Lithium-catalyzed hydroxide attack at the carbon atom of methyl phosphate

Article abstract of DOI:10.1021/ja0473586

The spontaneous hydrolysis of phosphate ester monoanions is relatively easy, but the reaction of water with simple phosphate ester dianion appears to be the slowest biomimetic reaction whose spontaneous rate has been measured in water, with an estimated half time of _～10¹² years at room temperature in the absence of a catalyst. Here, we report an alternative mode of cleavage of methyl phosphate that involves hydroxide attack at the carbon atom of methyl phosphate and proceeds at a rate proportional to the square of the concentration of lithium hydroxide. Copyright

Full text of DOI:10.1021/ja0473586

Published on Web 06/25/2004
Lithium-Catalyzed Hydroxide Attack at the Carbon Atom of Methyl Phosphate
Richard Wolfenden* and Frances Zhao
Department of Biochemistry & Biophysics, UniVersity of North Carolina, Chapel Hill, North Carolina 27599
Received May 5, 2004; E-mail: water@med.unc.edu
The spontaneous hydrolysis of phosphate ester monoanions is
relatively easy,1 but the reaction of water with simple phosphate
ester dianion appears to be the slowest biomimetic reaction whose
spontaneous rate has been measured in water,2 with an estimated
half-time of ∼1012 years at room temperature in the absence of a
catalyst.3 So unreactive is the methyl phosphate dianion that the
monoanion remains a major target of water attack even in 1 M
KOH, where it constitutes approximately one part in 108 of the
total methyl phosphate that is present. Because water attack on
phosphate ester dianions is so sluggish, enzymes that catalyze water
attack at the phosphorus atom of dianionic forms of their substrates
(fructose 1,6-bisphosphatase, protein phosphatase-1, and inositol
1-phosphatase, for example) exceed other known enzymes in the
rate enhancements that they produce. Here, we report an alternative
mode of cleavage of methyl phosphate that involves hydroxide
attack at the carbon atom of methyl phosphate and proceeds at a
rate proportional to the square of the concentration of lithium
hydroxide.
Figure 1. Apparent first-order rate constants (s-1) for hydrolysis of methyl
phosphate at 137 °C, plotted as a function of increasing concentrations of
LiOH (2), NaOH (9), KOH (0), or RbOH (O) up to their limits of
solubility.
Using PTFE-lined reaction vessels to examine the hydrolysis of
methyl phosphate in strongly alkaline solutions at elevated tem-
peratures as described earlier,3 we observed no significant variation
in rate as the concentrations of KOH or RbOH were raised to their
solubility limits at room temperature. Pronounced increases in rate
were observed in the presence of NaOH or LiOH (Figure 1).
The activity of water is much reduced at high concentrations of
NaOH,4 and the hydrolysis of some phosphate ester dianions has
been shown to proceed much more rapidly in aqueous solutions
that contain aprotic solvents.5,6 Thus, the enhanced rates observed
in concentrated sodium hydroxide might be considered to arise, at
least in part, as an indirect effect of changes in the activity of solvent
water. However, water activity has been shown to be very much
less depressed in LiOH than in equivalent molalities of NaOH at
elevated temperatures.7 We confirmed that result by direct mano-
metric measurements of the vapor pressure of water, observing a
water activity coefficient of 0.87 over a saturated solution of LiOH
at 25 °C. Thus, a reduction in water activity seemed unlikely to
explain the rate-enhancing effect of LiOH.
The hydrolysis of methyl phosphate was found to proceed at a
rate that was roughly proportional to the square of the concentration
of LiOH (Figure 2). To test the possibility that two lithium ions
might be involved in this reaction, we examined the effects of
varying the component ions of lithium hydroxide independently
by varying the concentrations of LiCl and KOH, neither of which
catalyzes methyl phosphate hydrolysis by itself. The rate of hydro-
lysis was found to vary with the first power of the concentration
of lithium at constant hydroxide ion concentration and, with the
first power of the concentration of hydroxide ion, at constant con-
centrations of lithium or sodium (data not shown). Thus, hydrolysis
in concentrated LiOH appears to proceed by hydroxide attack on
the monolithium complex of the methyl phosphate dianion. The
more rapid hydrolysis of the lithium than the sodium salt (Figure
1) may arise from a difference between the dissociation constants
Figure 2. Apparent first-order rate constants (s-1) for hydrolysis of methyl
phosphate, at 137 °C, plotted as a logarithmic function of increasing
concentrations of LiOH. The slope of the regression line is 2.1.
of the complexes formed by these cations with the methyl phosphate
dianion in water, from a difference between the inherent rates of
reaction of their complexes with the hydroxide ion, or from both.
Plotted as a function of reciprocal temperature (Kelvin), the
logarithm of the apparent rate constant for hydrolysis in the presence
of 4.4 M LiOH yielded a linear Arrhenius plot (Figure 3) that could
be extrapolated to yield an apparent first-order rate constant (3 ×
10-9 s-1) at 25 °C. Adopting the rate expression in eq 1, that
apparent first-order rate constant is equivalent to an actual third-
order rate constant (k3) of 1.5 × 10-10 M-2 s-1, at 25 °C. The
catalytic effect of lithium hydroxide shows no evidence of saturation
even at its solubility limit (Figures 1 and 2), and thus the reactivity
of the lithium complex can be estimated only as a lower limit. Even
at that lower limit, at which the presumed complex is incompletely
formed, the apparent first-order rate for hydroxide attack at the
carbon atom of lithiated methyl phosphate (Scheme 1c) is 3 × 10-9
s-1 at 25 °C, exceeding the first-order rate constants for P-O
cleavage of either the monoanion or the dianion of methyl phosphate
(Table 1).
-d[MeP]/dt ) k3[MeP][Li+][OH-]
(1)
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10.1021/ja0473586 CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Rates of Methyl Phosphate Reactions in Scheme 1
q
q
k
25 (s-1
)
k100 (s-1
)
∆H (kcal/mol)
T∆S 25 (kcal/mol)
(a)a
(b)b
(c)c
(c)d
4 × 10-10
>3 × 10-9
1.5 × 10-10
8 × 10-6
1.4 × 10-13
>8 × 10-7
4.4 × 10-8
+30
+47
+17
+17
-0.2
+2.1
<-12
-14
2 × 10-20
a
b
References 1 and 2. These values were estimated by extrapolation
from the behavior of aryl phosphates (ref 2) and from the behavior of methyl
c
phosphate in 1 M KOH as described in ref 3. Nominal values of apparent
first-order rate constants observed in 4.4 M LiOH (this work). The
dissociation constant of the Li+ complex of the methyl phosphate dianion
d
is unknown, but exceeds 4.4 M (see text). Third-order rate constants (M-2
s-1) calculated from values of apparent rate constants in footnote c, using
eq 1.
Figure 3. Arrhenius plot of the common logarithm of the apparent first-
order rate constant (s-1) for hydrolysis of methyl phosphate, plotted as
function of the reciprocal of temperature (Kelvin).
glucose 1-phosphate enhances its intrinsic susceptibility toward
attack by oxygen nucleophiles at C-1, in view of the extremely
sluggish rate of spontaneous P/O cleavage of methyl phosphate
dianion1 and the fact that C/O cleavage of methyl phosphate must
be slower still. Somewhat more closely resembling the present
reaction, in that the carbon atom under attack is not anomeric, is
the reaction catalyzed by methionine adenosyltransferase (E. C.
2.5.1.6), in which the sulfur atom of methionine reacts with the
C-5′ atom of ATP to form S-adenosylmethionine. These reactions,
like the decarboxylation of OMP, appear to be among the more
formidable challenges that have been overcome during the evolution
of modern enzymes.
Scheme 1. Methyl Phosphate Reactions
Acknowledgment. We thank J. P. Guthrie, A. J. Kirby, and N.
H. Williams for helpful discussions. This work was supported by
Grant #GM-18325 from the National Institute of General Medical
Sciences.
References
(1) Bunton, C. A.; Llewellyn, D. R.; Oldham, K. G.; Vernon, C. A. J. Chem.
Soc. 1958, 1958, 3574.
(2) Kirby, A. J.; Varvoglis, A. G. J. Am. Chem. Soc. 1967, 89, 415.
(3) Lad, C.; Williams, N. H.; Wolfenden, R. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 5607.
(4) Robinson. R. A.; Stokes, R. H. Electrolyte Solutions, 2nd ed.; Butter-
worths: London, 1959; pp 510-511.
(5) Abell, K. W. Y.; Kirby, A. J. Tetrahedron Lett. 1986, 27, 1085.
(6) Grzyska, P. K.; Czyryca, P. G.; Golightly, J.; Small, K.; Larsen, P.; Hoff,
R. H.; Hengge, A. C. J. Org. Chem. 2002, 67, 1214.
(7) Stephan, E. F.; Miller, P. D. J. Chem. Eng. Data 1962, 7, 501.
(8) It would be of interest to compare the leaving group activity of the
presumed lithium phosphate dianion with the leaving group activities of
other anions, with respect to their ease of displacement from methane by
the hydroxide ion. But since the catalytic effect of lithium hydroxide shows
no sign of saturation even at its solubility limit (Figures 1 and 2), that
reactivity lex can be estimated only as a lower limit.
(9) Keq for glycogen phosphorylation is approximately unity (Hestrin, S. J.
Biol. Chem. 1949, 179, 943), but under the conditions that typically prevail
in mammalian tissue, the concentration of inorganic phosphate so greatly
exceeds the concentration of glucose 1-phosphate that this reaction
proceeds entirely in the direction of phosphorolysis.
(10) Pyridoxal phosphate is an essential cofactor for glycogen phosphorylase.
Its position in the crystal structure suggests that it is (just) close enough
to the reacting substrates to furnish a phosphoryl group that acts as a
general base: (a) Sygusch, J.; Madsen, N. B.; Kasvinski, P. J.; Fletterick,
R. J. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 4757. (b) Weber, I. T.;
Johnson, L. N.; Wilson, K. S.; Yeates, D. G.; Wild, D. L.; Jenckins, J. A.
Nature (London) 1978, 274, 433. Other evidence suggests that this may
be an oversimplification however: (c) Withers, S. G.; Shechosky, S.;
Madsen, N. B. Biochem. Biophys. Res. Commun. 1982, 108, 322.
After hydrolysis had been carried to completion in 4.4 M LiOH
containing 90% H218O, at 130 °C, the methanol product was found
to be 88% enriched in 18O, indicating that hydrolysis proceeds by
cleavage between the oxygen and carbon atom of the monolithium
complex (Scheme 1c). As expected for nucleophilic displacement
at carbon, no reaction (<1%) could be detected for isopropyl or
neopentyl phosphate under the same conditions, and ethyl phosphate
was found to be ∼20-fold less reactive than methyl phosphate.8
Many enzymes act on phosphoric ester substrates by facilitating
oxygen attack at the phosphorus atom, rather than at the carbon
atom. One of the few exceptions is glycogen phosphorylase (E. C.
2.4.1.1) reacting with glucose 1-phosphate to add single glucose
residues at the 4-OH group at the terminus of glycogen, in the
direction of reaction that is favored thermodynamically.9 No metal
ion is involved in the action of glycogen phosphorylase, and no
covalent intermediate has been detected thus far.10 It would be of
interest to learn the extent to which the anomeric structure of
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