multinuclear lanthanide–hydroxide complexes form spontaneously in
water,7a and dimeric lanthanide complexes cleave phosphodiester bonds
up to 1,000-times faster than analogous monomers.5a,18 In contrast to the
trend observed for the hydrolysis of P8, aqueous solutions of Ce(IV)
became less active for RNA hydrolysis over time (Table 2). In contrast to
Ce(IV), the activities of Ce(III), La(III), and Lu(III) did not appreciably
change over 100 days.
selectivity is consistent with the well-established relationship
between reaction rate and leaving group pKa.13
The rates of Ce(IV)-mediated dephosphorylation of P8–P10
were up to 10,000-fold faster than those reported for metal-
mediated RNA and DNA cleavage.5 To provide a direct
comparison of these differences under our conditions, the cleavage
rates of an RNA (CpG) and DNA (dCpdG) dinucleotide were
measured in the presence of 1 mM Ce(IV), Ce(III), La(III), or Lu(III)
(Table 2). The rates for Ce(IV)-mediated phosphomonoester
hydrolysis of P9 and P10 were more than 1,000-times faster than
that of dCpdG phosphodiester hydrolysis (Table 2). These
differences are consistent with a report that 59-deoxyadenosine
monophosphate (pdA) was hydrolyzed by Ce(IV) up to 500-times
faster than the adenosine dinucleotide dApdA.14 Interestingly,
Ce(IV)-mediated dephosphorylation of P8 is over one million times
faster than dCdpG hydrolysis and up to 27,000 times faster than
CpG hydrolysis (Table 2). Along with the noteable progress made
towards the site-selective cleavage of nucleic acids using mixtures
of hydrolytic metal ions and various chelating/targeting agents,15
these results suggest it may be possible to effect site-specific
dephosphorylation of phosphoproteins using lanthanide ions in
conjunction with high affinity ligands for protein surfaces.
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In summary, we have developed a solid-phase-based assay for
phosphoester cleavage suitable for screening large numbers of
different lanthanide ions, peptide sequences, and chelating agents.
Using this assay, we found that certain lanthanide ion–BTP
complexes dephosphorylate phosphopeptides under near-
physiological conditions of pH, temperature, and salt. Control
experiments conducted in solution confirmed the trends from
the solid-phase assay and, unexpectedly, revealed that trivalent
lanthanide ions can mediate b-elimination of phosphate from
phosphomonoesters. In agreement with previous studies using
other types of substrates,5b,16 Ce(IV) was the most reactive ion
tested, in all cases generating a single product consistent with
phosphomonoester hydrolysis. For Ce(III), La(III), and Lu(III),
b-elimination of the phosphate group from P3, P9, and P10
was faster than hydrolysis, generating dehydroalanine- and
methyldehydroalanine-containing peptides. Dephosphorylation of
phosphotyrosine-containing peptides by Ce(IV) was particularly
rapid with apparent second-order rate constants approaching
10 M21 21. This value is 106-fold lower than typical kcat/Km
s
values for phosphotyrosine protein phosphatases, but is 1013-fold
higher than the estimated hydrolysis rate of phenylphosphate at
high pH.17
{
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This work was supported by the NIH (GM65453 and
GM59843), the National Foundation for Cancer Research, and
in part by a grant to Yale University, in support of A. S., from the
Howard Hughes Medical Institute. N. W. L. was supported by
NIH-NRSA GM074401.
Notes and references
{ A 40-fold increase in the apparent rate of P8 dephosphorylation (see
Table 2 and ESI) was observed for Ce(IV) stock solutions of increasing age
(prepared at 100 mM in deionized water and stored in borosilicate glass at
RT). This observation may indicate that in the absence of BTP, a slow-
forming multimeric complex is the most reactive Ce(IV) species. Indeed,
17 R. Kluger and L. L. Cameron, J. Am. Chem. Soc., 2002, 124, 3303.
18 (a) P. Hurst, B. K. Takasaki and J. Chin, J. Am. Chem. Soc., 1996, 118,
9982; (b) M. Yashiro, A. Ishikubo and M. Komiyama, J. Biochem.
(Tokyo), 1996, 120, 1067.
5428 | Chem. Commun., 2005, 5426–5428
This journal is ß The Royal Society of Chemistry 2005