Remarkably rapid cleavage of a model phosphodiester by complexed ceric ions in aqueous micellar solutions

Full text of DOI:10.1021/ja971448b

J. Am. Chem. Soc. 1997, 119, 9323-9324
9323
mitigate the metal’s activity. Komiyama has used a weak
complex of Ce(IV) and γ-cyclodextrin to obtain homogeneous,
neutral, aqueous solutions for the cleavage of nucleoside 3′,5′-
cyclic monophosphates13a and peptides.13d Now, we describe
three new ligands which provide stabilized, highly reactive Ce-
(IV) in nonionic Brij-35 micelles.14 These systems mediate the
cleavage of BNPP over a broad pH range (4.0-12.0) at rates
clearly surpassing those previously reported for other Ln cations.
The ligands we employed included N-octanoyl-N-methyl-D-
glucamine (2), palmitic acid/palmitate (3), and 4-(1-hexadec-
ynyl)-2,6-pyridinedicarboxylate (4). Ligands 2 and 3 were
Remarkably Rapid Cleavage of a Model
Phosphodiester by Complexed Ceric Ions in
Aqueous Micellar Solutions
Kathryn Bracken, Robert A. Moss,* and
Kaliappa G. Ragunathan
Department of Chemistry, Rutgers
The State UniVersity of New Jersey
New Brunswick, New Jersey 08903
ReceiVed May 5, 1997
The 1990’s have witnessed intense interest in the use of
lanthanide (Ln) cations to facilitate the hydrolysis of phos-
phodiesters. “Early” explorations of model DNA and RNA
hydrolyses, accelerated by various lanthanides,1 were followed
by comparative kinetic studies focused on bis(p-nitrophenyl)-
phosphate (1, BNPP), which has become an informal “standard”
phosphodiester substrate.2,3 Using BNPP, one can readily
commercially available, whereas 4 was readily prepared by Cu/
Pd(PPh3)2 catalyzed coupling of hexadecyne with diethyl
4-bromo-2,6-pyridinedicarboxylate15 (75 °C, 3 h, 72%), followed
by saponification (aq KOH, MeOH, 40 °C, 36 h, 84%). Ligand
4 was characterized by NMR and elemental analysis.
compare the accelerations provided by simple complexes of Ln
cations,2 La3+/H2O2,3,4 Ln/non-Ln clusters,5 hydroxyl-function-
alized azamacrocyclic Ln complexes,6 and paired Ln cations in
macrocylic complexes.7 Other recent applications of Ln ion
mediation have been reported for hydrolyses of DNA,8 RNA,9
hydroxyquinoline phosphodiesters or phosphonates,10 and lipo-
somal phosphodiesters.11
Mechanistically, the Ln cations serve a dual purpose: as
Lewis acids to bind and charge-neutralize the phosphodiester’s
P-O-, while simultaneously furnishing a metal-bound OH
nucleophile to attack the substrate’s phosphonyl group. Increas-
ing the metal cation’s charge density should enhance both its
Lewis acid proclivity and the acidity of its waters of hydration,
so that particular attention centers on Ce(IV), the only lanthanide
with a readily available +4 oxidation state. Applications of
Ce(IV) to DNA cleavage have been emphasized.12,13
Hydrolytic media were prepared in 2 × 10-3 M (for 2) or 5
× 10-3 M sonicated (for 3 or 4) aqueous Brij-35 solutions that
also contained 1.0 mM ligand and 0.01 M KCl. The solutions
were buffered with 0.01 M MES, HEPES, CHES, or CAPS
according to pH requirements, but solutions at pH 4, 5, or 12
were unbuffered. To initiate hydrolyses, 1.0 or 2.0 mM Ce-
(NH4)2(NO3)6 was added, the pH was adjusted with KOH, and
then 5 × 10-5 M BNPP was added. Importantly, stock solutions
of Ce(IV) were prepared at pH e1.5 to avoid hydroxide gel
formation and an accompanying reactivity decrease. Delivery
of the Ce(IV) into the ligand/Brij medium appears to stabilize
the Ce(IV) against hydroxide at higher pH.
Control experiments demonstrate that both ligand and Brij
are required to provide homogeneous Ce(IV) solutions above
pH 5.16 The critical micelle concentration of Brij-35 is 0.06-
0.09 mM,14 so that our reaction solutions are micellar. For
example, dynamic light scattering analysis of 2 + Brij or 2 +
Brij + Ce(IV) solutions reveal aggregates of ∼15-nm diameter,
consistent with micellar aggregation.
Unfortunately, above pH 4, the formation and precipitation
of Ce(IV)-hydroxide gels hinders kinetic studies of Ce(IV)-
mediated phosphodiester hydrolyses. Complexation of the Ce-
(IV) could solve this problem, but strongly bound ligands would
Rate constants for the hydrolyses of BNPP mediated by Ce-
(IV) and ligands 2-4 at various pH’s appear in Table 1. At
pH >6, kinetics were monitored at 400 nm, following released
p-nitrophenylate ions. (HPLC demonstrated that p-nitrophenol
was the sole, final organic product.) Below pH 6, p-nitrophenol
formation (317 nm) and BNPP disappearance (290 nm) were
(1) Breslow, R.; Huang, D. L. Proc. Natl. Acad. Sci. U.S.A. 1991, 88,
4080. Morrow, J. R.; Buttrey, L. A.; Shelton, V. M.; Berback, K. A. J. Am.
Chem. Soc. 1992, 114, 1903. Morrow, J. R.; Buttrey, L. A.; Berback, K.
A. Inorg. Chem. 1992, 31, 16. Komiyama, M.; Matsumura, K.; Matsumoto,
Y. Chem. Commun. 1992, 640.
(2) (a) Schneider, H-J.; Rammo, J.; Hettich, R. Angew. Chem., Int. Ed.
Engl. 1993, 32, 1716. (b) Rammo, J.; Schneider, H-J. Liebigs Ann. 1996,
1757.
(3) (a) Takasaki, B. K.; Chin, J. J. Am. Chem. Soc. 1993, 115, 9337. (b)
Takasaki, B. K.; Chin, J. J. Am. Chem. Soc. 1995, 117, 8582.
(4) Breslow, R.; Zhang, B. J. Am. Chem. Soc. 1994, 116, 7893.
(5) Takeda, N.; Irisawa, M.; Komiyama, M. Chem. Commun. 1994, 2773.
(6) Morrow, J. R.; Aures, K.; Epstein, D. Chem. Commun. 1995, 2431.
(7) Ragunathan, K. G.; Schneider, H-J. Angew. Chem., Int. Ed. Engl.
1996, 35, 1219.
(8) Hashimoto, S.; Nakamura, Y. Chem. Commun. 1995, 1413. Rammo,
J.; Hettich, R.; Roigk, A.; Schneider, H-J. Chem. Commun. 1996, 105.
Hashimoto, S.; Nakamura, Y. J. Chem. Soc., Perkin Trans. 1 1996, 2623.
(9) Hurst, P.; Takasaki, B. K.; Chin, J. J. Am. Chem. Soc. 1996, 118,
9982.
(10) Tsubouchi, A.; Bruice, T. C. J. Am. Chem. Soc. 1994, 116, 7893.
Tsubouchi, A.; Bruice, T. C. J. Am. Chem. Soc. 1995, 117, 7399.
(11) Moss, R. A.; Park, B. D.; Scrimin, P.; Ghirlanda, G. Chem. Commun.
1995, 1627.
(12) (a) Takasaki, B. K.; Chin, J. J. Am. Chem. Soc. 1994, 116, 1121.
(b) Komiyama, M.; Kodama, T.; Takeda, N.; Sumaoka, J.; Shiiba, T.;
Matsumoto, Y.; Yashiro, M. J. Biochem. 1994, 115, 809. (c) Komiyama,
M.; Shiiba, T.; Kodama, T.; Takeda, N.; Sumaoka, J.; Yashiro, M. Chem.
Lett. 1994, 1025.
(13) (a) Sumaoka, J.; Miyama, S.; Komiyama, M. Chem. Commun. 1994,
1755. (b) Komiyama, M.; Takeda, N.; Takahashi, Y.; Uchida, H.; Shiiba,
T.; Kodama, T.; Yashiro, M. J. Chem. Soc., Perkin Trans. 2 1995, 269.
(c)Takeda, N.; Imai, T.; Irisawa, M.; Sumaoka, J.; Yashiro, M.; Shigekawa,
H.; Komiyama, M. Chem. Lett. 1996, 599. (d) Yashiro, M.; Takarada, T.;
Miyama, S.; Komiyama, M. Chem. Commun. 1994, 1757. (e) Komiyama,
M.; Takeda, N.; Shiiba, T.; Takehashi, Y.; Matsumoto, Y.; Yashiro, M.
Nucleosides & Nucleotides 1994, 13, 1297.
(14) Previous use of Brij micelles for the solubilization of complexed
metal ions for phosphate ester hydrolysis includes: Gellman, S. H.; Petter,
R.; Breslow, R. J. Am. Chem. Soc. 1986, 108, 2388. Weijnen, J. G. J.;
Engbersen, J. F. J. Recl. TraV. Chim. 1993, 112, 351.
(15) Takalo, H.; Kankare, J. Acta Chem. Scand. 1987, B41, 219.
(16) Brij oxygens should assist the ligands in binding Ce(IV); crown
and cryptand ether oxygens bind Ln cations weakly in water; for example,
K ) 4 for the binding of Eu3+ by sorbitol.2b Carboxylate groups (as in 3 or
4) are more effective: Arnaud-Neu, F. Chem. Soc. ReV. 1994, 235. In
addition, Ce-ligand coordination energies might be altered in the Brij
micelles, and the reactivity of water could be enhanced. The micellar
pseudophase will also concentrate the substrate and Ce complex into a small
reaction volume, thus further enhancing the rate.
S0002-7863(97)01448-0 CCC: $14.00 © 1997 American Chemical Society

9324 J. Am. Chem. Soc., Vol. 119, No. 39, 1997
Communications to the Editor
Table 1. Rate Constants for Ce(IV)-Mediated Hydrolyses of
1:1 Ce(IV)/4 is 32 times more reactive than Ce(III) under
comparable conditions at pH 11.
BNPPa,b
4d
The pH dependences of the several Ce(IV)-ligand combina-
tions vary with both ligand identity and the Ce/ligand ratio. With
glucamine 2, ceric reactivity decreases with increasing pH,
presumably due to (microscopic) hydroxide gel formation.
However, 2:1 Ce(IV)/3 exhibits a reactivity maximum at pH 7,
whereas the reactivities of 1:1 Ce(IV)/3 and Ce(IV)/4 continue
to increase up to pH 11-12. The pH titration of Ce(IV) in the
presence of 1 equiv of 3 requires almost 3 equiv of base between
pH 8 and 11, suggesting the deprotonation of several water
molecules coordinated to the same metal ion. Similar phenom-
ena presumably attend Ce(IV)/4.
The high reactivity of 2:1 Ce(IV) with ligands 3 or 4 (but
not 2) may reflect electrophilic/nucleophilic cooperativity
between the 2 Ce(IV) centers. Related synergism is known with
other lanthanides,5,7 as well as Ce(IV),13c and, of course, with
phosphatase enzymes.3,10,20 In the present case, the 2:1 systems
might involve hyroxide bridged Ce(IV) centers,21 additionally
stabilized by Brij and carboxylate oxygens.16
2
3
no
pH
ligandc
1:1e
2:1f
1:1e
2.5
2:1f
44
1:1e
2:1f
4.0g
5.0g
6.0
7.0
8.0
173
107
h
h
h
h
h
162
102
43.6
18.1
11.0
10.0
79.8
39.7
21.9
11.2
7.8
3.9
2.4
180
260
190
97
6.0
7.0
9.5
15.5
22.0
7.5
9.0
13.1
22.0
64.1
98.0
9.0
51
160
200
10.0
11.0
12.0g
74
1.9
a Rate constants are multiplied by 104, units are s-1 b Conditions:
.
[Brij] ) 2 × 10-3 M (for 2) or 5 × 10-3 M (for 3, 4); [Ce(IV)] ) 1
or 2 × 10-3 M; [ligand] ) 1 × 10-3 M; [BNPP] ) 5 × 10-5 M; [buffer]
) 0.01 M (see text for buffers); 0.01 M KCl, 37 °C. c Brij and other
components are present. d Rate constant for BNPP to nitrophenylphos-
phate. e Ce(IV):ligand ) 1:1, [Ce(IV)] ) 1 mM. f Ce(IV):ligand ) 2:1,
[Ce(IV)] ) 2 mM. g No buffer. h Ce(IV) precipitates above pH 5 in
the absence of ligand.
Both 1:1 Ce(IV)/2 and Ce(IV)/3 mediate the hydrolysis of
BNPP with the release of 2 equiv of p-nitrophenol; the first
step is slower than the cleavage of mononitrophenylphosphate
(MNPP), and is rate-determining. In the former case, 2-fold
excess BNPP can be completely cleaved at pH 7.5, 45 °C, with
k ) 9.6 × 10-4 s-1 and turnover. In the latter case (pH 11, 37
°C), 2 p-nitrophenols are liberated from a single substrate
followed. Pseudo-first-order rate constants (>8 half-lives17
)
were obtained as means of triplicate runs with r > 0.997 and
reproducibilities within (10%.
Table 1 indicates that, at selected pH’s, 1-2 mM Ce(IV)
provides rate constants exceeding 0.01 s-1 for the cleavage of
BNPP in Brij micelles. At pH 4, the cation itself affords k )
1.7 × 10-2 s-1, and the Ce(IV)-glucamine (2) complex is
comparable. The palmitate (3) preparation provides k ) 2.6 ×
10-2 s-1 for 2:1 Ce(IV)/ligand at pH 7, whereas k ) 2.0 ×
10-2 s-1 for 2:1 Ce(IV)/4 at pH 11.18
molecule with k ) 2.2 × 10-3 s-1 22
,
but binding of inorganic
phosphate16 inhibits subsequent reactions (as demonstrated by
controls). Surprisingly, 1:1 Ce(IV)/4 releases only 1 equiv of
p-nitrophenol from BNPP at pH 11 (k ) 6.4 × 10-3 s-1);
cleavage of the MNPP is 105 times slower. These differences
in selectivity and reactivity between Ce(IV) complexed with
ligands 3 or 4 illustrate the sensitivity of the hydrolytic processes
to the identity and disposition of ligand donor atoms around
the Ce(IV) catalytic center(s) within the Brij micelles.
In conclusion, dilute aqueous solutions of Ce(IV), solubilized
with simple ligands in Brij micelles, provide extraordinary
accelerations in the hyrolysis of BNPP, the most commonly
employed model phosphodiester for DNA. The observed kinetic
behavior is very sensitive to ligand structure and pH, so that
further development and optimization of these systems should
be possible.
A rate constant of 2.6 × 10-2 s-1 represents an enhancement
of about 2.4 billion in BNPP cleavage, relative to k0 ∼ 1.1 ×
10-11 at 25 °C.3b This appears to be the largest metal ion
induced acceleration reported for BNPP. Ce(IV), either alone
in Brij at pH 4, or at optimal pH in the presence of ligands
2-4, is g50 times more reactive toward BNPP than any of the
other lanthanides, including Eu3+ 2b,3b,6 La3+ 3b,5 or Pr3+ 7
, . It is
,
also somewhat more reactive than the La3+/H2O2 systems
studied by Chin3b and Breslow.4
The enormous accelerations of BNPP hydrolyses fostered by
Ce(IV) are surely related to the cation’s high charge/size ratio.
The +4 oxidation state makes Ce(IV) a “hard” Lewis acid that
efficiently interacts with the substrate’s P-O- and acidifies
coordinated water molecules so that they are available as OH
nucleophiles even under acidic conditions.19 As anticipated,12b
Ce(IV) is more reactive toward BNPP than Ce(III); for example,
Acknowledgment. We are grateful to the U.S. Army Research
Office for financial support.
JA971448B
(17) Infinity titers reflected stoichiometric cleavage of both p-nitrophenol
moieties with Ce(IV) and ligands 2 and 3, but of only a single p-nitrophenol
with ligand 4; see below.
(18) Cleavage is extraordinarily slow at pH 11 in the absence of Ce(IV).
(19) The pKa of CeIV-OH2 is 0.7, whereas that of Ce(III) is 9.0: Burgess,
J. Metal Ions in Solution; Halstead Press: New York, 1978; p 267.
(20) For leading references, see: Strater, N.; Lipscomb, W. N.; Klabunde,
T.; Krebs, B. Angew. Chem., Int. Ed. Engl. 1996, 35, 2024. Wilcox, D.
Chem. ReV. 1996, 96, 2435.
(21) Samuni, A.; Czapski, G. J. Chem. Soc., Dalton Trans. 1973, 487.
(22) Controls show that 1:1 CeIV/3 cleaves mononitrophenylphosphate
1.4 times faster than BNPP at pH 11 in Brij.