Cleavage and isomerization of UpU promoted by dinuclear metal ion complexes

Article abstract of DOI:10.1021/ja711347w

The catalysis of phosphoryl transfer by metal ions has been intensively studied in both biological and artificial systems, but the status of the transient pentacoordinate phosphoryl species (as transition state or intermediate) is the subject of considerable debate. We report that dinuclear metal ion complexes that incorporate second sphere hydrogen bond donors not only promote the cleavage of RNA fragments just as efficiently as the activated analogue HPNPP but also provide the first examples of metal ion catalyzed phosphate diester isomerization close to neutral pH. This observation implies that the reaction catalyzed by these complexes involves the formation of a phosphorane intermediate that is sufficiently long-lived to pseudorotate. Copyright

Full text of DOI:10.1021/ja711347w

Published on Web 03/07/2008
Cleavage and Isomerization of UpU Promoted by Dinuclear Metal Ion
Complexes
Heidi Linjalahti,† Guoqiang Feng,‡ Juan C. Mareque-Rivas,§ Satu Mikkola,*,† and
Nicholas H. Williams*,‡
Department of Chemistry, FI-20014 UniVersity of Turku, Turku, Finland, Centre for Chemical Biology, Department
of Chemistry, UniVersity of Sheffield, Sheffield, U.K. S3 7HF, and School of Chemistry, UniVersity of Edinburgh,
Edinburgh, U.K. EH9 3JJ
Received December 21, 2007; E-mail: satu.mikkola@utu.fi; N.H.Williams@Sheffield.ac.uk
Chart 1. Metal Ion Complex and Substrates
The catalysis of phosphoryl transfer by metal ions has been
intensively studied in biological, artificial, and theoretical systems,
yet considerable debate still surrounds the status of the transient
pentacoordinate phosphoryl species.1 For potential di- or trianionic
anionic intermediates, a central question is whether the reaction
passes through a transition state or a formal intermediate and,
perhaps more importantly, whether such intermediates are stable
enough to allow the reaction pathways to follow an alternative
course to direct in-line displacement at phosphorus. We report that
dinuclear metal ion complexes 1.M not only promote the cleavage
of RNA fragments just as efficiently as the activated analogue
2-hydroxypropyl p-nitrophenyl phosphate (HPNPP) but also provide
the first examples of metal ion catalyzed phosphate diester
isomerization close to neutral pH (Chart 1). This observation implies
that these complexes stabilize the formation of a phosphorane
intermediate sufficiently to allow it to pseudorotate.
mixture and observing that it efficiently inhibits catalysis by 1.Zn.
This much tighter binding compared to similar mononuclear
complexes suggests that the phosphates bridge the two metal ion
centers, as we have previously reported.3b
When UpU (0.05 mM) is incubated with 1 mM 1.Zn (1.M with
M ) Zn; formed in situ from 1 mM ligand and 2 mM Zn(II) ions)
at pH 6.5 at 25 °C, it is efficiently cleaved with a half-life of ∼7
h. This corresponds to a rate acceleration of ∼106-fold,2 making
this complex the most efficient Zn(II) based catalyst for promoting
RNA cleavage under mild aqueous conditions reported to date.3
The reaction proceeds Via the usual intramolecular transesterification
where the 2′ oxyanion on the ribose ring attacks the phosphate and
expels the 5′OH of the nucleoside leaving group. The 2′,3′cUMP
that initially forms is rapidly hydrolyzed to 2′UMP and 3′UMP
monophosphates; we confirmed this by observing that, under the
same conditions, an authentic sample of 2′3′cUMP was consumed
within a few minutes. We ruled out alternative cleavage pathways
by testing a 13-mer oligo-uridine substrate in which all but one
site contained 2′OMe groups.4 Two products were observed by
capillary zone electrophoresis analysis, consistent with cleavage at
the normal ribose linkage to give a leaving group 6-mer and a 7-mer
terminated in a phosphorylated ribose. Furthermore, only the 7-mer
product reacted when a sample was treated with alkaline phos-
phatase showing that it contained a terminal phosphomonoester
group.
Previous studies have shown that the catalytic activity of
transition metal ion complexes generally depends on the identity
of the metal ion, with complexes of Cu(II) usually being the most
active catalysts,5,6 so we screened several late transition metal ions
for activity. However, the rate constants collected in Table 1 show
that, of the complexes studied in this work, 1.Co and 1.Zn are the
most efficient catalysts. Zn(II) complexes have frequently been
studied as potential catalysts for the cleavage of RNA, but few
reports on the catalysis by Co(II) exist despite the fact that Co(II)
can substitute for Zn(II) in many metalloenzyme catalyzed reactions
in Vitro.7 As simple aquo ions, Zn(II) and Co(II) only modestly
promote the cleavage of UpU, with the effect of Co(II) being only
one-tenth of that of Zn(II).8 Bound in complex 1.M, this reactivity
is greatly enhanced and the difference is reversed to give a 4-fold
advantage in favor of Co(II).
Remarkably, in addition to efficiently promoting the cleavage
of the 5′ phosphodiester bond in 3′5′UpU, we observed that
2′,5′UpU appeared in the chromatograms, showing that 1.M also
catalyzes isomerization of the diester. This reaction is slower than
cleavage, and the signal area of the 2′,5′ isomer remained low
throughout the whole reaction (2′5′UpU cleavage is also promoted
by the complex), so reliable rate constants for isomerization of UpU
could not be calculated directly from these data. The observation
was therefore verified using a nucleoside phosphonate (2) as a
substrate. This substrate cannot be cleaved by intramolecular
transesterification, but previous studies have shown that it isomer-
izes in the same way as dinucleoside monophosphates, that is, with
pH-independent and acid-catalyzed components but without base
catalysis.9,10 The rate constants for isomerization of 2 are also shown
in Table 1 and are consistent with the extent of 2′5′UpU that appears
in the original experiments. The relative rate enhancement is modest
in comparison to that observed for the cleavage reaction but
Varying the concentration of 1.Zn (1-5 mM) has no effect on
the rate of the cleavage of 3′,5′UpU at this pH. This suggests that
the complex binds the monoanionic phosphate very strongly, with
a binding constant > 104 M-1 so that saturation is achieved at the
millimolar concentrations used in our experiments. The final
monophosphate products (2′UMP and 3′UMP), which under the
experimental conditions are dianionic, should bind even more
strongly. This was confirmed by adding 3′UMP to the reaction
† FI-20014 University of Turku.
‡ University of Sheffield.
§ University of Edinburgh.
9
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J. AM. CHEM. SOC. 2008, 130, 4232-4233
10.1021/ja711347w CCC: $40.75 © 2008 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Rate Constants for Reactions Promoted by 1.Ma
approximate upper limit of a rate enhancement that can be achieved
by neutralization of the charge on the phosphate, without additional
catalysis of the nucleophilic attack by the alkoxy anion or of the
departure of the leaving group, can be estimated from the reactions
of neutral phosphotriesters. Previous results obtained with nucleo-
side phosphotriesters have shown that the spontaneous cleavage of
nucleoside phosphotriesters under neutral conditions is about 105
fold faster than that of corresponding phosphodiesters.14 Considering
that 1.M binds the phosphate through two coordinate bonds and
four hydrogen bonds, the rate enhancement observed could well
be attributed to strong interactions between the catalyst and the
phosphate group. However, data obtained with nucleoside phos-
photriesters also show that under neutral conditions nucleophilic
attack on a neutral phosphate results in extremely fast phosphate
migration relative to cleavage, via a monoanionic phosphorane. In
the present case the catalysis of the cleavage is efficient, whereas
the isomerization is only modestly enhanced. This shows that, in
addition to electrophilic catalysis, the complexes of ligand 1a direct
the reaction of the phosphorane toward the cleavage products, but
it is not possible to say whether this results from catalysis of the
cleavage reaction or from the inhibition of pseudorotation. Enzymes
or ribozymes that catalyze RNA cleavage do not appear to catalyze
isomerization as well, but presumably in these cases, the confines
of the active site even more firmly direct the reaction toward
cleavage if similar phosphoranes are involved. These data do clearly
demonstrate that dinuclear metal ion complexes with second sphere
hydrogen bond donors stabilize a phosphorane intermediate enough
to allow pseudorotation.
metal ion M
Zn(II)
Co(II)
Cu(II)
Ni(II)
kobs/10-6 s-1
(3′,5′ UpU
cleavage)
26 ( 3
95 ( 2
3.9 ( 0.1
0.70 ( 0.05
ki/10-6 s-1
1.25 ( 0.02b 0.92 ( 0.02b 0.35 ( 0.01b ND
(isomerization 0.87 ( 0.03c 1.05 ( 0.03c 0.31 ( 0.04c
of 2)
a
1 mM ligand, 2 mM metal ion, 50 mM MOBS buffer, pH 6.5, 25 °C.
b
c
Interconversion of 3′ isomer to 2′ isomer. Interconversion of 2′ isomer
to 3′ isomer.
Scheme 1. Possible Mechanism for Cleavage and Isomerization
of UpU Promoted by 1.Zna
Acknowledgment. We thank the BBSRC for financial support
and Dr I. Rosenberg for generously providing compound 2.
Supporting Information Available: Experimental methods for
kinetic analysis and representative chromatograms showing reaction
progress for isomerization of 2. This material is available free of charge
via the Internet at http://pubs.acs.org.
a 1.Zn is only represented by the Zn ions for clarity.
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