Acceleration of acid-catalyzed transesterification of 2-hydroxypropyl-p-nitrophenyl phosphate by organic solvents

Article abstract of DOI:10.1021/ol991321m

(Formula Presentation) Transesterification of 2-hydroxypropyl-p-nitrophenyl phosphate in the presence of 0.092 M HClO₄ is 50-5000 times faster in acetonitrile, 1,4-dioxane, methanol, ethanol, N,N-dimethylformamide, or dimethyl sulfoxide than in

Full text of DOI:10.1021/ol991321m

ORGANIC
LETTERS
2
000
Vol. 2, No. 3
77-380
Acceleration of Acid-Catalyzed
Transesterification of
3
2
-Hydroxypropyl-p-nitrophenyl
Phosphate by Organic Solvents
In Seok Hong and Junghun Suh*
Department of Chemistry, DiVision of Chemistry and Molecular Engineering, and
Center for Molecular Catalysis, Seoul National UniVersity, Seoul 151-742, Korea
jhsuh@snu.ac.kr
Received December 8, 1999
ABSTRACT
4
Transesterification of 2-hydroxypropyl-p-nitrophenyl phosphate in the presence of 0.092 M HClO is 50−5000 times faster in acetonitrile, 1,4-
dioxane, methanol, ethanol, N,N-dimethylformamide, or dimethyl sulfoxide than in water. This demonstrates the importance of tuning the
microenvironments in designing synthetic nucleases.
The drive to develop synthetic nucleases has stimulated
intensive studies in catalysis of hydrolysis of phosphate
diesters in recent years. Phosphate diesters are very difficult
to hydrolyze. The half-life for spontaneous hydrolysis of
phosphate diester linkages of DNA at 25 °C and pH 7 has
and lanthanide complexes.2
6-28
In view of the high stability
of DNA and RNA, various activated analogues of DNA
(7) Breslow, R.; Huang, D. L.; Anslyn, E. Proc. Natl. Acad. Sci. U.S.A.
1
989, 86, 1746.
(
8) Stern, M. K.; Bashkin, J. K.; Sall, E. D. J. Am. Chem. Soc. 1990,
11
1
been estimated as 10 years. Hydrolysis of phosphate diester
linkages of RNA is much easier than those of DNA as it
involves the intermediacy of cyclic phosphate diester formed
by the nucleophilic attack of 2′-hydroxyl group at the
phosphorus atom. Yet, half-life for the spontaneous hydroly-
sis of phosphate diester linkages of RNA at 25 °C and pH
112, 5357.
(9) Chin, J. Acc. Chem. Res. 1991, 25, 145.
10) Vance, D. H.; Czarnik, A. W. J. Am. Chem. Soc. 1993, 115, 12165.
11) Wall, M.; Hynes, R. C.; Chin, J. Angew. Chem., Int., Ed. Engl. 1993,
(
(
32, 1633.
(
12) Bashkin, J. K.; Frolova, E. I.; Sampath, U. J. Am. Chem. Soc. 1994,
1
16, 5981.
(
13) Chapman, W. H., Jr.; Breslow, R. J. Am. Chem. Soc. 1995, 117,
is about 100 years.1
5462.
7
(
(
(
14) Young, M. J.; Chin, J. J. Am. Chem. Soc. 1995, 117, 10577.
15) Linkletter, B.; Chin, J. Angew. Chem., Int. Ed. Engl. 1995, 34, 472.
16) Wahnon, D.; Lebuis, A.-M.; Chin, J.Angew. Chem., Int. Ed. Engl.
Artificial nucleases designed to date include nonmetallic
2
-6
7-25
compounds
as well as transition metal complexes
1
995, 34, 2412.
(
17) Koike, T.; Inoue, M.; Kimura, E.; Shiro, M. J. Am. Chem. Soc.
(1) Williams, N. H.; Takasaki, B.; Wall, M.; Chin, J. Acc. Chem. Res.
1996, 118, 3091.
1
999, 32, 485.
(18) Ragunathan, K. G.; Schneider, H.-J. Angew. Chem., Int. Ed. Engl.
1996, 35, 1219.
(19) Williams, N. H.; Chin, J. J. Chem. Soc., Chem. Commun. 1996,
131.
(2) Breslow, R.; Labelle, M. J. Am. Chem. Soc. 1986, 108, 2655.
3) Jubian, V.; Dixon, R. P.; Hamilton, A. D. J. Am. Chem. Soc. 1992,
(
1
14, 1120.
(
(
4) Barbier, B.; Brack, A. J. Am. Chem. Soc. 1992, 114, 3511.
5) G o¨ bel, M. W.; Bats, J. W.; D u¨ rner, G. Angew. Chem., Int., Ed. Engl.
(20) Molenveld, P.; Kapsabelis, S.; Engberson, J. F. J.; Reinhoudt, D.
N. J. Am. Chem. Soc. 1997, 119, 2948.
1
992, 31, 207.
6) Smith, J.; Ariga, K.; Anslyn, E. V. J. Am. Chem. Soc. 1993, 115,
62.
(21) Hettich, R.; Schneider, H.-J. J. Am. Chem. Soc. 1997, 119, 5638.
(22) Liu, S.; Hamilton, A. D. Tetrahedron Lett. 1997, 38, 1107.
(23) Liu, S.; Hamilton, A. D. Bioorg. Med. Chem. Lett. 1997, 7, 1779.
(
3
1
0.1021/ol991321m CCC: $19.00 © 2000 American Chemical Society
Published on Web 01/20/2000

and RNA have been used as substrates for artificial
nucleases. For example, bis(p-nitrophenyl)phosphate
or HCl in MeCN, 1,4-dioxane, MeOH, EtOH, DMF, or
DMSO containing 1% (v/v) water as well as water are
summarized in Figure 1. Transesterification of HPNPP with
(BNPP) has been regarded as a doubly activated analogue
of DNA, and 2-hydroxypropyl-p-nitrophenyl phosphate
HPNPP),1
3,19,20,22-25,28,29
which undergoes transesterification
(
(
eq 1), has been frequently exploited as an activated analogue
of RNA.
To design effective artificial nucleases, knowledge on
various aspects of phosphate diester hydrolysis is needed.
Enzymes optimize the microenvironment of their active sites
30
Figure 1. log k
o
measured for transesterification of HPNPP with
to maximize the catalytic capability. Similarly, adjustment
of microenvironments around catalytic centers of artificial
enzymes is important for improvement of the catalytic
efficiency. In the case of phosphate diester hydrolysis, little
is known of the solvent effects. This is mainly due to the
difficulties encountered in the kinetic measurement of the
very slow hydrolysis of phosphate diesters. BNPP, the
aforementioned activated DNA analogue, has a half-life of
about 2000 years for spontaneous hydrolysis at 25 °C and
0
1
.092 M HClO
%(v/v) water.
4
or HCl in various solvents. Organic solvents contain
0
4
.092 M HClO or HCl is 50-5000 times faster in the
organic solvents than in water. The rates measured in water,
MeOH, or EtOH are not affected significantly by the nature
of the acid used. On the other hand, the reaction with HClO
is faster than that with HCl by 3-4 times in MeCN or 1,4-
dioxane. This difference might be related to stronger acidity
4
3
1
pH 7. This can be compared with the half-life of 500-
1
000 years for hydrolysis of unactivated peptides at 25 °C
of HClO
both HCl and HClO
4
. In basic solvents such as water, MeOH, and EtOH,
are converted to the conjugate acid of
32-34
and pH 7.
In this regard, we chose HPNPP as an
4
activated analogue of not only RNA but also DNA and
examined solvent effects on its transesterification to obtain
information on designing microenvironments of effective
artificial nucleases.
the solvent. If the acid is not fully ionized in less basic
organic solvents, the different acidity of the acid would be
reflected in the rate. The k values measured in MeCN-
water cosolvents with varying composition are illustrated in
36
o
In the present study, kinetic data were obtained for the
Figure 2.
4
acid (HCl or HClO )-catalyzed transesterification of HPNPP
-
5
(
(
5 × 10 M) at 25 °C in various solvents such as acetonitrile
MeCN), 1,4-dioxane, methanol (MeOH), ethanol (EtOH),
N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),
and water by spectrophotometric measurement (324 nm) of
the release of p-nitrophenol. Products of the reactions were
3
1
identified as the cyclic phosphate ester on the basis of
NMR measurement.35
P
The values of pseudo-first-order rate constants (k
o
) for
transesterification of HPNPP measured with 0.092 M HClO
4
(24) Molenveld, P.; Engberson, J. F. J.; Kooijman, H.; Speck, A. L.;
Reinhoudt, D. N. J. Am. Chem. Soc. 1998, 120, 6726.
(
(
25) Suh, J.; Hong, S. H. J. Am. Chem. Soc. 1998, 120, 12545.
26) Morrow, J. R.; Buttrey, L. A.; Shelton, V. M.; Berback, K. A. J.
Am. Chem. Soc. 1992, 114, 1903.
27) Schneider, H.-J.; Rammo, J.; Hettich, R. Angew. Chem., Int. Ed.
Engl. 1993, 32, 1716.
(
Figure 2. log k for transesterification of HPNPP in the presence
o
(28) Kalesse, M.; Loos, A. Bioorg. Med. Chem. Lett. 1996, 6, 2063.
29) Perrault, D. M.; Anslyn, E. V. Angew. Chem., Intl. Ed. Engl. 1997,
of 0.092 M HClO in MeCN-water cosolvents.
4
(
3
6, 432.
30) For structures and mechanisms of phosphoester hydrolases, see:
(
Hannon, C. L.; Anslyn, E. V. In Bioorganic Chemistry Frontiers; Dugas,
H., Ed.; Springer-Verlag: Berlin, 1993; Vol. 3, pp 193-255.
In Figure 3 is illustrated the dependence of k
o
on the
concentration of HClO measured in various solvents. Slopes
4
(
31) Chin, J.; Banaszczyk, M.; Jubian, V.; Zou, X. J. Am. Chem. Soc.
1
989, 111, 186.
of the straight lines of Figure 3 are 1.0, within experimental
error for organic solvents. The kinetic data measured in
organic solvents are consistent with Scheme 1 which assumes
(32) Bryant, R. A. R.; Hansen, D. E. J. Am. Chem. Soc. 1996, 118, 5498.
(33) Radzicka, A.; Wolfenden, R. J. Am. Chem. Soc. 1996, 118, 6105.
(34) Smith, R. M.; Hansen, D. E. J. Am. Chem. Soc. 1998, 120, 8910.
378
Org. Lett., Vol. 2, No. 3, 2000

form of HPNPP. In this regard, the data points obtained in
water were analyzed again in terms of Scheme 2, considering
Scheme 2
ionization of HPNPP. Results of the analysis are summarized
in Figure 4. Much better fit is obtained by analysis with
Figure 3. Plot of log k
of HPNPP in 99% (v/v) organic solvents (a (b), MeCN; b (9),
,4-dioxane; c (O), MeOH; d (0), EtOH; e (2) DMF; f (4), DMSO)
or in water (g ([), ionic strength maintained at 0.5 M with NaClO ).
o 4
against log [HClO ] for transesterification
1
4
Slopes of the straight lines are 0.97 ( 0.04 for a, 1.06 ( 0.09 for
b, 0.98 ( 0.03 for c, 1.01 ( 0.12 for d, 1.02 ( 0.04 for e, 0.98 (
0.08 for f, and 1.41 ( 0.02 for g.
Scheme 1
Figure 4. Plot of log k
with ionic strength maintained at 0.5 M with NaClO
o
against log [HClO
4
] measured in water
. Regression
4
participation of one acid (HA; HCl, HClO , or conjugate acid
of the solvent) and the neutral form of HPNPP in the rate-
determining step.
4
5
-
-1 -1
of the data points led to k ) (5.58 ( 0.96) × 10
2
M
s
for
-
5
-1 -1
Scheme 1 (curve a), and k
2
) (9.44 ( 0.46) × 10
M
s
and
pK ) 1.16 ( 0.05 for Scheme 2 (curve b).
a
The pKa1 values of monoalkyl phosphates and the pK
a
values of dialkyl phosphates are 1.3-1.9 in water and are
not much different from the pKa1 (2.1) of phosphoric acid,
Scheme 2 than with Scheme 1. The pK value (1.16 ( 0.05)
a
3
7
indicating that effects of alkyl moieties are not great. The
pK of phosphoryl hydroxy group of HPNPP in water,
estimated from analysis with Scheme 2 is consistent with
that estimated above. When the reactivity of the un-ionized
form of HPNPP in acid-catalyzed transesterification is
considered, the reaction is 30-3000 times faster in organic
solvents than in water (Figure 1).
On the basis of the kinetic data, the mechanism of 1 may
be proposed for the acid-catalyzed transesterification of
HPNPP in water and organic solvents. Here, the protonated
species is formed by equilibration between HPNPP and HA
of Scheme 1 or 2. In solvents such as alcohols and water,
another solvent molecule may participate as a general base
to assist the intramolecular attack by the hydroxy group.
a
therefore, may be taken as near 1.3-1.9. Then, HPNPP can
ionize at the acid concentration employed for kinetic
measurements in water. Ionization of HPNPP should be
suppressed in organic solvents compared with water, and
HPNPP would be present mostly as neutral species in the
organic cosolvents in the presence of strong acids.
For the reaction carried out in water, the slope (1.41 (
.02) of the straight line drawn in Figure 3 is considerably
0
lager than 1. This can be attributed to ionization of the
phosphoryl hydroxyl group of HPNPP at lower acid con-
centrations which reduces the molar fraction of the neutral
(35) Chemical shift (85% phosphoric acid used as the external standard)
for the product obtained in D2O with 0.092 M HClO4 was 18.8 ppm in
25
agreement with that for the authentic cyclic ester, whereas that for HPNPP
in D2O was -4.2 ppm. The product obtained in an organic solvent was
compared with that obtained in water. For example, when the product
solutions obtained in 99% (v/v) CD3CN or D2O with 0.092 M HClO4 were
diluted with equal volume of D2O or CD3CN, respectively, identical
chemical shifts (0.55 ppm) were obtained for the phosphorus atom of the
product. In the same solvent, chemical shift for HPNPP was -5.2 ppm.
(36) In MeCN, HCl is partially ionized, whereas HClO4 is fully
ionized: Kolthoff, I. M.; Bruckenstein, S.; Chantooni, M. K. Jr. J. Am.
Chem. Soc. 1961, 83, 3927.
Many organic reactions respond sensitively to changes in
media. Various sets of parameter values have been proposed
for quantitative analysis of solvent effects on rates of organic
(
37) Jencks, W. P.; Regenstein, J. In Handbook of Biochemistry and
Molecular Biology, 3rd ed.; Fasman, G. D., Ed.; CRC Press: Cleveland,
976; Vol. 1, pp 305-351.
1
Org. Lett., Vol. 2, No. 3, 2000
379

reactions. Attempts to correlate kinetic data summarized in
Figure 1 with the solvent parameters38 such as dielectric
constant, Z, E , DN, AN, and Swain A/B were unsuccessful.
T
This suggests that the solvents effects examined in the present
study are not directly related to the physical phenomena
associated with those parameter sets. When charge becomes
more dispersed in the transition state as in some nucleophilic
substitution reactions, the rate is enhanced in less polar
media. It is noteworthy that the charge is more dispersed
in the transition state than in the protonated HPNPP for the
mechanism of 1.
the metal-catalyzed transesterification may be also subject
3
8
3
9
to considerable solvent effects. The high catalytic power
II
of the PEI derivative containing Ni TP has been partly
attributed to the hydrophobic microenvironment created in
Several effective catalysts have been designed for trans-
esterification of HPNPP by using various types of metal
complexes. The best synthetic catalyst reported to date for
transesterification of HPNPP is a poly(ethylenimine) (PEI)
derivative containing Ni(II) complexes of terpyridine (TP)
II
25
the vicinity of the Ni TP centers. It is also possible that
the effective catalysis by the calixarene-based binuclear metal
20,24
complexes
originates partly from the hydrophobic micro-
environment created in water. Complexes of transition metal
ions or lanthanides are the most effective synthetic catalysts
reported so far for hydrolysis of phosphate diester linkages
25
and lauryl pendants. The highest catalytic rate was observed
II
when the contents of Ni TP and lauryl pendants were 5%
8
,13,14,21,26
of DNA and RNA.
Results of the present study
and 12%, respectively, of the amino groups of PEI. Effective
implicate that adjustment of microenvironments of the
catalytic metal centers is important in designing effective
artificial nucleases.
catalysis was also achieved by binuclear metal complexes
derived from a calixarene.2
0,24
The catalytic action of the
metal complexes in transesterification of HPNPP has been
explained in terms of the activation of the two phosphoryl-
oxygen bonds as indicated by 2.1
1,19,23
Acknowledgment. This work was supported by a grant
from Korea Science and Engineering Foundation through
Center for Molecular Catalysis, Seoul National University.
In some cases, the
general base assistance for the intramolecular attack of the
hydroxyl group by a metal-bound hydroxide ion is also
2
3
assumed. The mechanism of 2 is analogous to that of 1
since the two P-O oxygen atoms are bound by Lewis acids
in both mechanisms.
OL991321M
The remarkable acceleration of the acid-catalyzed trans-
esterification of HPNPP in organic solvents suggests that
(39) The degree of acceleration of HPNPP transesterification in the
presence of bis(guanidinium) receptors was higher in acetonitrile than in
water (Oost, T.; Filippazzi, A.; Kalesse, M. Liebigs Ann. 1997, 1005). In
this case, strengthening the hydrogen bonding between HPNPP and the host
by the organic solvent would make a major contribution to the observed
solvent effect.
(38) Isaacs, N. S. Physical Organic Chemistry, 2nd ed.; Longman: Burnt
Mill, 1995; Chapter 5.
380
Org. Lett., Vol. 2, No. 3, 2000