Lanthanide catalyzed cyclization of uridine 3′-p-nitrophenyl phosphate

Article abstract of DOI:10.1006/bioo.2000.1179

Steady state kinetics and ¹⁵N isotope effects have been used to study the cyclization reaction of uridine 3′-p-nitrophenyl phosphate. The cyclization reaction is catalyzed by transition metal ions and lanthanides, as are substitution reactions of many phosphate esters. Kinetic analysis reveals that the erbium-catalyzed cyclization reaction involves the concerted deprotonation of the 2′-OH group and departure of the leaving group. The transition state is very late, with a very large degree of bond cleavage to the leaving group, which could be due to a large degree of polarization of the P-O bonds by erbium.

Full text of DOI:10.1006/bioo.2000.1179

Bioorganic Chemistry 28, 283–292 (2000)
doi:10.1006/bioo.2000.1179, available online at http://www.idealibrary.com on
Lanthanide Catalyzed Cyclization of Uridine
3Ј-p-Nitrophenyl Phosphate
Mark A. Rishavy, Alvan C. Hengge,1 and W. W. Cleland2
Institute for Enzyme Research, University of Wisconsin at Madison, Wisconsin 53705
Received February 25, 2000
Steady state kinetics and 15N isotope effects have been used to study the cyclization reaction
of uridine 3Ј-p-nitrophenyl phosphate. The cyclization reaction is catalyzed by transition metal
ions and lanthanides, as are substitution reactions of many phosphate esters. Kinetic analysis
reveals that the erbium-catalyzed cyclization reaction involves the concerted deprotonation of
the 2Ј-OH group and departure of the leaving group. The transition state is very late, with a
very large degree of bond cleavage to the leaving group, which could be due to a large degree
of polarization of the P–O bonds by erbium.
᭧ 2000 Academic Press
Nucleophilic substitution reactions of phosphate esters have been the subject of
much research (1). Of special interest is the hydrolysis of phosphate diesters, for this
class of compounds includes DNA and RNA, and hydrolysis of these compounds is
the function of many enzymes. Metal ions have been found to be important in both
enzymatic and nonenzymatic reactions of phosphate esters (2,3). Metal ions can
catalyze substitution reactions of phosphates in several ways and understanding the
role of metal ions in these reactions is critical to understanding the entire mechanism.
Metal ions in the lanthanide series have received some attention as exceptional
catalysts of phosphate ester substitution reactions in several systems (4,5). Lanthanide
complexes have been used as catalysts of phosphate ester substitutions with the
eventual goal of preparing complexes that can cleave specific nucleic acids sequences
(6–11). Under certain conditions, a lanthanide ion dimer has been observed with
incredible catalytic capability toward phosphate ester substitution reactions of a dinu-
cleotide (12). In this work, we have studied the cyclization reaction of uridine-3Ј-p-
nitrophenyl phosphate, a model for RNA with an activated leaving group, by lanthanide
ion catalysts. We report the results of steady state kinetics and 15N isotope effects
using several lanthanides as well as Znϩ2 and imidazole as catalysts for comparison.
The results provide insight into the transition state of this lanthanide ion catalyzed
phosphate ester substitution reaction.
1 Present address: Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322.
2 To whom correspondence and reprint requests should be addressed.
283
0045-2068/00 $35.00
Copyright ᭧ 2000 by Academic Press
All rights of reproduction in any form reserved.

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RISHAVY, HENGGE, AND CLELAND
EXPERIMENTAL PROCEDURES
Materials. Uridine and dibutyl tin oxide from Aldrich were used without further
purification. Benzoyl chloride, triethylamine, dioxane, pyridine, methanol, and dihy-
dropyran from Aldrich were distilled prior to use. p-Toluenesulfonic acid from Aldrich
was recrystallized prior to use. Zinc sulfate was from MCB. Lanthanides were from
Alfa except for europium which was from Aldrich. All lanthanides were nitrates
except lutetium which was the chloride. p-Nitrophenyl phosphorodichloridate was
prepared as previously described (13). Triethylammonium bicarbonate was prepared
by bubbling CO2 through a solution of 200 ml of triethylamine in 1080 ml of water
until the pH of the solution was 8.0.
3Ј-O-Benzoyluridine. Uridine (4.86 g) was dissolved in 600 ml of distilled methanol
in a dried flask and the solution was heated to reflux under nitrogen. Dibutyl tin
oxide (4.92 g) was added and the solution was refluxed for about 1 h, after which
the cloudy solution had clarified. The solution was cooled to room temperature and
triethylamine (8.4 ml) was added followed by benzoyl chloride (7.2 ml). After 10
min, the reaction was evaporated in vacuo to a viscous oil. The oil was partitioned
between water and ether, and the water was evaporated in vacuo until a precipitate
was observed. The water fraction was refrigerated overnight and the crude precipitate
was collected by filtration. The white powder was recrystallized from ethanol.
2Ј,5Ј-Di-tetrahydropyranyluridine. A solution of 3Ј-O-benzoyluridine (3.2 g), dihy-
dropyran (17.3 ml), and p-toluenesulfonic acid (0.45 g) in 60 ml of dry dioxane was
stirred at room temperature. After 90 min, saturated NH4OH (0.16 ml) was added
and the solution was evaporated in vacuo to a viscous oil. The oil was taken up in
methanol (50 ml) and the solution was cooled to 4ЊC. Ammonia was bubbled through
the solution until the solution was saturated, and the solution was stirred for 64 h.
Silica gel was added to the solution, which was evaporated to dryness in vacuo. The
silica was dry loaded onto a silica column (4 ϫ 30 cm) and the column was eluted with
19:1 chloroform: methanol. Fractions were analyzed by TLC in the chromatography
solvent. Peaks exhibiting UV absorbance were pooled, evaporated to dryness, and
1
analyzed by H NMR. The light orange glassy product was dried in a vacuum
desiccator over P2O5.
2Ј,5Ј-Di-tetrahydropyranyluridine 3Ј-p-nitrophenyl phosphate. p-Nitrophenyl
phosphorodichloridate (2.98 g) was dissolved in 10 ml of dry dioxane with pyridine
(1.8 ml). A solution of 2Ј,5Ј-di-tetrahydropyranyluridine (2.3 g) in 10 ml of dry
dioxane was added to the first solution by syringe over 20 min. After stirring for 2
h, a solution of pyridine (1.8 ml) in water (8 ml) was added rapidly and the solution
was stirred for 5 min. The solution was evaporated in vacuo and the crude oil was
taken up in 25 ml of water. The aqueous solution was extracted with chloroform
(3 ϫ 50 ml) and the organic layers were dried over MgSO4, filtered, and evaporated
to a green foam. The foam was taken up in 2 ml of chloroform and this solution was
loaded onto a silica column (5 ϫ 15 cm). The column was eluted with 5:1 ethyl
acetate: methanol. Fractions were analyzed by TLC in the chromatography solvent.
Peaks exhibiting UV absorbance were pooled, evaporated to dryness, and analyzed
1
by H NMR.
The crude product was taken up in 250 ml of water, titrated to pH 7, and loaded

LANTHANIDE CYCLIZATION OF PHOSPHATE DIESTERS
285
onto a Sephadex A25 anion exchange column (2.5 ϫ 10 cm), which had been
equilibrated with 0.1 M triethylammonium bicarbonate, pH 8.0, and washed with
deionized water until the eluate was free of buffer salt. The column was eluted with
a linear gradient from 0 to 0.25 M TEAB and the eluate was monitored at 280 nm.
Fractions containing product were evaporated to dryness, and the crude oil was
repeatedly taken up in methanol and evaporated to dryness. The TEAB-free oil was
taken up in 200 ml of water and passed through a Sephadex C25 cation exchange
column (2.5 ϫ 10 cm), which had been equilibrated with 1 M sodium acetate, pH
5.5, and washed with deionized water until the eluate was free of buffer salts. The
column was washed with water and the eluate was monitored at 280 nm. Fractions
containing product were titrated to pH 6.0 and lyophilized.
Uridine 3Ј-p-nitrophenylphosphate. The amount of 2Ј,5Ј-di-tetrahydropyranyluri-
dine 3Ј-p-nitrophenyl phosphate required for a particular experiment was taken up in
water sufficient to make a 10 mM solution and 6 N HCl was added to make the
solution 0.1 N in HCl. After 40 min the solution was placed on ice and used as needed.
Determination of product. A sample of 2Ј,5Ј-di-tetrahydropyranyluridine 3Ј-p-ni-
trophenyl phosphate (6 mg) was taken up in 0.5 ml of D2O and deblocked by addition
of 25 ␮l of 1 N HCl. This sample was scanned by 31P NMR at 202.34 MHz. The
sample was titrated to pH 5.5 by addition of 50 mM MES at pH 6.0, and 0.09 ml of
10 mM Euϩ3 was added. The sample was again scanned by 31P NMR after 1 h.
Determination of cyclization rates. The rates of cyclization of uridine 3Ј-p-nitrophe-
nyl phosphate were determined by spectrophotometric assay at 330 nm. The extinction
coefficient was determined by complete reaction of a known amount of substrate
with ribonuclease. Reactions with lanthanides were performed in 3 ml quartz cuvettes
at 37ЊC in 5 mM MES, pH 5.5. The pH of reactions was found to remain constant
to any extent of reaction.
pH Variation of cyclization rates. The rates of cyclization of uridine 3Ј-p-nitrophe-
nyl phosphate were determined with erbium as above except that 5 mM MES was
replaced by 25 mM Homopipes (pH 4–5.5) or 25 mM MES (pH 5.5–6), and that
reactions were performed in 1 ml quartz cuvettes. The pH of reactions was found to
remain constant to any extent of reaction. As ionization of p-nitrophenol becomes
significant at the higher pH values, the extinction coefficients were determined at
330 nm at each individual pH.
Nitrogen isotope effects with lanthanides and zinc. All of the procedures described
below were performed in triplicate for a given experiment. 2Ј, 5Ј-Di-tetrahydropyrany-
luridine 3Ј-p-nitrophenyl phosphate (60 mg) was deblocked in 10 ml of dilute HCl
and the solution was mixed with an equal volume of 0.5 M MES, pH 6.0. To this
solution was added 1 ml of 50 mM erbium nitrate or zinc sulfate, or 0.5 ml of 50
mM europium nitrate. After 15 min, the erbium reactions were quenched by titration
with 5 N sulfuric acid to give a pH of 3–4. The europium reactions were likewise
quenched after 8 min, while the zinc reactions were quenched after 60 min. The
acidified reaction was extracted three times with an equal volume of ether. The
aqueous layer was made basic with 1 ml of 5 N NaOH. The ether layers were
combined and evaporated to dryness and the extracted p-nitrophenol was taken up
in water and made basic with 1 ml of 5 N NaOH. The concentration of p-nitrophenol
in both samples was determined by spectrophotometric assay at 400 nm. Both samples

286
RISHAVY, HENGGE, AND CLELAND
were extracted three times with equal volumes of ether and the aqueous layers were
acidified with 5 N sulfuric acid until the solutions were colorless. The solutions were
extracted three times with equal volumes of ether. The ether layers were combined,
dried over MgSO4, and filtered. The ether was removed by evaporation in vacuo and
the remaining p-nitrophenol was purified by vacuum sublimation. The p-nitrophenol
was washed into quartz tubes (24 cm ϫ 9 mm o.d., 7 mm i.d.) with ether. The ether
was removed by aspiration and copper oxide (10 g), copper (0.2 g), and silver (0.2
g) were placed in the tube. The quartz tube was evacuated on a vacuum line, sealed
with a torch, and heated to 850ЊC for 2 h. The quartz tubes were cracked on a high
vacuum line and the nitrogen gas was distilled through dry ice-2-propanol and liquid
nitrogen traps and collected on molecular sieves chilled with liquid nitrogen. The
purified nitrogen gas was analyzed on a Finnegan MAT Delta isotope ratio mass
spectrometer.
2Ј,5Ј-Di-tetrahydropyranyluridine 3Ј-p-nitrophenylphosphate (30 mg) was de-
blocked in 5 ml dilute HCl. This solution was made basic with 0.2 ml of 5 N NaOH
and the reaction was monitored at 400 nm until no further increase was observed.
The solution was acidified and extracted three times with equal volumes of ether.
The ether layers were combined, dried over MgSO4, and further worked up as described
above. The isotopic abundance determined by this procedure is that of the nitro group
in the starting material.
Nitrogen isotope effects with imidazole. 2Ј,5Ј-Di-tetrahydropyranyluridine 3Ј-p-
nitrophenylphosphate (60 mg) was deblocked in 10 ml of dilute HCl and this solution
was added to 40 ml of imidazole buffer (0.625 N, pH 6.75). After 20 min, the reaction
was quenched by addition of 5 N sulfuric acid to pH 3–4. All of the procedures
above were followed for the imidazole reactions except that the final ether extraction
of the basic aqueous solutions was replaced by an extraction with dichloromethane.
The pH of the aqueous layer was checked to ensure that it was greater than 12 before
the basic ether extraction.
Data analysis. The abundances of 15N in unreacted starting material (Ro), in re-
maining substrate (Rs), and product (Rp) were determined as described, as was the
fraction of reaction ( f ). For comparisons between Ro and Rs, the isotope effects were
calculated using Eq. [1].
Isotope effect ϭ log(1 Ϫ f )/log((1 Ϫ f )(Rs/Ro))
[1]
For comparisons between Ro and Rp, the isotope effects were calculated using Eq. [2].
Isotope effect ϭ log(1 Ϫ f )/log(1 Ϫ f )(Rp/Ro))
[2]
RESULTS
Determination of product. The reaction of uridine 3Ј-p-nitrophenyl phosphate was
scanned before and after addition of metal ion catalyst by 31P NMR. The peak observed
after addition of metal ion was at 17.8 ppm, which is in the region where cyclic
phosphates are observed, and no other peaks were observed. The only detectable
product of the metal catalyzed cyclization was uridine 2Ј,3Ј-cyclic phosphate.

LANTHANIDE CYCLIZATION OF PHOSPHATE DIESTERS
287
FIG. 1. Cyclization reaction of uridine 3Ј-p-nitrophenyl phosphate.
Spectrophotometric rate determinations. At pH 5.5, the product of the cyclization
reaction (Fig. 1) is protonated p-nitrophenol. The difference in the UV-vis spectrum
between this product and the substrate is greatest at 330 nm, so this wavelength was
selected for rate determinations. As the change in extinction coefficient varies some-
what with pH, ribonuclease was used to completely cyclize a known amount of 1
and the change in extinction coefficient was measured. The rate of cyclization of 1
was determined at two different concentrations of metal ion and substrate for every
lanthanide and compared to the uncatalyzed rate (Table 1).
TABLE 1
Rate Enhancement of Cyclization by Lanthanides
Background cyclizationa
Metal ion
2.49 ϫ 10Ϫ5 sϪ1
0.1 mMb (kcat/kuncat
)
0.5 mMc (kcat/kuncat
)
Laϩ3
Ceϩ3
Prϩ3
26.6
37.0
41.2
34.5
50.7
57.4
46.0
53.0
54.5
43.0
48.6
42.8
46.5
43.0
51.6
81.0
81.0
76.6
107.6
106.1
78.1
81.0
79.5
56.0
61.9
72.2
113.5
70.7
Ndϩ3
Smϩ3
Euϩ3
Gdϩ3
Tbϩ3
Dyϩ3
Hoϩ3
Erϩ3
Tmϩ3
Ybϩ3
Luϩ3
a pH 5.5, 9 mM MES, 37ЊC. [Uridine 3Ј-p-nitrophenyl phosphate] ϭ 0.291 mM.
b pH 5.5, 9 mM MES, 37ЊC. [Uridine 3Ј-p-nitrophenyl phosphate] ϭ 0.291 mM.
c pH 5.5, 5 mM MES, 37ЊC. [Uridine 3Ј-p-nitrophenyl phosphate] ϭ 0.454 mM.

288
RISHAVY, HENGGE, AND CLELAND
TABLE 2
Kinetic Constants for Lanthanide Catalyzed Cyclization
Metal
kcat ϫ 100 (sϪ1
)
KD (mM)
Euϩ3
Ybϩ3
Ceϩ3
Dyϩ3
Erϩ3
1.01
1.000
0.115
0.196
0.434
1.724
0.485
0.290
0.382
1.08
Five lanthanides were selected for a more detailed determination of kinetic constants
(Table 2). For these experiments, the concentration of 1 was constant at 454 ␮M,
while metal ion concentration was varied from 50 to 500 ␮M. The apparent dissociation
constants, Kapp, and rate constants, kcat, for cyclization of the metal–substrate complex
were determined by fitting the data to the hyperbolic equation
v ϭ kcatSM/(Kapp ϩ M),
[3]
where v was the observed rate, S was the concentration of substrate, and M was the
concentration of lanthanide. When metal ion and substrate are present in similar
concentrations, the true dissociation constant KD is related to the calculated dissociation
constant Kapp as shown in Eq. [4].
KD ϭ Kapp Ϫ S
[4]
pH Variation of rate constants. Erbium was selected for determination of the pH
variation of kinetic constants. Kinetic constants were determined as above at half-
unit pH intervals from 4 to 6. The concentration of substrate was constant at 491
␮m, while erbium was varied from 50 to 500 ␮m (MES pH 6), 100 ␮m to 1 mM
(MES pH 5.5), or 250 ␮m to 2 mM (Homopipes, pH 5.5 and less). The rates with
Homopipes buffer at pH 5.5 were about a factor of three less than the rates in MES
under the same conditions. A plot of log(kcat/KD) vs pH is in Fig. 2.
FIG. 2. Plot of log(kcat/KD) vs. pH.

LANTHANIDE CYCLIZATION OF PHOSPHATE DIESTERS
289
15N Isotope effects. Erbium and europium were selected for determination of 15N
isotope effects on the cyclization of 1. For comparison, isotope effects were also
determined with zinc ion and imidazole as catalysts. Reactions were run to completion
to determine the abundance of 15N in the starting material, and this number was
compared to the abundance of 15N in the remaining substrate and the product of
reactions run to partial completion. Typically, six experiments were performed for
each catalyst, each resulting in two independent determinations of the isotope effect
(Table 3).
DISCUSSION
Metal ions can catalyze substitution reactions of phosphate esters in three ways.
Metal ions can coordinate to a nucleophile, lowering the pKa of the nucleophile so
that it can be deprotonated and attack the phosphate (14,15). As a phosphate ester is
subjected to nucleophilic attack, a pentavalent transition state develops. Metal ions
can ligate to the nonesterified oxygens, stabilizing the transition state by electrostatic
interactions (15,16). Finally, as the bond to the leaving group is broken, negative
charge appears on the oxygen in the scissile bond. For poor leaving groups, metal
ligation can stabilize the negative charge developing on the leaving oxygen (14,17–19).
Kinetic evidence that complexed zinc can both activate a nucleophile and coordinate
to a phosphodiester substrate has been observed (20), while lanthanide complexes
with only one open coordination site are either inactive or less active than similar
complexes with two open coordination sites in phosphate ester substitution reactions
(7,8). Thus, metal ions act in more than one role in these reactions.
Lanthanide catalysis of 1 will probably not arise from coordination of the leaving
group, as p-nitrophenol is an excellent leaving group and requires little stabilization.
Activation of the 2Ј-oxygen nucleophile and coordination of the nonesterified oxygens,
however, should result in catalysis. At pH 5.5, there will only be a small fraction of
hydroxide ligands bound to lanthanides (5), so either this portion of the total metal
ion present is responsible for catalysis by acting as a general base or the lanthanides
may coordinate with the 2Ј-oxygen directly, lowering the pKa and promoting nucleo-
philic attack. The lanthanide catalyzed reaction is about 400 times faster than the
uncatalyzed reaction at pH 5.5, which corresponds to a decrease in the pKa of the 2Ј-
hydroxyl of 2.6 units if the full catalytic effect were due to activation of the nucleophile.
Measurement of 15N isotope effects at the nitro group nitrogen can give information
about the transition state of the cyclization reaction (21). Although this nitrogen is
far removed from the atoms involved in the reaction, the nitrogen is coupled to the
TABLE 3
15N Isotope Effects on Cyclization
Catalyst
15k
Erϩ3
1.0030 Ϯ 0.0002
1.0027 Ϯ 0.0001
1.0015 Ϯ 0.0004
1.0017 Ϯ 0.0003
Euϩ3
Znϩ2
Imidazole

290
RISHAVY, HENGGE, AND CLELAND
scissile oxygen by the aromatic ring. As negative charge develops on the scissile
oxygen, it is delocalized into the nitrophenol ring, causing an increasing contribution
from the quinonoid resonance form (Fig. 3). As the double-bond character of the
nitrogen–oxygen bond in the nitro group is decreased and double bond character is
increased in the carbon–nitrogen bond to the ring, the fractionation factor of the
nitrogen is lowered. This is due to the stiffer vibrational modes of bonds to oxygen
as opposed to bonds to carbon. Thus, P–O bond cleavage in the transition state results
in a normal secondary isotope effect at this nitrogen (22).
The observation of an 15N isotope effect with erbium and europium is evidence
that departure of the leaving group is highly rate-determining in the cyclization
reaction. The pH dependence of the cyclization reaction, however, shows that there
is a deprotonation in the rate-determining step. This deprotonation can only be that
of the 2Ј-OH nucleophile, either by a metal-bound hydroxide or as a result of direct
metal coordination to this oxygen. Since deprotonation of the nucleophile and depar-
ture of the leaving group are both part of the rate-determining step, the cyclization
reaction must take place by a concerted reaction.
The nitrogen isotope effects observed with the lanthanides erbium and europium
are among the largest observed in aqueous solution for the nitrophenol system. Effects
as large as these have only been observed in cases where phosphate ester hydrolysis
involves a transition state that is very close to a metaphosphate intermediate. In these
cases, the large nitrogen isotope effect of 1.0034 is a result of the negative charge
on the metaphosphate pushing electron density into the leaving group (23). The 15N
equilibrium isotope effect for deprotonation of p-nitrophenol is only 1.0023, although
the equilibrium effect for dephosphorylation will be larger (22). The transition state
for the lanthanide catalyzed cyclization reaction appears to be very late, with high
electron density developed on the leaving group. This type of transition state is
somewhat unusual for phosphate diester substitution reactions, which typically employ
more associative transition states (24). The unusual transition state could be due to
the influence of the highly charged lanthanide ion, which can polarize the P–O bonds
to a high degree. This would result in the development of positive charge on the
phosphorus in the transition state, which would lead to a transition state with a large
degree of bond cleavage to the leaving group as the positive phosphorus resists loss
of this bond (Fig. 4). Similar experiments involving hydrolysis of ethyl-4-nitrophenyl
phosphate by a copper complex also revealed a concerted reaction, but the observed
15N isotope effects were only about half as large as those observed with the lanthanides
(25). The smaller charge on copper will result in less polarization of any P–O bond
involving coordination to the metal ion and therefore the phosphorus will be less
positive and the transition state will be earlier. Similarly, the observed 15N isotope
effects with zinc ion as the catalyst of 1 are about half as large as with erbium and
europium (Table 3).
FIG. 3. Resonance forms of p-nitrophenolate anion.

LANTHANIDE CYCLIZATION OF PHOSPHATE DIESTERS
291
FIG. 4. Transition state of lanthanide-catalyzed cyclization of uridine 3Ј-p-nitrophenyl phosphate.
The background rate of cyclization of 1 is significant even at pH 5.5 (Table 1),
necessitating the blocking of the 2Ј-oxygen until one is ready to carry out reactions.
The observed background rate agrees within a factor of 4 to that expected from the
determined rate constant for hydroxide reaction of 840 MϪ1 sϪ1(26). The background
rate of cyclization of the p-nitrophenyl phosphate ester of propylene glycol is over
two orders of magnitude smaller at pH 6.85 (27,28). If this rate is corrected to the
expected rate at pH 5.5, the difference in reactivity between the two compounds is
over three orders of magnitude. This is presumably due to the freedom of rotation
in the glycol so that the nucleophile is not held in the correct position to undergo the
cyclization. In 1, the 2Ј-hydroxyl is held in the proper position for nucleophilic attack
by the closed ribose ring. However, the rate constant for breakdown of the metal ion-
substrate complex (Table 2) is only two orders of magnitude more favorable for 1
than for the glycol after correction for pH (27). The difference between the advantage
of 1 over the glycol in the uncatalyzed and catalyzed reactions can be explained by
the ability of the metal ion to organize the substrate for nucleophilic attack, which
is less important for 1. This organization would be brought about by simultaneous
coordination of the metal to the nucleophile and the phosphate group.
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