5-(β-Cyclodextrinylamino)-5-deoxy-α-D-riboses as models for nuclease, ligase, phosphatase, and phosphorylase

Article abstract of DOI:10.1002/(SICI)1521-3773(20000117)39:2<347::AID-ANIE347>3.0.CO;2-Q

β-Cyclodextrin derivatives crowned with ribose rings (such as 1) catalyzed the hydrolysis, esterification, and phosphorylation of catechol- derived phosphate esters. The vicinal cis-diols on the ribose groups appear to play a major role in the catalytic activity of these enzyme models by the formation of hydrogen bonds which activate the phosphorus atom to nucleophilic attack.

Full text of DOI:10.1002/(SICI)1521-3773(20000117)39:2<347::AID-ANIE347>3.0.CO;2-Q

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[15] N. Okabe, M. Koizumi, Acta Crystallogr. C: Cryst. Struct. Commun.
1998, 54, 288; R. Faure, H. Loiseleur, G. Thomas-David, Acta
Crystallogr. B: Struct. Crystallogr. Cryst. Chem. 1973, 29, 1890; S. L.
Wang, J. W. Richardson, S. J. Brigg, R. A. Jacobson, W. P. Jensen,
Inorg. Chim. Acta 1986, 111, 67.
because there are plenty of biopolymers containing ribose
rings. This prompted us to further study the mechanism of
action by synthesizing enzyme models containing riboses
capping b-cyclodextrin (CD) and then performing model
reactions with phosphate substrates. The models showed
catalytic activity for the hydrolysis of phosphodiester (nucle-
ase activity), the hydrolysis of phosphomonoester (phospha-
tase activity), the esterification of phosphomonoester to
phosphate diester (ligase activity), and the phosphorylation
of alcohols with phosphate ions (phosphorylase activity).
6-Monotosyl-b-cyclodextrin was prepared as described in
the literature.[4] It was treated with 5-amino-5-deoxy-1,2-O-
isopropylidene-a-d-ribose to yield 5-(b-cyclodextrinylami-
no)-5-deoxy-1,2-O-isopropylidene-a-d-ribose (R12), which
was hydrolyzed to form 5-(b-cyclodextrinylamino)-5-deoxy-
a-d-ribose (R0) (Scheme 1). The derivatives R1 and R3, in
[16] X-ray structure analysis: Enraf-Nonius CAD-4 diffractometer. Unit
cell parameters were determined from automatic centering of 25
reflections (4 < 2q < 608) and refined by least-squares methods. Data
were collected with graphite-monochromatized MoKa radiation (l ˆ
0.71069 Š) by using the w scan technique. Lorentzian, polarization,
and extinction corrections were made. The structure was solved by
direct methods and successive Fourier difference syntheses with the
SHELXL-93 computing program. Crystal data for 1:
C23H24ClN7Ni2O10 (Mr ˆ 711.36), monoclinic, space group P21/c, a ˆ
13.456(3), b ˆ 10.906(2), c ˆ 19.990(7) Š, b ˆ 91.59(3), Vˆ
3
1
2932.4(13) Š3, Z ˆ 4, 1calcd ˆ 1.611 gcm
,
m ˆ 14.40 cm
,
F(000) ˆ
1456. The refinements by full-matrix least-squares methods gave final
R1(Fo) ˆ 0.074, wR2(Fo2† ˆ 0.184 and S ˆ 0.999 for 7643 reflections
with I ꢀ 2s(I) and 389 variables. All non-hydrogen atoms were refined
anisotropically. Crystallographic data (excluding structure factors) for
the structure reported in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary publica-
tion no. CCDC-102206. Copies of the data can be obtained free of
charge on application to CCDC, 12 Union Road, Cambridge
CB21EZ, UK (fax: (‡44)1223-336-033; e-mail: deposit@ccdc.cam.
ac.uk).
[17] The calculations were made with the computer program CLUMAG,
which uses the irreducible tensor operator formalism (ITO): D.
Gatteschi, L. Pardi, Gazz. Chim. Ital. 1993, 123, 231.
5-(b-Cyclodextrinylamino)-5-Deoxy-a-d-
Riboses as Models for Nuclease, Ligase,
Phosphatase, and Phosphorylase**
Man Jung Han,* Kyung Soo Yoo, Ji Young Chang, and
Tae-Kyu Ha
The synthesis and activity testing of enzyme models have
drawn attention to the elucidation of enzyme structures and
mechanisms.[1] Several models have been synthesized and
their activities have been tested for nuclease, ligase, phos-
phatase, and phosphorylase.[2] We recently reported that
ribose-containing polymers show catalytic activity for the
cleavage of DNA.[3] The study of various polymer structures
strongly suggested that the vic-cis-diol groups of the ribose
rings play a key role in the catalysis, which is surprising
Scheme 1. b-Cyclodextrinyl compounds and reactions of phosphate esters.
which 1-OH and 3-OH of the ribose ring are blocked by
methyl groups, were synthesized by similar methods.[5] The N-
methylpyridinium salt of the cyclic phosphate (CP)[5] and the
1-phosphate (1P) and 2-phosphate (2P) of 4-tert-butylcate-
chol (BC) were also synthesized as substrates (Scheme 1).
Compounds BC[6] and CP are well known to form inclusion
complexes with CD, and CD capped with imidazole has been
investigated as a nuclease model using CP as substrate.[2]
Reactions 1 (and 5), 2, 3, and 4 (Scheme 1) are the model
reactions for nuclease, ligase, phosphatase, and phosphoryl-
ase, respectively. The rates of the reactions were measured at
pH7.4 (Tris buffer), ionic strength (m) of 0.2 (KCl), and 258C
by HPLC in the presence of the cyclodextrinyl compounds
(4 mm) and the substrates (0.4 mm). This concentration ratio
was chosen for the activity measurements since no significant
rate acceleration was observed by further increasing the
concentration of the former compound.
[*] Prof. Dr. M. J. Han, Dr. K. S. Yoo
Department of Molecular Science and Technology
Ajou University, Suwon 442-749 (Korea)
Fax : (‡ 82)331-214-8918
E-mail: mjhan@madang.ajou.ac.kr
Prof. Dr. J. Y. Chang
Department of Fiber and Polymer Science
College of Engineering, Seoul National University
Kwanak-Ku, Seoul 151-742 (Korea)
Dr. T.-K. Ha
Laboratory of Physical Chemistry
Swiss Federal Institute of Technology
ETH Zentrum, 8092 Zürich (Switzerland)
[**] This work was supported by the Korean Science and Engineering
Foundation and the Korean Ministry of Education. The computer
center of ETH Zürich is also gratefully acknowledged for the use of
computing facilities.
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The hydrolysis rates of CP were measured in the buffer
alone and also in the presence of ribose, CD, R0, R1, R3, or R12.
The time-dependent concentration changes of CP are shown
in Figure 1a. The hydrolysis was accelerated by R0, R1, and R3
in the order of R0 > R3 > R1, whereas ribose, CD, and R12
showed no catalytic activity above the activity of the buffer,
of R1 are shown in Figure 1c. The reaction reached equili-
brium after 100 hours. Similar reactions occurred in the
presence of R0 or R3.[8]
When BC, cyclodextrinyl compounds (R0, R1, or R3), and
KH2PO4 were mixed in the mole ratio of 1:10:15 at pH7.4 (Tris
buffer), esterification to form 1P occurred, which was
followed by cyclization to yield CP. In order to make this
reaction first order, phosphate was added in excess. These
reactions did not occur in the buffer solution or in the
presence of CD or R12. The concentration change in the
presence of R3 versus time of the reaction is plotted in
Figure 1d. Phosphate compound 1P formed immediately at
the beginning of the reaction and then CP began to form. The
reaction reached equilibrium after 140 hours. Similar reac-
tions occurred in the presence of R0 and R1.[9]
In order to investigate whether the sec-amino groups on the
cyclodextrinyl compounds contribute to the catalysis as a
general base or, more likely at the operating pH of 7.4, as a
general acid, we synthesized 6-monomethylamino-b-cyclo-
dextrin as a model compound. The reactions with CP, 1P, and
BC were repeated in the presence of the 6-monomethylami-
no-b-cyclodextrin (instead of the enzyme models R0, R1, and
R3) using the same conditions, and the rates of reaction were
measured. The rate constant for the hydrolysis of CP was
1
found to be 3.38 Â 10 4 h , nearly the same as that of the
1
uncatalyzed reaction (3.23 Â 10 4 h ), and reactions 2, 3, and
4 (Scheme 1) did not occur, indicating that the sec-amino
groups do not participate in the catalysis.
Based on the experimental results described above, the
mechanism of action can be postulated as follows: the vic-cis-
diols form hydrogen bonds with the two phosphoryl oxygen
atoms of the phosphate group when the substrate is in the CD
cavity of R0. This activates the phophorus atom to be attacked
by nucleophiles (H2O or OH) as shown in Scheme 2. In the
case of hydrolysis of CP (Scheme 2A), water could attack
either side of the activated phosphate to form 1P or 2P. The
hydrolysis and esterification of 1P (Scheme 2B) occur when
water or the 2-OH group of 1P attack the phosphorus atom
Figure 1. Concentration changes versus time: a) for hydrolysis of cyclic
*
*
phosphate (CP) in Tris buffer solution ( ) and in the presence of ribose ( ),
~
(
!
&
*
*
), R1
CD ( ), R12
(
), R3
(
), and R0 ( ); b) for the reaction of CP
~
&
&
*
( ) !1P ( ) ‡ 2P ( ) catalyzed by R0; c) for the reaction of 1P ( ) !CP
~
~
&
*
(
(
) ‡ BC ( ) catalyzed by R1; d) for the reaction of BC ( ) !1P ( ) !CP
*
) catalyzed by R3. (For reaction conditions, see the Experimental
Section.)
indicating clearly that the vic-cis-diol groups of riboses on
CDs are essential for the catalysis. As the reactions were
pseudo-first-order, the rate constants for the hydrolysis were
obtained by plotting logarithmic concentrations of CP against
1
time and were found to be 10.7, 1.41, and 2.03 Â 10 3 h for
R0, R1, and R3. These were, respectively, 33, 4.4, and 6.3 times
1
faster than for the uncatalyzed reaction (3.2 Â 10 4 h ). The
hydrolysis products of CP were found to be 1P and 2P as
previously reported.[2] The reactions in the presence of the
cyclodextrinyl compounds (R0, R1, and R3) reached equili-
brium after extended times, that is, the reactions are
reversible. In the hydrolysis of CP in the presence of R0
(Figure 1b), the concentration of CP decreased while 1P
and 2P simultaneously formed.[7] After 120 hours the reac-
tions reached equilibrium.
When 1P was mixed with R0, R1, or R3 in Tris buffer
solution at pH7.4, cyclization to CP and hydrolysis to BC
occurred simultaneously. Compound 1P was found to be
stable in the buffer solution and in the presence of CD or R12
under the same conditions. No significant reaction was found
after one month. The concentration changes of the starting
material and the products in the reaction of 1P in the presence
Scheme 2. Action mechanisms of b-cyclodextrinyl compounds. The 2,3-cis-
diol of ribose forms hydrogen bonds with the two phosphoryl oxygen atoms
of the phosphate to activate the phosphorus atom for attack by
nucleophiles (H2O or OH of catechol). a) The phosphorus atom of CP is
attacked by H2O 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 nucleo-
philic attack of the 1-OH group of BC to the phosphorus atom to eliminate
the OH group from the phosphate.
348
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with the aid of preparative liquid chromatography with 0.025m KCl
aqueous solution as the eluent. 6-Monomethylamino-b-cyclodextrin was
synthesized by a reaction of 6-monotosyl-b-cyclodextrin (1 g, 0.78 mmol)
with aqueous 40 wt% of methylamine (0.72 mL, 0.93 mmol) for 48 h at
room temperature (yield: 41.5%, m.p.: 219 ± 2208C).
Cyclodextrinyl compounds (4 Â 10 3 m) and phosphates (4 Â 10 4 m) were
dissolved in Tris-buffer solution (pH7.4) with an ionic strength of 0.2 (KCl)
in a vial, which was immersed in a water bath at 25 Æ 0.18C. Aliquots (80 ml)
of the reaction solution were taken by microsyringe at defined time
intervals and injected into a Waters liquid chromatograph with UV
detector at 285 nm (eluent: 0.025m KCl, flow rate: 0.8 mLmin 1). The
concentrations of the reactants and products were evaluated by peak areas,
which were corrected by the extinction coefficients of the compounds (e ˆ
2460 for CP, 3500 for 1P, 3490 for 2P, 2310 for BC at 285 nm and pH7.4).
and either break the P O ester bond or cause the OH group
of the phosphate to leave, respectively. The phosphorylation
of BC (Scheme 2C) occurs by nucleophilic attack of the 1-OH
group of BC at the activated phosphorus atom with the OH
group of the phosphate as the leaving group. Since the
cleavage of CP is occasionally regioselective, depending on
the directions of hydrogen bonds and attacking nucleo-
philes,[2] 2P is not formed for the reactions shown in Figure 1c
and d.
For catalytic activity, two hydrogen bonds should be formed
simultaneously between the vic-cis-diols of ribose and the two
phosphoryl oxygen atoms of the phosphate group. The order
of catalytic activity, R0 > R3 > R1, seems to depend on the
extent of hydrogen bond formation; in the case of R0,
hydrogen bonds can be formed either by the 1,2- or the 2,3-
diol of the ribose so its activity is the highest. As the arm
connecting ribose to CD in cyclodextrinyl compounds is
rather short and in a trans position to the diols, the 1,2-diol is
probably more easily accessible to the phosphate than the 2,3-
diol and thus, the activity of R3 was higher than that of R1. The
1-OH of BC (Scheme 2C) protrudes toward the phosphate so
that 1P was formed more favorably than 2P.
Received: May 3, 1999
Revised: October 12, 1999 [Z13367]
[1] A. J. Kirby, Angew. Chem. 1996, 108, 770 ± 790; Angew. Chem. Int. Ed.
Engl. 1996, 35, 707 ± 724.
[2] a) R. Breslow, J. B. Doherty, G. Guillot, C. Lipsey, J. Am. Chem. Soc.
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[5] T. A. Khwaja, C. B. Resse, J. C. M. Stewart, J. Chem. Soc. 1970, 2092 ±
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[6] R. Breslow, A. Graff, J. Am. Chem. Soc. 1993, 115, 10988 ± 10989.
[7] The rate constants k1 and k5 were measured to be 6.5 and 4.1 when
catalyzed by R0, 0.87 and 0.54 by R1 and 1.17 and 0.89 Â 10 3 h 1 by R3,
respectively.
The formation of strong hydrogen bonds was also born out
by a theoretical study of the interactions between 3,4-cis-
dihydroxytetrahydrofuran (DHTHF) and H2PO4 , and
2
DHTHF and HPO4 . Quantum-chemical calculations using
density functional theory at the B3LYP/6-31 ‡ G** level and
also at the MP2/6-31 ‡ G** level of theory[10] showed that:
1) the hydrogen bond distances are rather short (1.76 Š and
2
1.52 Š for the H2PO4 and HPO4 adducts, respectively),
2) the dissociation energies of the hydrogen bonds are quite
[8] The rate constants k2 and k3 were found to be 28.4 and 11.6 when
catalyzed by R0, 4.35 and 1.59 by R1, and 5.99 and 2.77 Â 10 3 h 1 by R3,
respectively.
1
large (19.3 and 42.5 kcalmol ), and 3) the red shifts of the
OH-stretching frequencies of DHTHF in the adducts are also
1
1
[9] The rate constant k4 was measured to be 10.2, 2.6, and 4.1 Â 10 3 h
very large (up to 300 and 650 cm , respectively). The
when catalyzed by R0, R1, and R3, respectively.
reduction of the charge density at the phosphate sites was
found to be appreciable (0.057 and 0.191 electrons).
[10] a) A. D. Becke, J. Chem. Phys. 1993, 98, 5648 ± 5652; b) J. A. Pople,
J. S. Binkley, R. Seeger, Int. J. Quantum Chem. Symp. 1976, 10, 1 ± 19.
[11] a) P. Chambon, J. D. Weill, J. Doly, M. T. Strosser P. Mandel, Biochem.
Biophys. Res. Commun. 1966, 25, 638; b) T. Sugimura, S. Fugimura, S.
Hasegawa, Y. Kawamura, Biochim. Biophys. Acta 1967, 138, 438.
[12] a) K. Ueda, O. Hayaishi, Ann. Rev. Biochem. 1985, 54, 73 ± 100; b) D.
Lautier, J. Lagueus, J. Thibodeau, L. Mennard, G. G. Poirier, Mol. Cell
Biochem. 1993, 122, 171 ± 193; c) R. Alvarez-Gonzalez, G. Pacheco-
Rodriguez, H. Mendoza-Alvarez, Mol. Cell Biochem. 1994, 138, 33 ±
37; d) H. Maruta, N. Matsumura, S. Tamura, Biochem. Biophys. Res.
Commun. 1997, 236, 265 ± 269.
[13] C. M. Simbulan-Rosenthal, D. S. Rosenthal, S. Iyer, A. H. Boulares
M. E. Smulson, J. Biol. Chem. 1998, 273, 13703 ± 13712.
[14] a) M. Malanga, F. R. Althaus, J. Biol. Chem. 1994, 269, 17691 ± 17696;
b) M. S. Satoh, G. G. Poirier, T. Lindahl, Biochemistry 1994, 33, 7099 ±
7106.
As the synthetic ribose-containing polymers have shown
nuclease activity,[3] biopolymers containing riboses may have
similar enzyme activities. One of the typical biopolymers
containing riboses with vic-cis-diols is poly(ADP-ribose)
‡
formed from NAD in chromatin. However, its functions
are not clear so far; since its discovery in 1966,[11] it has been
suggested to be involved in numerous biological reactions.[12]
Poly(ADP-ribose) forms during apoptosis[13] and DNA rep-
lication, transcription, and repair,[14] when nuclease, ligase,
phosphatase and/or phosphorylase actions will be required.
Further study on the enzyme functions of naturally occurring
biopolymers having riboses with free vic-cis-diols is necessary.
Experimental Section
Reaction of 6-monotosyl-b-cyclodextrin (0.5 g, 3.88 mmol) with 5-amino-5-
deoxy-1-O-methyl-d-ribose (0.15 g, 7.76 mmol) or 5-amino-5-deoxy-3-O-
methyl-1,2-O-isopropylidene-a-d-ribose (0.15 g, 7.51 mmol) in DMF
(5 mL) gave 5-(b-cyclodextrinylamino)-5-deoxy-1-O-methyl-a-d-ribose
(R1, yield: 72.4%) and 5-(b-cyclodextrinylamino)-5-deoxy-3-O-methyl-
1,2-O-isopropylidene-a-d-ribose (R12, yield: 74.9%), respectively. The
latter compound was hydrolyzed in 1n HCl to result in 5-(b-cyclodextri-
nylamino)-5-deoxy-a-d-ribose (R0, yield: 79.5%). 4-tert-Butylcatechol-1-
phosphate (1P) and 2-phosphate (2P) were obtained by separation of the
hydrolysis products of the cyclic phosphate of 4-tert-butyl catechol (CP)
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