Extraordinary acceleration of phosphodiester hydrolyses by thorium cations

Article abstract of DOI:10.1039/a703568c

Th^IV cations in aqueous Brij micelles strongly promote the hydrolyses of several phosphodiester substrates; in the case of bis(p-nitrophenyl)phosphate 1, the observed rate constant (0.028 s^-1) represents an acceleration of ca 2.8 bil

Full text of DOI:10.1039/a703568c

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Extraordinary acceleration of phosphodiester hydrolyses by thorium cations
Robert A. Moss,* Jing Zhang and Kathryn Bracken
Department of Chemistry, Rutgers University, New Brunswick, New Jersey 08903, USA
ThIV cations in aqueous Brij micelles strongly promote the
hydrolyses of several phosphodiester substrates; in the case
of bis(p-nitrophenyl)phosphate 1, the observed rate constant
2 mm Brij-35, 10 mm HEPES buffer, pH 6.0 (10 mm KCl,
37 °C). We employed a 20-fold excess of Th4 (1 mm) over
substrate (0.05 mm). Kinetics were followed spectropho-
tometrically, monitoring the disappearance of the substrate’s
p-nitrophenyl moiety (290 nm) and the concomitant appearance
of p-nitrophenol (317 nm). Reactions were observed for more
than eight half-lives and pseudo-first order rate constants were
obtained as the means of triplicate runs with r > 0.997 and
reproducibilities within ±5%.
+
2
1
(
0.028 s ) represents an acceleration of ca 2.8 billion.
Much attention is deservedly focused on the remarkable
acceleration of phosphodiester hydrolyses educed by lanthanide
1
(
Ln) cations. Acting as Lewis acids, they bind the substrate’s
2
P–O and mitigate its negative charge, while simultaneously
furnishing a metal-bound hydroxide nucleophile to attack the
phosphodiester’s phosphoryl group. Both Lewis acid strength
and the acidity of the cation’s water of hydration increase with
Rate constants appear in Table 1, where k for BNPP
2
1
hydrolysis, 0.028 s , represents an acceleration of ca. 2.8
1h
billion relative to the uncatalysed reaction at pH 7, whilst the
IV
increasing positive charge density, so that Ce , the only
entry for the intramolecularly assisted cleavage of RNA model
2
1
5
lanthanide with a readily accessible +4 oxidation state, is of
2, k = 0.013 s , is ca. 3.9 3 10 greater than in the absence of
2
1a
particular interest for the mediated hydrolysis of e.g. DNA.
metal cations at pH 7. These enhancements appear to be the
IV
Indeed, an excess of a 2:1 palmitate–Ce complex in aqueous
largest reported to date for Ln or actinide cations with these
commonly employed model substrates. We note, however, that
Brij micelles at pH 7 and 37 °C cleaves the model phosphodi-
ester substrate, bis(p-nitrophenyl)phosphate 1, (BNPP), with
4+
the 20-fold excess of Th is necessary; kobs slows markedly as
4+
[
Th ]:[BNPP] is reduced below 15:1 at pH 6. Control
O
experiments also reveal inhibition of the hydrolysis by (added)
inorganic phosphate.
The hydrolysis of BNPP was examined in detail: pH was
varied from 3.5–7.0 in water, aqueous HEPES buffer, or
HEPES–micellar Brij; added ligands included N-octyl-d-gluc-
O2N
OPO
O–
NO2
1
(BNPP)
2
2
21 3
amine.
21 at pH 3.5 to a maximum of 2.8 3 1022
s
3
In HEPES– Brij, kobs increased steadily from 9.3 3
k = 2.6 3 10
s , representing a record enhancement of ca.
9
1h
1024
s
21 at pH 6.0,
2
3 10 relative to the uncatalysed reaction at 25 °C.
Similarly, we examined hydrolyses mediated by the actinide
before declining slightly at pH 6.5 or 7.0. Above pH 7,
precipitation of OH-bridged Th oligomers was observed. A
similar pH dependence was seen in water, with k = 5.2 3
4
5
uranyl cation. Uranium offers a +6 oxidation state, but exists in
2+
IV
water as UO
2
, which is less reactive than Ce ; e.g. k = 9.5 3
obs
2
6
21
2+
1023 21 at pH 3.5 and 2.2 3 1022 21 at pH 6.0 (maximum).
s s
+
10
s
2
for hydrolysis of BNPP by excess UO –
The pK
depending on the exact conditions;
a
for water bound to Th4 varies from 2.4 to 5.0,
a recent report gives
N-hexadecyl-N,NA,NA,- trimethylethylenediamine (HTMED, pH
4
7
6
4
.9, 37 °C). Although this is equivalent to a rate enhancement
5
2+
pKa1 = 3.89 and pKa2 = 4.20. These data are consistent with
of at least 8.6 3 10 relative to the uncatalysed reaction, UO
2
IV
our observation of a reactivity maximum around pH 6.
is inferior to Ce by a kinetic factor of ca. 1100 in the cleavage
of BNPP.
4+
The Th -mediated hydrolysis of BNPP occurs with release
of both p-nitrophenyl groups. Kinetic experiments under the
conditions of Table 1 reveal that Th4 hydrolysis of the
presumptive intermediate, p-nitrophenylphosphate, is twice as
The actinide thorium, however, affords a stable +4 oxidation
state in aqueous solution with multiple coordinated water
+
molecules.5 The ‘hard acid’ Th4+ readily coordinates with
5
fast (k = 5.5 3 1022 21) as that of BNPP. The reported rate
s
oxygen ligands (including phosphates), so that we anticipate
6
constants therefore describe the hydrolysis of the first
p-nitrophenyl group of BNPP, which is the rate-limiting step.
Brij micelles enhance the Th4 reactivity: kobs increases
nonoxidative, hydrolytic potency toward phosphodiesters.
Indeed, Th4+ accelerates the hydrolysis of plasmid DNA and
nucleotides at pH 4 or 5, with rate constants approaching those
+
IV 6
steadily at pH 6 in HEPES buffer from 5.5 3 1023
s
21 in the
21 at 2.0 mm Brij.
s
of Ce .
In order to quantitatively evaluate Th4+ relative to UO
2+
absence of Brij, to a maximum of 2.8 3 1022
Not only does micellar Brij potentiate the reactivity of Th
2
and
4
+
various Ln promoters of phosphodiester cleavage, we have now
determined the reactivity of Th4+ toward BNPP and additional
(presumably by binding substrate molecules and cations), but its
oxygen atoms appear to solubilize the Th4 cations or oligomers
+
substrates 2–4. We are pleased to report conditions under which
O
Table 1 Kinetics of Th4+ mediated hydrolysesa
O2N
OPOR
O–
obs/102 s21
4
Substrate
k
2
3
4
R = CH2CH(OH)Me
R = C16H33
R = Et
1
2
3
4
282b
132
11.9
8.54
the reactivity of Th4 far exceeds that of UO
to that of Ce , and surpasses that of other metal cations.
Substrates 1–4 were hydrolysed readily in the presence of
3 4
aqueous Th(NO ) . Optimal conditions (see below) comprised
+
2+, is comparable
2
IV
a
At pH 6.0, 37 °C; other conditions are detailed in the text. Rate constants
are reported as means of three runs with r > 0.997 and reproducibilities
within ±5%. b Both p-nitrophenyl groups were cleaved.
Chem. Commun., 1997
1639

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‡ The minimal rate advantage for Th4+ cleavage of micellized substrate 3,
relative to nonmicellar analogue 4, contrasts with rate enhancements of
above pH 5. Brij-35 has been previously used to stabilize metal
4,8
cation catalysts for phosphodiester hydrolysis.
50–70 previously observed with Eu3 or UO
+
2+
toward aggregated
substrates (refs. 4, 14). The differing behaviour may be due to the Brij
IV
2
In contrast to Ce , which requires both a complexing ligand
IV
and micellar Brij to remain in solution above pH 4–5, Th is
comicelles employed with Th4
+
.
stabilized by Brij alone; added ligands merely sap its reactivity.
2
2
21
4+
For example, kobs = 2.8 3 10
s
(Th –BNPP in aqueous
s
2
3
21
Brij at pH 6) is reduced to 1.4 3 10
upon the addition of
1
(a) R. Breslow and D. L. Huang, Proc. Natl. Acad. Sci. USA, 1991, 88,
1
equiv. of N-octyl-d-glucamine, a ligand useful in the
IV 3
4080; (b) J. R. Morrow, L. A. Buttrey, V. M. Shelton and K. A. Berback,
J. Am. Chem. Soc., 1992, 114, 1903; (c) J. R. Morrow, L. A. Buttrey and
K. A. Berback, Inorg. Chem., 1992, 31, 16; (d) M. Komiyama,
K. Matsumura and Y. Matsumoto, J. Chem. Soc., Chem. Commun.,
stabilization of Ce .
4+
2+
Th is much more reactive than UO
2
toward substrates 1 or
2
. The rate constants of Table 1 can be compared with k(pH
2
6
21
2+
24
4
s
.9) = 9.5 3 10
s
for 1 (UO
2
–HTMED) or 2.2 3 10
1
992, 640; (e) H.-J. Schneider, J. Rammo and R. Hettich, Angew.
2
1
2+
(UO
2
) for 2.† Using BNPP hydrolysis as a reactivity
Chem., Int. Ed. Engl., 1993, 32, 1716; (f) J. Rammo and H.-J. Schneider,
Liebigs Ann., 1996, 1757; (g) B. K. Takasaki and J. Chin, J. Am. Chem.
Soc., 1993, 115, 9337; (h) B. K. Takasaki and J. Chin, J. Am. Chem.
Soc., 1995, 117, 8582; (i) R. Breslow and B. Zhang, J. Am. Chem. Soc.,
1994, 116, 7893.
2 B. K. Takasaki and J. Chin, J. Am. Chem. Soc., 1994, 116, 1121;
M. Komiyama, T. Kodama, N. Takeda, J. Sumaoka, T. Shiiba,
Y. Matsumoto and M. Yashiro, J. Biochem., 1994, 115, 809;
M. Komiyama, T. Shiiba, T. Kodama, N. Takeda, J. Sumaoka and
M. Yashiro, Chem. Lett., 1994, 1025; J. Sumaoka, S. Miyama and
M. Komiyama, J. Chem. Soc., Chem. Commun., 1994, 1755;
M. Komiyama, N. Takeda, Y. Takahashi, H. Uchida, T. Shiiba,
T. Kodama and M. Yashiro, J. Chem. Soc., Perkin Trans. 2, 1995,
269.
4+
IV
measure, Th is comparable to Ce –palmitate in Brij (k = 2.6
2
2
3
3+
24
3
10 , pH 7), but more reactive than Eu (k = 1.7 3 10
1e,f 3+
2
1
s
, pH 7, 50 °C), or various Eu complexes (2.0–6.7 3
4 1f,9
2
21
, pH 7–7.4, 25–50 °C), La3+ (1.4 3 10
27 21 1g
1
0
s
s
)
or
3+
23 21
1g
3+
La –H
2
O
2
(4.8 3 10
s , both at pH 7, 25 °C), 1:1 La –
s , pH 7, 50 °C), or binuclear azamacro-
3+
24 21
10
Fe (2.8 3 10
3+
23 21
3+
cyclic complexes of Eu (1.4 3 10
s
, pH 7, 50 °C) or Pr
2
4
21
11
(8.4 3 10
s , pH 7, 50 °C).
It is clear from these comparisons that Th and CeIV are the
IV
most reactive actinide or lanthanide cations yet reported for the
acceleration of BNPP hydrolysis. A similar conclusion follows
IV
IV
from the reactivity of Th toward substrate 2. Indeed, Th and
CeIV appear to be more reactive toward BNPP or 21c than any
3 K. Bracken, R. A. Moss and K. Ragunathan, unpublished work.
4
5
R. A. Moss, K. Bracken and J. Zhang, Chem. Commun., 1997, 563.
S. Cotton, Lanthanides and Actinides, Oxford University press, New
York, 1991, pp. 115–121.
other metal cations thus far described, including (complexes of)
cobalt12 and copper.
13
Toward long-chain phosphodiester substrate 3, comicellized
with 2 mm Brij-35 (critical micelle concentration, 0.06–0.09
6
T. Ihara, H. Shimura, K. Ohmori, H. Tsuji, J. Takeuchi and M. Takagi,
Chem. Lett., 1996, 687.
8
a
4+
23 21
mm ), excess Th affords kobs = 1.2 3 10
s
. Compar-
7 J. Burgess, Metal Ions in Solution, Halsted Press, New York, 1978,
pp. 27–28.
8 (a) S. H. Gellman, R. Petter and R. Breslow, J. Am. Chem. Soc., 1986,
108, 2388; (b) J. G. J. Weijnen and J. F. J. Engbersen, Recl. Trav. Chim.
Pays-Bas, 1993, 112, 351.
2+
isons are lacking except for UO
2
–HTMED where kobs = 1.1
2
4
21 4
4+
3
10
s
. Perhaps a fairer comparison of Th –Brij vs.
2
+
4+
UO
2
–HTMED is with the short-chain substrate 4, where Th
2+
(
(
Table 1) manifests a 500-fold rate advantage over UO
2
2
6
21
9 J. R. Morrow, K. Aures and D. Epstein, J. Chem. Soc., Chem. Commun.,
k = 1.7 3 10
s ).‡
1
995, 2431.
IV
In conclusion, Th cations are an extraordinarily reactive,
1
1
0 N. Takeda, M. Irisawa and M. Komiyama, J. Chem. Soc., Chem.
Commun., 1994, 2773.
1 K. G. Ragunathan and H.-J. Schneider, Angew. Chem., Int. Ed. Engl.,
1996, 35, 1219.
readily employed promoter of the hydrolysis of a variety of
model phosphodiester substrates. We are continuing to probe
their application to challenging problems in this area.
We are grateful to the U.S. Army Research Office for
financial support.
12 J. Chin, Acc. Chem. Res., 1991, 24, 145 and references cited therein.
13 E. K o¨ vari and R. Kr a¨ mer, J. Am. Chem. Soc., 1996, 118, 12 704;
K. A. Deal and J. N. Burstyn, Inorg. Chem., 1996, 35, 2792.
1
4 R. A. Moss, B. D. Park, P. Scrimin and G. Ghirlanda, J. Chem. Soc.,
Chem. Commun., 1995, 1627.
Footnotes and References
†
No attempt is made in the kinetic comparisons to correct for differing
conditions or cation–substrate ratios. Rather, the maximum rate constants
available in the literature are compared to those of Th4+
.
Received in Corvallis, OR, USA, 22nd May 1997; 7/03568C
1640
Chem. Commun., 1997