavage of ds
ribozyme and deoxyribozyme10,11 especially, has drawn much
attention to the investigation of synthetic models that catalyze
phosphate ester hydrolysis.
Recently, we reported that the ribose ring capping on
β-cyclodextrin, an enzyme model, showed catalytic activity for
the hydrolysis of phosphodiester (nuclease) and phosphomono-
ester (phosphatase), the esterification of phosphomonoester
DNA and RNA, which was reported in our preceding short
communication.14
Herein, we report a full account of our studies on the syn-
thesis of ribose-containing polymers, their catalytic activities
for phosphate substrates, and the probable reaction mechanism.
to diester (ligase) and the phosphorylation of alcohols with
phosphate ions (phosphorylase).12 The catalytic activity is attri-
Results and discussion
butable to the vic-cis-diols of riboses, which form hydrogen
bonds with two phosphoryl oxygen atoms of the phosphate so
as to activate the phosphorus atom for attack by nucleophiles
(H2O or alcohol).13 These results prompted us to prepare a
polymer containing riboses with free vic-cis-diols, poly(5Ј-acryl-
amido-5Ј-deoxy-α--ribose), as an enzyme mimic. Very inter-
Synthesis of monomers and polymers
Monomer 5, 5Ј-acrylamido-5Ј-deoxy-1Ј,2Ј-O-isopropylidene-α-
-ribose, was prepared according to Scheme 1. 3Ј-O-Acetyl-
1Ј,2Ј-O-isopropylidenene-5Ј,6Ј-diol-α--allofuranose (1) was
Scheme 1 Synthesis of monomers: (i) NaIO4, silica gel, 25 ЊC, 40 min; (ii) NH3 in MeOH, 25 ЊC, 24 h; (iii) Pd/C, 4 atm H2, 25 ЊC, 48 h; (iv) acryloyl
chloride, 25 ЊC, 2 h; (v) PPh3, I2, Py., dioxane, 25 ЊC, 24 h; (vi) NaN3, 110 ЊC, 24 h; (vii) PPh3, THF–H2O, 25 ЊC, 24 h; (viii) acrylic anhydride, 0 ЊC,
11 h.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 7 6 – 2 2 8 2
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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obtained by deprotection of 5Ј,6Ј-acetonide groups after the
acetylation of 1Ј,2Ј:5Ј,6Ј-di-O-isopropylidene-α--allofuran-
ose.15 Compound 1 was oxidized with the aid of NaIO4 in a
suspension of silica gel in methylene chloride to yield 3Ј-O-
acetyl-1Ј,2Ј-O-isopropylidene-α--ribo-pentoaldo-1Ј,4Ј-furan-
ose (2). Treatment of 2 with ammonia saturated in MeOH
at room temperature for 24 h gave the imine compound (3).
5Ј-Amino-5Ј-deoxy-1Ј,2Ј-O-isopropylidene-α--ribose (4) was
obtained by hydrogenation of 3 with the aid of Pd on charcoal
in MeOH by stirring under an H2 atmosphere at room temper-
ature. The corresponding monomer 5 was obtained by a substi-
tution reaction with acryloyl chloride in anhydrous THF at
room temperature.
The synthesis of another monomer, 5Ј-acrylamido-5Ј-deoxy-
1-O-methyl--ribose (10), was accomplished in four steps
starting from 1Ј-O-methyl--ribose (6) as shown in Scheme 1.
Compound 6 was obtained by methylation of α--ribose
according to the literature.16 Treatment of 6 with two equiv-
alents of triphenylphosphine and iodine in dioxane at room
temperature for 24 h afforded the iodo-ribose compound (7),
which was reacted with sodium azide in DMF at 110 ЊC to give
5Ј-azido-5Ј-deoxy-1Ј-O-methyl--ribose (8). Reduction of the
azide compound (8) with triphenylphosphine in THF–H2O
afforded the corresponding amine (9). Monomer 10 was
obtained by the reaction of 9 with one equivalent of acrylic
anhydride in THF at 0 ЊC for 11 h.
The polymerization of monomers 5 and 10 was carried out
in H2O by initiation with K2S2O8 at 80 ЊC and at 70 ЊC to
yield poly(5Ј-acrylamido-5Ј-deoxy-1Ј,2Ј-O-isopropylidene-α--
ribose) (11) and poly(5Ј-acrylamido-5Ј-deoxy-1Ј-O-methyl--
ribose) (13) respectively (Scheme 2). The polymers were purified
by precipitation in acetone. Number-average molecular weights
of polymers 11, 12, and 13 measured by GPC with poly-
(ethylene glycol)s as standards in an aqueous 0.1 M NaNO3
Fig. 1 1H NMR spectra of (a) monomer 5 in CDCl3 and (b) polymer
11 in D2O.
room temperature. After neutralization with a dilute NaOH
solution, the desired poly(5Ј-acrylamido-5Ј-O-deoxy-α--
ribose) (12) was isolated by dialysis through a cellulose mem-
brane with a molecular weight cut off of 1000 and then by
freeze-drying.
1
solution were 19300, 18000, and 18500, respectively.14 The H
NMR spectra of monomer 5 and polymer 11 are shown in
Fig. 1. The proton signals of the acryl groups appeared at 6.3–
6.8 ppm in Fig. 1a, which are changed to a broad peak at 2.0–
2.6 ppm of ethylene groups on the polymer chain in Fig. 1b.
Hydrolysis of polymer 11 was accomplished with 1 M HCl at
Catalytic activity for ethyl p-nitrophenyl phosphate (ENPP)
Ethyl p-nitrophenyl phosphate (ENPP) was used as the sub-
strate. The hydrolysis reaction of the substrate in the presence
of polymer 12 was confirmed by 31P NMR spectroscopy. After
reaction for 24 h in Tris-buffer solution under the conditions:
[substrate] = 4.67 × 10Ϫ4 M and [polymer 12]† = 5.26 × 10Ϫ6 M at
pH = 7.4, 50 ЊC, and ionic strength = 0.02 (KCl), the reaction
mixture was examined by 31P NMR spectroscopy. Two peaks at
2.69 ppm for ethyl phosphate and Ϫ5.97 ppm for ethyl p-nitro-
phenyl phosphate (substrate) relative to phosphoric acid at
0 ppm were observed in the 31P NMR spectrum. The possibility
of transphosphorylation on the hydroxyl groups of the polymer
was also investigated. The polymer was separated from the
reaction mixture by dialysis through a cellulose membrane with
a molecular weight cut off of 1000 and subjected to 31P NMR
analysis. Phosphorus atoms were not detected on the polymer,
indicating that the hydrolysis reaction took place exclusively.
Hydrolysis rates of the substrate were determined in Tris-
buffer (pH = 7.4, ionic strength = 0.02, KCl) at 50 ЊC in the
presence of the monomers and the polymers. The rates were
followed by the measurement of the ultraviolet absorption
(ε400 nm = 10268) of the conjugate anion of p-nitrophenol evolved.
The hydrolysis of a phosphate substrate is often catalyzed by
metal ions with various ligands.17 To exclude this possibility, we
used deionized water (resistivity > 18 MΩ cmϪ1) and Tris-buffer
materials crystallized from water–ethanol three times. The con-
centrations of p-nitrophenol produced during the hydrolysis in
thepresenceofmonomer5, monomer10, ribose, orpolymers11–
13, at pH 7.4, 50 ЊC and µ = 0.02, as a function of time are plotted
in Fig. 2. Polymers 12 and 13 with free vic-cis-diols of furanose
rings accelerated the hydrolysis, while the catalytic activity was
negligible in the presence of polymer 11, whose diol groups were
Scheme 2 Synthesis of polymers: (i) K2S2O8, H2O, 10 h, 80 ЊC; (ii) HCl,
18 h; (iii) K2S2O8, H2O, 15 h, 70 ЊC.
† Concentration of polymer chains.
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Table 1 Fractionation results of polymer 12
No.a
Membrane cut offb
Mn
c
1
2
3
4
3500
8000
15000
25000
2500
6400
9200
17800
27100d
a The order of cellulose membrane tubes used for successive dialyses.
b Molecular weight cut off of the cellulose membrane tube. c Number-
average molecular weight of the polymer in the filtrate after dialysis.
d Number-average molecular weight of the polymer in the cellulose
membrane tube with a molecular cut off of 25000 after dialysis.
The initial hydrolysis rates of the substrate (ENPP) in the
presence of polymer 12 ([polymer] = 5.26 × 10Ϫ6 M, [substrate]
= 1.86 × 10Ϫ3 M) were measured at pH 7.4 (Tris-buffer),
50 ЊC, and µ = 0.02 (KCl). The rates were dependent on the
number-average molecular weights of the polymers. The initial
hydrolysis rate increased above an Mn of 17800, and therefore
we presumed that one active center formed on each polymer
chain with a molecular weight higher than 17800.
Polymer 12 with Mn = 17800 was used for the kinetic study.
From Fig. 3, Km and Vmax were obtained and kcat was calculated
from eqn. (1). Kinetic parameters of the catalysis are summar-
ized in Table 2.
Fig. 2 Concentration of p-nitrophenol evolved during hydrolysis of
ethyl p-nitrophenyl phosphate in the presence of polymers 11, 12, 13,
and monomers 5, 10, and ribose as a function of time at pH 7.4 (Tris-
buffer), 50 ЊC, µ = 0.02 (KCl). [substrate] = 3.73 × 10Ϫ3 M, [polymer] =
5.26 × 10Ϫ6 M, [monomer 5] = [monomer 10] = [ribose] = 4.67 × 10Ϫ4 M
(same as the ribose residue concentration of the polymers).
blocked by isopropylidene groups. It is also noteworthy that
the monomeric species (5 and 10) and ribose showed negligible
catalytic activity.
In hydrolysis kinetics, the subtraction of vu (uncatalyzed
reaction) from the measured rate (vm) is v (catalyzed reac-
tion).18 The initial v was obtained at a constant concentration
of polymers 12 and 13 by changing the substrate concen-
trations. Michaelis–Menten kinetics for 12 and 13 were con-
firmed by plotting the double reciprocal form of Lineweaver
kcat = Vmax / [E]o
(1)
The rate constants (kcat) for polymers 12 and 13 were found to
be about 103 higher than that of the uncatalyzed reaction.
Polymer 12 showed higher catalytic activity than 13, probably
because the former contained two vic-cis-diol groups (1Ј 2Ј and
2Ј 3Ј) while the latter had one (2Ј 3Ј OH).
Competitive inhibition was found in the presence of acetate
ions and its dissociation constant (KI) was measured to be
3.49 × 10Ϫ4 in the catalysis by polymer 12 (Fig. 4a).
Non-competitive inhibition was also observed by addition of
K2HPO4 (KI = 9.89 × 10Ϫ4 M) (Fig. 4b). Both inhibitions
seemed to occur because the vic-cis-diols were blocked by form-
ation of hydrogen bonds with the inhibitors. The acetate ions
seemed to compete with the substrate for forming hydrogen
bonds with the polymeric catalyst. The phosphate ion, as a
dianion, could form stronger hydrogen bonds with vic-cis-diols
of the polymer catalyst than the substrate, leading to non-
competitive inhibition.
and Burk (1/v vs. 1/s) (Fig. 3), which gave Km and Vmax
.
Enzyme mimics for nuclease, ligase, phosphatase, and
phosphorylase
The catalytic activities of the polymers for the model reactions
of N-methylpyridinium 4-tert-butylcatechol cyclic phosphate
(CP) were investigated (Scheme 3). The reactions of the
Fig. 3 Reciprocals of the initial rates as a function of reciprocals of
the substrate concentrations (1/v vs. 1/[S]) in the presence of polymers
12 and 13.
Since only the polymers showed catalytic activity, we pre-
sume that active centers were formed where the substrate was
bound, through chain folding. The extent of active center
formation will depend on the polymer chain length, i.e. molecu-
lar weight. Polymers with different molecular weights were
obtained by the successive dialysis of polymer 12 through cellu-
lose membrane tubes with different molecular weight cut offs.
The four filtrates obtained by dialyzing through the series of
tubes (molecular weight cut off = 3500, 8000, 15000, and 25000)
and the polymer solution remaining in the last tube were freeze-
dried to give five polymer portions with different molecular
weights as summarized in Table 1.
Scheme 3 Reactions of cyclic phosphate substrate CP.
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Table 2 Kinetic parameters of the catalysis for hydrolysis of ethyl p-nitrophenyl phosphate at 50 ЊC
Polymer no.
k (hϪ1
)
Km (M)
Vmax (M hϪ1
)
kcat (hϪ1
)
Blank
12
13
9.12 × 10Ϫ4
—
—
—
—
—
1.44 × 10Ϫ3
6.14 × 10Ϫ4
5.19 × 10Ϫ6
2.37 × 10Ϫ6
9.43 × 10Ϫ1
4.33 × 10Ϫ1
Fig. 5 Concentration changes vs. time (a) for the hydrolysis of cyclic
phosphate (CP) in Tris-buffer solution (᭺) and in the presence of ribose
and 11 (᭺) and 12 (᭿), (b) for the reaction of CP (ᮀ)
1P (ꢀ) ϩ 2P
(᭞) catalyzed by 12, (c) for the reaction of 1P (ᮀ) CP (ꢀ) ϩ BC (᭺)
Ϫ
catalyzed by 12, and (d) for the reaction of BC (ᮀ) ϩ H2PO4
(᭺) ϩ 2P (ꢀ) CP (᭞) catalyzed by 12.
1P
As observed in the reaction of ethyl p-nitrophenyl phosphate,
only polymer 12 showed catalytic activity for the hydrolysis of
CP. Ribose and polymer 11 did not accelerate the reaction at all
within experimental error. As the reactions were pseudo-first
order, the rate constants for the hydrolysis were obtained by
plotting logarithmic concentrations of CP against time and
found to be 3.09 × 10Ϫ3 hϪ1 for 12, which was about 10 times
higher than that of the uncatalyzed reaction (3.2 × 10Ϫ4 hϪ1). In
the hydrolysis in the presence of 12, the concentration of CP
decreased while phosphoric acid mono(4-t-butyl 2-hydroxy-
phenyl) ester (1P) and phosphoric acid mono(5-t-butyl-2-
hydroxyphenyl) ester (2P) formed simultaneously as reported
by others (Fig. 5b).20,21 The rate constants of reactions 1 and 5
in Scheme 3 were found to be 1.86 and 1.23 × 10Ϫ3 hϪ1
respectively.
Fig. 4 Reciprocals of the initial rates as a function of reciprocals of
the substrate concentrations (1/v vs. 1/[S]) measured at different
concentrations of inhibitors, (a) sodium acetate or (b) K2HPO4, in the
presence of polymer 12 in Tris-buffer (pH = 7.4) at ionic strength of
0.02 (KCl) at 50 ЊC. [polymer 12] = 5.26 × 10Ϫ6 M. KI determinations are
shown in the upper left plots.
1P was stable in the buffer solution. When 1P was mixed with
12 in Tris-buffer solution at pH 7.4 however, its cyclization to
CP and hydrolysis to t-butylcatechol (BC) occurred simul-
taneously. The concentration changes of the products and the
reactants in the cyclization and hydrolysis of 1P in the presence
of 12 are shown in Fig. 5c. The rate constants for reactions 2
and 3 were found to be 3.19 and 1.79 × 10Ϫ3 hϪ1 respectively.
When 12, BC, and KH2PO4 were mixed in the mole ratio of
1 : 10 : 20 at pH 7.4 (Tris-buffer), esterification either to 1P or to
2P occurred, which was followed by cyclization to CP. In order
to lead this reaction to the first order, phosphate ion was added
in excess. The concentration changes vs. time of the reaction in
the presence of 12 are plotted in Fig. 5d. At the beginning of
phosphate substrate were carried out in Tris-buffer (pH 7.4) at
25 ЊC and an ionic strength of 0.02 (KCl). The starting concen-
trations of the substrate and the polymers were 4 × 10Ϫ4 M and
2 × 10Ϫ5 M respectively. The concentrations of reactants and
products were analyzed by liquid chromatography with a UV
detector at 285 nm. Under the reaction conditions, the reaction
rate was moderate enough to be measured and no significant
interference of the polymers was observed in the LC chromato-
grams.
The hydrolysis rates of CP were measured in the buffer solu-
tion alone and in the presence of ribose,19 11 or 12. The time-
dependent concentration changes of CP are shown in Fig. 5a.
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the reaction, 1P and 2P formed, and thereafter CP. The rate
constants for reactions 4 and 6 were found to be 3.41 and 1.70 ×
10Ϫ3 hϪ3, respectively.
phosphate as a leaving group. The catalytic activities for
reactions 1 (and 5), 2, 3, and 4 (and 6) (Scheme 3) correspond to
the enzyme activities for nuclease, ligase, phosphatase, and
phosphorylase respectively.
In order to investigate whether the reactions described above
could occur on the hydroxyl groups of the ribose rings on the
polymer, the polymers used for the reactions were separated by
dialysis through cellulose membranes with a molecular weight
cut off of 1000 and were subjected to 31P NMR analysis. How-
ever, P atoms were not detected on the polymers, indicating that
the reactions on the hydroxyl groups did not occur.
In conclusion, the polymers containing vic-cis-diols of
riboses catalyzed the hydrolysis of ethyl p-nitrophenyl phos-
phate with a rate acceleration of 103 compared with that of the
uncatalyzed reaction. They also showed catalytic activity for
the hydrolysis of phosphodiester and phosphomonoester, the
esterification of phosphomonoester and the phosphorylation
of alcohols with phosphate ions. In biological systems there are
plenty of biopolymers containing riboses. One of the typical
biopolymers containing riboses with vic-cis-diols is poly(ADP-
ribose) formed from NADϩ in chromatin. Since its discovery in
1966,22,23 the polymer has been suggested to be involved in
numerous biological reactions,24–27 although its functions are
not clear so far. Poly(ADP-ribose) forms during apoptosis28
and DNA repair,29,30 where a nuclease will be required. More
interestingly, the ribozyme (RNA) also contains vic-cis-diols of
ribose at the 3Ј-OH termini or at apurinic sites, which might
catalyze the cleavage of nucleic acids when the vic-cis-diols of
ribose anchored inside the active center formed by chain folding
through base pairing. We hope that this report will contribute
to the elucidation of their functions and further investigations
are in progress along these lines.
Based on the experimental results described above and our
previous results,12 the mechanism of catalysis can be postulated
as follows: since no activity was found for ribose, the polymer
backbone was likely to form an active site to hold a substrate
therein by chain folding. It seems possible that the polymer-
pendent ribose rings with vic-cis-diol groups were located inside
the active site, where the phosphate substrate was also accom-
modated. The vic-cis-diol groups form hydrogen bonds with the
two oxygen atoms of the phosphate so as to activate the phos-
phorus atoms to be attacked by nucleophiles (H2O or hydroxy
groups) as shown in Scheme 4. Either 1Ј,2Ј or 2Ј,3Ј-diols
of riboses can form hydrogen bonds although only the bonds
with 2Ј,3Ј- diols are illustrated in Scheme 4. The formation of
strong hydrogen bonds was borne out by a theoretical study
of the interaction between 3,4-dihydroxytetrahydrofuran and
Ϫ1 13
H2PO4
.
Experimental
Materials and instrumentation
Chemicals were purchased from Sigma-Aldrich. Tris-buffer
materials (TRIZMA® Base and TRIZMA® HCl) were
recrystallized from deionized water and ethanol three times to
exclude metal ions. THF was dried over sodium metal and
distilled. Dimethyl formamide was dried over anhydrous
MgSO4 and distilled. Pyridine was refluxed over KOH and dis-
tilled. Other commercially available reagent chemicals were
used without purification. The substrates, ethyl p-nitrophenyl
phosphate (ENPP),18 N-methyl pyridinium 4-tert-butylcatechol
cyclic phosphate (CP),20 and 1-phosphate (1P)12 and 2-phos-
phate (2P)12 of 4-tert-butylcatechol were prepared according to
the literature.
1H and 13C NMR spectra were recorded on a Varian Gemini
200 spectrometer. IR spectra were obtained with a Nicolet
Magna IR-550 spectrophotometer. The reaction rates were
determined with a Hitachi (Model 200–20) thermostatted
spectrophotometer ( 0.1 ЊC). Measurement of molecular
weights was carried out by gel permeation chromatography,
Waters 150-Cplus with a RI detector. Elemental analysis was
performed at Korea Research Institute of Chemical
Technology.
Scheme 4 Action mechanism of polymer 12 for the catalysis: (a) The
phosphorus atom of CP is attacked by H2O either side of the phosphate
to form 1P or 2P. (b) The hydrolysis and esterification of 1P occur
when H2O attacks the phosphorus atom to break the P–O ester bond or
the 2-OH group of 1P attacks to eliminate the OH group of the
phosphate, respectively. (c) The phosphorylation of BC occurs by
nucleophilic attack of the 1-OH group of BC to the phosphorus atom
to eliminate the OH group from the phosphate.
3Ј-O-Acetyl-1Ј,2Ј-O-isopropylidene-5Ј,6Ј-diol-ꢀ-D-allofuranose
(1)
Compound 1 was synthesized according to the literature.15
In the case of hydrolysis of CP (Scheme 4a), water can attack
either side of the activated phosphate to form 1P or 2P. The
hydrolysis and esterification of 1P (Scheme 4b) occur when
water or the 2-OH group of 1P attack the phosphorus atom
and either break the P–O ester bond or cause the OH group of
the phosphate to leave, respectively. The phosphorylation of BC
(Scheme 4c) occurs by nucleophilic attack of 1-OH or 2-OH
of BC at the activated phosphorus atom with the OH of the
3Ј-O-Acetyl-1Ј,2Ј-O-isopropylidene-ꢀ-D-ribo-pentoaldo-1Ј,4Ј-
furanose (2)
A solution of 1 (1 g, 3.83 mmol) in methylene chloride (10 mL)
was dropped slowly into a suspension of silica gel (8 g) in
methylene chloride (80 mL) containing 10 mL of a 0.65 M
NaIO4 aqueous solution. The reaction mixture was stirred for
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40 min at room temperature. The solids were removed by fil-
tration and the filtrate was concentrated by evaporation. Com-
pound 2 (0.9 g, 97%) was isolated by column chromatography
on silica gel (eluent; ethyl acetate–hexane = 1 : 1, v/v). (Found:
C, 51.97; H, 5.93. Calc. for C10H14O6: C, 52.17; H, 6.13%);
δH (200 MHz, CDCl3): 1.37, 1.57 (ss, 6H, acetonide), 2.16
(s, 3H, acetyl), 4.44 (d, 1H, H3, J = 8.6 Hz), 4.86 (d, 1H, H2,
J = 2.4 Hz), 4.90 (t, 1H, H4, J = 4.6 Hz), 5.93 (d, 1H, H1,
J = 3.6 Hz), 9.67 (d, 1H, aldehyde, J = 2.2 Hz); δC (50 MHz,
CDCl3) 20.35 (CH3CO2), 26.54 (CCH3), 26.62 (CCH3), 72.07
(C3), 77.47 (C2), 80.84 (C4), 104.75 (CCH3), 113.91 (C1), 169.97
(CH3CO2), 197.29 (CHO).
C6H11IO4: C, 26.30; H, 4.05%); δH (200 MHz, CDCl3): 3.29 (m,
2H, H5), 3.39 (s, 1H, methoxy), 4.18 (m, 2H, H2,3), 4.23 (q, 1H,
H4, J = 6 Hz), 4.87 (s, 1H, H1); δC (50 MHz, CDCl3): 38.03
(CH2I), 55.28 (OCH3), 75.45 (C3), 75.59 (C4), 82.68 (C2),
108.13 (C1).
5Ј-Azido-5Ј-deoxy-1Ј-O-methyl-D-ribose (8)
Compound 7 (4 g, 14.6 mmol) and sodium azide (3 g, 46.1
mmol) were dissolved in N,N-dimethylformamide (70 mL) and
the solution was stirred at 110 ЊC for 24 h. After evaporation of
the solvent, the residue was dissolved in H2O (150 mL) and
extracted by diethyl ether 5 times. After drying with anhydrous
MgSO4, the solvent was evaporated to give syrupy 8 (2.7 g,
97%). (Found: C, 37.94; H, 5.88; N, 21.92. Calc. for C6H11N3O4:
C, 38.10; H, 5.86; N, 22.21%); δH (200 MHz, CDCl3): 3.37 (s,
3H, methoxy), 3.32, 3.47 (dd, dd, 2H, H5, J = 6.2 Hz, 3.2 Hz),
3.99 (d, 1H, H3, J = 5.2 Hz), 4.08 (dd, 1H, H2, J = 3.2 Hz, 3.2
Hz), 4.18 (q, 1H, H4, J = 5.2 Hz), 4.83 (s, 1H, H1); δC (50 MHz,
CDCl3): 51.12 (CH2N3), 55.73 (OCH3), 75.24 (C3), 75.03 (C4),
81.61 (C2), 107.3 (C1).
5Ј-Imino-5Ј-deoxy-1Ј,2Ј-O-isopropylidene-ꢀ-D-ribose (3)
Compound 2 (0.9 g, 3.91 mmol) was dissolved in ammonia
saturated in methanol (10 mL) and stirred for 24 h at room
temperature. After evaporation of the solvent under reduced
pressure, column chromatography on silica gel (eluent; ethyl
acetate–hexane = 2 : 1, v/v) gave 3 (0.7 g, 96%). (Found: C,
51.08; H, 6.92; N, 7.24. Calc. for C8H13NO4: C, 51.33; H, 7.00;
N, 7.48%); δH (200 MHz, CDCl3): 1.36, 1.57 (ss, 6H, acetonide),
3.89 (d, 1H, H2, J = 8.8 Hz), 4.03 (s, 1H, H5), 4.20 (dd, 1H, H3,
J = 5 Hz, J = 5 Hz), 4.54 (t, 1H, H4, J = 4.3 Hz), 5.78 (d, 1H, H1,
J = 3.6 Hz); δC (50 MHz, CDCl3): 26.34 (CCH3), 26.47 (CCH3),
70.74 (C3), 79.24 (C4), 81.52 (C2), 104.08 (CCH3), 112.73 (C1),
5Ј-Amino-5Ј-deoxy-1Ј-O-methyl-D-ribose (9)
Compound 8 (2.7 g, 14.3 mmol) and triphenylphosphine (5.2 g,
19.8 mmol) were dissolved in tetrahydrofuran (50 mL) and the
solution was stirred for 24 h at room temperature. To the solu-
tion was added 100 mL of H2O and triphenylphosphine oxide
was removed by washing with diethyl ether three times. The
solution was concentrated by evaporation and the syrupy resi-
due was dissolved in H2O. After filtration and evaporation,
compound 9 (1.5 g, 64%) was obtained. (Found: C, 43.89; H,
7.99; N, 8.36. Calc. for C6H13NO4: C, 44.16; H, 8.03; N, 8.58%);
δH (200 MHz, D2O): 2.53 (s, 2H, –OH), 2.91 (m, 2H, H5), 3.37
(s, 3H, methoxy), 3.93 (d, 1H, H2, J = 4.8 Hz), 3.98 (d, 1H,
H3, J = 4.8 Hz), 4.14 (t, 1H, H4, J = 5.0 Hz), 4.82 (s, 1H, H1);
δC (50 MHz, D2O): 39.97 (CH2NH2), 55.04 (OCH3), 71.43 (C3),
74.55 (C4), 80.54 (C2), 106.92 (C1).
139.81 (NH᎐CH).
᎐
5Ј-Amino-5Ј-deoxy-1Ј,2Ј-O-isopropylidene-ꢀ-D-ribose (4)
Compound 3 (0.7 g, 3.74 mmol) was dissolved in methanol
(20 mL). After addition of Pd on charcoal (600 mg), the solu-
tion was stirred under H2 atmosphere (4 atm.) for two days at
room temperature. After filtration through celite twice and
evaporation, the product was isolated by column chromato-
graphy on silica gel (eluent; methanol–chloroform = 1 : 7, v/v)
(0.5 g, 71%).
(Found: C, 50.84; H, 8.04; N, 7.19. Calc. for C8H15NO4: C,
50.78; H, 7.99; N, 7.40%); δH (200 MHz, CDCl3): 1.36, 1.57 (ss,
6H, acetonide), 2.85, 3.00 (dd, dd, 2H, H5, J = 5.1, 5.3 Hz, 3.0,
3.6 Hz), 3.85 (m, 2H, H2,3), 4.55 (t, 1H, H4, J = 4.6 Hz), 5.77 (d,
1H, H1, J = 3.8 Hz); δC (50 MHz, CDCl3): 26.36 (CCH3), 26.62
(CCH3), 49.13 (CH2NH2), 75.38 (C3), 79.04 (C4), 87.74 (C2),
103.91 (CCH3), 113.10 (C1).
5Ј-Acrylamido-5Ј-deoxy-1Ј-O-methyl-D-ribose (10)
Compound 9 (0.4 g, 2.45 mmol) and acrylic anhydride (0.247 g,
1.96 mmol) were dissolved in THF (20 mL) and stirred for
20 min at 0 ЊC and for 11 h at room temperature. After evapor-
ation of the solvent, compound 10 (0.25 g, 47%) was isolated by
column chromatography on silica gel (eluent; acetone : CCl4 =
3 : 3, v/v). (Found: C, 49.51; H, 6.85; N, 6.19. Calc. for
C9H15NO5: C, 49.76; H, 6.96; N, 6.45%); δH (200 MHz,
[D6]DMSO): 3.39 (s, 3H, methoxy), 3.58 (t, 2H, H5, J = 4.2 Hz),
4.01 (d, 1H, H3, J = 4.6 Hz), 4.12 (m, 2H, H2,4), 4.82 (s, 1H, H1),
5.86 (d, 1H, H8c, J = 10 Hz), 6.19 (m, 2H, H7,8t); δC (50 MHz,
[D6]DMSO): 41.50 (CH2NH), 54.19 (OCH3), 71.63 (C3), 73.96
(C4), 80.84 (C2), 107.71 (C1), 124.75 (CH2=CH), 130.48
(CH2=CH), 164.95 (CONH).
5Ј-Acrylamido-5Ј-deoxy-1Ј,2Ј-O-isopropylidene-ꢀ-D-ribose (5)
Acryloyl chloride (0.4 mL) and 4 (0.3 g, 1.59 mmol) were dis-
solved in THF (10 mL) and stirred for 2 h at room temperature.
After evaporation of the solvent, compound 5 (0.21 g, 54%) was
isolated by column chromatography on silica gel (eluent: ethyl
acetate). (Found: C, 53.98; H, 6.84; N, 5.47. Calc. for C11H17-
NO5: C, 54.31; H, 7.04; N, 5.76%); δH (200 MHz, CDCl3): 1.34,
1.56 (ss, 6H, acetonide), 3.56 (m, 2H, H5), 3.83 (m, 2H, H2,3),
4.58 (t, 1H, H4, J = 4.2 Hz), 5.76 (d, 1H, H1, J = 4.4 Hz), 6.37 (d,
1H, H8c, J = 14 Hz), 6.71 (d, 1H, H8t, J = 10 Hz), 6.79 (d, 1H,
H7, J = 10 Hz); δC (50 MHz, CDCl3): 26.35 (CCH3), 26.48
(CCH3), 54.97 (CH2NH2), 74.59 (C3), 79.23 (C4), 87.94 (C2),
104.05 (CCH3), 112.98 (C1), 125.91 (CH ᎐CH), 131.25
Polymerization
Monomer (5: 2.47 mmol LϪ1 or 10: 1.15 mmol LϪ1) and potas-
sium persulfate (2 mol%) were dissolved in water in a poly-
merization tube. After three freeze–thaw cycles under N2, the
tube was sealed and placed in a water bath at 80 ЊC for 15 h. The
polymerization solution was precipitated in acetone to give 11
(yield: 84%) or 13 (yield: 68%). Polymer 11. δH (200 MHz,
CDCl3): 1.38, 1.59 (ss, 6H, acetonide), 1.39–2.31 (3H, ethylene),
3.89 (4H, H2,3,5), 4.55 (1H, H4), 5.74 (1H, H1).
᎐
2
(CH2=CH), 165.17 (CONH).
1Ј-O-Methyl-D-ribose (6)
Compound 6 was prepared according to the literature.16
5Ј-Iodo-5Ј-deoxy-1Ј-O-methyl-D-ribose (7)
Polymer 13. δH (200 MHz, [D6]DMSO): 1.32–2.32 (3H, ethyl-
ene), 3.29 (3H, methyl), 3.82 (3H, H2,3,5), 4.29 (1H, H4), 4.69
(1H, H1).
To a solution of 6 (5.2 g, 31.6 mmol) in 1,4-dioxane (50 mL),
triphenylphosphine (17 g), iodine (17.2 g), and pyridine (6 mL)
were added, and the solution was stirred for 24 h at room
temperature. After evaporation of the solvent, compound 7
(5.6 g, 61%) was isolated by column chromatography on silica
gel (eluent; diethyl ether). (Found: C, 26.17; H, 4.03. Calc. for
Hydrolysis of polymer 11
Polymer 11 (60 mg) was dissolved in 2 M HCl and stirred for
18 h at room temperature. The resulting polymer (12) was
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 7 6 – 2 2 8 2
2281

View Article Online
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7060–7064.
isolated by precipitation in acetone and dried (32.4 mg, 65%).
δH (200 MHz, D2O): 1.32–2.32 (3H, ethylene), 3.35–4.2 (2H,
H5), 4.07 (1H, H3), 4.19 (2H, H2,4), 5.25 (1H, H1); δC (50 MHz,
D2O): 39.21 (chain –CH2–), 52.04 (C5), 56.19 (chain –CH–),
74.92 (C3), 76.84 (C4), 82.97 (C2), 104.03 (C1), 179.92 (C᎐O).
᎐
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Kinetic measurements for ethyl p-nitrophenyl phosphate (ENPP)
The solutions of the catalysts (7.3 × 10Ϫ5 M) and the solutions
of ethyl p-nitrophenyl phosphate (7.0 × 10Ϫ3 M) in water
buffered with 0.02 M Tris (pH 7.4) were prepared. The ionic
strength was adjusted to 0.02 (KCl). The definite portions of
the catalyst and the substrate solutions were mixed in a measur-
ing cell. The reference cell was filled with a solution of the same
substrate concentration buffered with Tris (pH 7.4) at the ionic
strength of 0.02 (KCl). The reaction rates were determined by
measuring the absorption (At) of the conjugate anion of
p-nitrophenol (400 nm) as a function of time (t) with a Hitachi
(Model 200–20) thermosttated spectrophotometer ( 0.1 ЊC).
16 F. Weygand and F. Wirth, Chem. Ber., 1952, 85, 1000–1007.
17 (a) R. Breslow and D.-H. Huang, Proc. Natl. Acad. Sci. U. S. A.,
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H.-J. Schneider, Liebigs Ann. Chem., 1996, 1757–1767.
18 (a) M. J. Han, K. S. Yoo, T. J. Cho, J. Y. Chang, Y. J. Cha and
S. H. Nam, Chem. Commun., 1997, 163–164; (b) M. J. Han, K. S.
Yoo, K. H. Kim, G. H. Lee and J. Y. Chang, Macromolecules, 1997,
30, 5408–5415.
19 The mole concentration of ribose is same as the ribose residue
concentration of the polymer.
20 R. Breslow, J. B. Doherty, G. Guillot and C. Lipsey, J. Am. Chem.
Soc., 1978, 100, 3227–3229.
Kinetic measurements for N-methyl pyridinium 4-tert-butyl-
catechol cyclic phosphate (CP)
Polymer 12 (2 × 10Ϫ5 M) and CP (4 × 10Ϫ4 M) were dissolved in
Tris-buffer (pH 7.4) with ionic strength of 0.2 (KCl) at 25 ЊC.
80 µL of the reaction solution was taken by a microsyringe
at definite time intervals and injected into a Waters LC
(Conditions: Waters Ultrahydrogel 120 GPC column, UV
detector λ = 285 nm, eluent; 0.025 M KCl, flow rate; 0.8 mL
minϪ1). The concentrations of the reactants and products were
evaluated by peak areas, which were corrected by the extinction
coefficients of the compounds (ε = 2460 for CP, 3500 for 1P,
3490 for 2P and 2310 for BC at 285 nm and pH 7.4).
21 R. Breslow, P. Bovy and C. L. Hersh, J. Am. Chem. Soc., 1980, 102,
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22 P. Chambon, J. D. Weill, J. Doly, M. T. Strosser and P. Mandel,
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25 D. Lautier, J. Lagueux, J. Thibodeau, L. Menard and G. G. Poirier,
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Acknowledgements
This work was supported by a grant from the Korean Ministry
of Education and Korean Science and Engineering
Foundation.
26 R. Alvarez-Gonzalez, G. Pacheco-Rodriguez and H. Mendoza-
Alvarez, Mol. Cell. Biochem., 1994, 138, 33–37.
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28 C. M. Simbulan-Rosenthal, D. S. Rosenthal, S. Iyer, A. H. Boulares
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O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 2 7 6 – 2 2 8 2
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