Kinetic isotope effects and stereochemical studies on a ribonuclease model: Hydrolysis reactions of uridine 3'-nitrophenyl phosphate

Article abstract of DOI:10.1006/bioo.2000.1170

The reactions of a ribonuclease model substrate, the compound uridine-3'-p-nitrophenyl phosphate, have been examined using heavy-atom isotope effects and stereochemical analysis. The cyclization of this compound is subject to catalysis by general base (by imidazole buffer), specific base (by carbonate buffer), and by acid. All three reactions proceed by the same mechanistic sequence, via cyclization to cUMP, which is stable under basic conditions but which is rapidly hydrolyzed to a mixture of 2'- and 3'-UMP under acid conditions. The isotope effects indicate that the specific base-catalyzed reaction exhibits an earlier transition state with respect to bond cleavage to the leaving group compared to the general base-catalyzed reaction. Stereochemical analysis indicates that both of the base-catalyzed reactions proceed with the same stereochemical outcome. It is concluded that the difference in the nucleophile in the two base-catalyzed reactions results in a difference in the transition state structure but both reactions are most likely concerted, with no phosphorane intermediate. The ¹⁵N isotope effects were also measured for the reaction of the substrate with ribonuclease A. The results indicate that considerably less negative charge develops on the leaving group in the transition state than for the general base-catalyzed reaction in solution. (C) 2000 Academic Press.

Full text of DOI:10.1006/bioo.2000.1170

Bioorganic Chemistry 28, 119–133 (2000)
doi:10.1006/bioo.2000.1170, available online at http://www.idealibrary.com on
Kinetic Isotope Effects and Stereochemical Studies on a
Ribonuclease Model: Hydrolysis Reactions of Uridine
3
Ј-Nitrophenyl Phosphate
Alvan C. Hengge,*,1,2 Karol S. Bruzik,† Aleksandra E. Tobin,*
,3
W. W. Cleland,* and Ming-Daw Tsai,†,1
*
Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin 53705; and
†Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio 43210
Received December 15, 1999
The reactions of a ribonuclease model substrate, the compound uridine-3Ј-p-nitrophenyl
phosphate, have been examined using heavy-atom isotope effects and stereochemical analysis.
The cyclization of this compound is subject to catalysis by general base (by imidazole buffer),
specific base (by carbonate buffer), and by acid. All three reactions proceed by the same
mechanistic sequence, via cyclization to cUMP, which is stable under basic conditions but
which is rapidly hydrolyzed to a mixture of 2Ј- and 3Ј-UMP under acid conditions. The isotope
effects indicate that the specific base-catalyzed reaction exhibits an earlier transition state with
respect to bond cleavage to the leaving group compared to the general base-catalyzed reaction.
Stereochemical analysis indicates that both of the base-catalyzed reactions proceed with the
same stereochemical outcome. It is concluded that the difference in the nucleophile in the two
base-catalyzed reactions results in a difference in the transition state structure but both reactions
are most likely concerted, with no phosphorane intermediate. The 15N isotope effects were also
measured for the reaction of the substrate with ribonuclease A. The results indicate that
considerably less negative charge develops on the leaving group in the transition state than for
the general base-catalyzed reaction in solution.
᭧ 2000 Academic Press
INTRODUCTION
Ribonucleases catalyze the cleavage of the (5Ј–O–P bond of RNA. Because of the
biological importance of this reaction its mechanistic details have been the object of
numerous studies, involving both the enzyme itself and model systems (1). Williams
and co-workers have conducted linear free energy studies on a ribonuclease model,
uridine 3Ј-aryl phosphates (2). These compounds undergo the first step of the ribo-
nuclease reaction, namely cleavage of the P–O bond, with formation of a 2Ј,3Ј cyclic
phosphate. In that work, the Brønsted parameters were measured for the general base
reaction catalyzed by imidazole buffer and the specific base reaction in carbonate
1
To whom correspondence should be addressed.
Present address: Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322.
Present address: Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy,
2
3
University of Illinois at Chicago, Chicago, IL 60612.
1
19
0
045-2068/00 $35.00
Copyright ᭧ 2000 by Academic Press
All rights of reproduction in any form reserved.

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HENGGE ET AL.
buffer. It was concluded that both the general and the specific base-catalyzed reactions
were concerted, with bond cleavage to the leaving group and bond formation to the
nucleophilic 2Ј hydroxyl group each occurring in the transition state (2). In this paper,
we describe the results of heavy-atom isotope effects and of stereochemical studies
with uridine 3Ј-p-nitrophenyl phosphate (Scheme 1) under conditions of general base
and specific base catalysis. In addition, the isotope effects were measured for the
15
acid-catalyzed hydrolysis reaction, and the N isotope effects were measured for the
ribonuclease-A-catalyzed reaction of the substrate at pH 6.2 and 7.6.
MATERIALS AND METHODS
Pyridine was distilled from calcium hydride under nitrogen before use. DMSO was
˚
stored over KOH and 3-A molecular sieves. Chloroform and hexane were dried in
evacuated ampoules over P O and lithium aluminum hydride, respectively, and were
2
5
distilled directly into reaction vessels under vacuum. Other reagents and solvents
were purchased from commercial sources and used as received unless otherwise
specified. Ribonuclease A was purchased from Sigma.
The product identification NMR experiments were performed on a Bruker 500-
MHz instrument, operating at 202.34 MHz for 31P experiments. These P spectra
31
were referenced to 80% phosphoric acid in D O (external reference). Thin layer
2
chromatography was performed on precoated silica gel plates (silica gel 60, F254
,
Merck) using phosphomolybdic acid/ethanol reagent for visualization. Column chro-
matography was performed on silica gel 60 (40–63 mm, Merck).
14
15
18
[
N]-p-Nitrophenol (3) and [ N, phenolic- O]-p-nitrophenol (4) were prepared
SCHEME 1

STUDIES ON A RIBONUCLEASE MODEL
121
as previously described. 2Ј,5Ј-bis(tert-Butyldiphenylsilyl)uridine (2) was prepared
18
according to Ogilvie et al. (5). [ O]-Phosphorus oxychloride was prepared by hydroly-
18
18
sis of phosphorus pentachloride with 48% [ O]-H O. Phosphoryl-[ O]-p-nitrophenyl
2
phosphorodichloridate was prepared from this material by reaction of an excess of
phosphorus oxychloride with p-nitrophenol as described (6).
Preparation of natural abundance 2Ј,5Ј-bis(tetrahydropyranyl)-uridyl-3Ј-p-ni-
trophenyl phosphate (1a). This compound was prepared by the general procedure of
Williams et al. (2). Uridine was benzoylated at the 3Ј position using dibutyltin oxide
and benzoyl chloride (7), followed by protection of the 2Ј- and 5Ј-hydroxy groups
as their tetrahydropyranyl ethers using 5,6-dihydro-2H-pyran, with subsequent re-
moval of the benzoyl group with ammonia. The resulting 2Ј,5Ј-bis(tetrahydropyranyl)
uridine (900 mg) was dissolved in 4.5 mL of dry dioxane. This solution was added
to a solution of p-nitrophenyl phosphorodichloridate (725 mg) and pyridine (450 ␮L)
in 3 mL of dry dioxane, and the mixture was stirred under nitrogen for 6 h. Then a
mixture of water (4 mL) and pyridine (1 mL) was added, and the reaction mixture
was stirred for 15 min. After concentration in vacuo, the crude product was extracted
from water with methylene chloride, purified by reverse phase chromatography eluting
with methanol/water, and finally converted into the sodium salt by ion exchange. The
compound was stored as the 2Ј,5Ј-bis(tetrahydropyranyl) ether and was deblocked in
dilute HCl solution just before use.
Preparation of remote labeled p-nitrophenyl phosphorodichloridate (Scheme
1
4
15
18
2
). Quantities of [ N]-p-nitrophenol and [ N, phenolic- O]-p-nitrophenol were
15
mixed in proportion to closely duplicate the 0.365% natural abundance of N. The
isotopic ratio was confirmed by isotope ratio mass spectrometry. This mixture was then
used to prepare remote labeled p-nitrophenyl phosphorodichloridate by the method
previously described (3).
Preparation of remote labeled 2Ј,5Ј-bis(4-methoxytetrahydropyran-4-yl)-uridyl-3Ј-
p-nitrophenyl phosphate (1b). Uridine was protected at the 2Ј- and 5Ј-positions as
the bis (4-methoxytetrahydropyran-4-yl) ether by the same reaction sequence described
above for the preparation of 2Ј,5Ј-bis(tetrahydropyranyl) uridine, except that
4
-methoxy-5,6-dihydro-2H-pyran was used in place of 5,6-dihydro-2H-pyran (8). This
compound was reacted with remote labeled p-nitrophenyl phosphorodichloridate and
purified as described above for the natural abundance compound. The compound was
SCHEME 2

122
HENGGE ET AL.
stored as the 2Ј,5Ј-bis(4-methoxytetrahydropyran-4-yl) ether and deblocked in dilute
18
HCl solution just before use in measurements of bridge- O isotope effects. The
achiral 4-methoxytetrahydropyran-4-yl ether protecting group was used to avoid differ-
ential rates in the acid-catalyzed removal of the protecting groups which could occur
with the chiral tetrahydropyranyl ether.
Preparation of 3Ј-O-[2Ј,5Ј-bis(tert-butyldiphenylsilyl)uridyl]-O-methyl-O-p-ni-
1
8
trophenyl O-phosphate (3) (Fig. 1). Protected uridine 2 (948 mg, 2 mmol) and
pyridine (230 ␮L) were dissolved in hexane/chloroform (5:1 v/v, 5 mL) and the
18
resulting solution was treated with a solution of p-nitrophenyl-[ O]-phosphorodichlor-
idate (517 mg, 2 mmol) in 1 mL of the same solvent at 0ЊC. A 0.5-mL aliquot of the
reaction mixture was transferred into an NMR tube and the progress of the reaction
3
1
was monitored by P NMR. The formation of two diastereomers of the product
giving rise to a doublet at Ϫ3 and Ϫ4 ppm was observed, along with unidentified
by-products. The reaction was continued to reach the optimum yield of the product.
The reaction mixture was then mixed with 230 ␮L of pyridine and 120 ␮L of methanol.
TLC (hexane/acetone, 3:1) of the reaction mixture showed formation of a distinct
product spot along with some unreacted protected uridine. The mixture was concen-
trated and purified by chromatography eluting with hexane/acetone (5:1) to give a
2
:1 mixture of the diastereomers of 3, along with some p-nitrophenyl dimethyl phos-
phate. This mixture was inseparable by TLC and was subjected to demethylation
3
1
16
without further purification. P NMR (C D ) diastereomer a: Ϫ8.13 [ O], Ϫ8.17
6
6
18
1
8
16
[
O]; diastereomer b: Ϫ8.18 [ O], Ϫ8.22 [ O]; p-nitrophenyl dimethyl phosphate:
16
18
Ϫ6.57 [ O], Ϫ6.71 [ O].
Preparation of 3Ј-O-[2Ј,5Ј-bis(tert-butyldiphenylsilyl)uridyl] O-p-nitrophenyl
phosphate (4). The triesters 3a,b (141 mg, 0.205 mmol) and tetra-n-butylammonium
iodide (150 mg, two-fold excess) were dissolved in dry acetonitrile (0.5 mL), and
the solution was stored at room temperature. After 12 h TLC could detect no remaining
substrate. The mixture was chromatographed on silica gel, eluting first with chloro-
form/methanol (20:1) to elute excess tetrabutylammonium iodide and some p-nitrophe-
31
nol and then with chloroform/methanol (7:1) to give pure product (178 mg). P NMR
(
CD OD) ␦ Ϫ7.70, Ϫ7.73 ppm.
3
Desilylation of 4 to diester 5 and subsequent cyclization to cyclic 2Ј,3Ј-uridine
phosphate 6 (Fig. 1). Cleavage of the TBDMS groups in 4 with tetra-n-butylammon-
ium fluoride in THF or with HCl in aqueous methanol were found to give unsatisfactory
results due to the rapid formation of 2Ј,3Ј-cyclic UMP following deprotection of the
2
Ј-hydroxyl group. The tetra-n-butylammonium salt of diester 4 (120 mg) from the
previous step was converted to the potassium salt using the potassium form of Dowex
0-X8 cation exchange resin. This salt was dissolved in methanol/water (5:3) and
5
31
1
the solution was treated with Dowex 50-X8 resin in the proton form. P and H
NMR were used to monitor the progress of deprotection of the 5Ј-hydroxyl group,
and the reaction was judged complete within approximately 0.5 h as revealed by an
upfield shift of the P chemical shift by 0.25 ppm. Deprotection of the 2Ј-hydroxyl
was slower and required several hours and resulted in a further upfield shift of 0.18
ppm. In parallel with deprotection, cyclization and hydrolysis of 2Ј,3Ј-cyclic UMP
proceeded which manifested itself by the appearance of two new signals at 2.9 and
3
1
2
.7 ppm, which were ascribed to 2Ј- and 3Ј-UMP. After 3 h the concentration of

STUDIES ON A RIBONUCLEASE MODEL
123
FIG. 1. Outline of the sequence used to produce the diastereomeric mixture of 5, its subsequent
31
cyclization to cUMP with imidazole or carbonate buffer, and subsequent methylation for P NMR
+
Ϫ
+
analysis. i: Pyridine; ii: methanol, pyridine; iii: Bu
4 2 3
N I ; iv: H -Dowex 50-X8; v: K CO (pH 9.6) or
imidazole (pH 6.7) buffers; vi: CH I, dibenzo-18-crown-6.
3
diester 5 reached a maximum and began to decline. At this point the mixture was
split into two equal portions. The first was added to imidazole–HCl buffer (20 mL,
0
0
.2 M, pH 6.7), and the second was added to potassium carbonate buffer (20 mL,
.2 M, pH 9.6). The pH of the solutions was checked immediately and found to be
31
within 0.1 unit of the pH of the original buffer. P NMR spectra recorded shortly
after mixing showed formation of the cyclic UMP at 19.2 ppm. The sample in
+
carbonate buffer was passed through a Dowex 50-X8 column in the Et NH form,
3
the eluate concentrated under vacuum, and the residue was chromatographed on
DEAE–Sephadex eluting with triethylammonium bicarbonate buffer. The pure cUMP
obtained was freed of the residual buffer salt by absorbption on charcoal, washes
with excess water, and elution with ethanol/aqueous ammonia (100:3). The eluate

124
HENGGE ET AL.
was concentrated, passed through a Dowex 50-X8 column in the potassium form,
3
1
16
and concentrated to give cUMP as its potassium salt. P NMR ␦ 19.10 [ O], 19.07
[
1
8
O]. The sample treated with imidazole buffer was worked up in a similar manner
except that it was treated sequentially with charcoal, DEAE–Sephadex chromatogra-
+
phy, and cation exchange with K -Dowex.
16
18
Configurational analysis of [ O, O] cUMP. An aqueous mixture of the foregoing
1
6
18
potassium salts of [ O, O] cUMP (half of the total quantity) was transferred to a
-mm NMR tube and freeze-dried. The solid residue was made anhydrous by repeated
5
dispersion in warm, dry dioxane and freeze-drying. The anhydrous solid was dissolved
in dry DMSO (0.4 mL), dibenzo-18-crown-6 (15 mg) was added, and the homogeneous
sample was treated with methyl iodide (100 ␮L) at room temperature. The progress
31
of the reaction to form 6 and 7 was followed by P NMR. The reaction was complete
within 3 h, as evidenced by the appearance of two groups of signals at 19.5 and 18.8
ppm. In addition to the two groups of signals arising from exo- and endomethyl esters
16
18
of [ O, O] cUMP, the formation of a minor quantity of upfield (carbonate sample)
and down field (imidazole sample) was observed (Fig. 2). These signals displayed a
similar intensity pattern as the major peak clusters and are probably the product of
31
further alkylations occurring on the uridine moiety. 7: P NMR (DMSO) ␦ 19.476
1
6
16
18
16
16
18
16
16
(
O–Pϭ O), 19.459 ( O–Pϭ O), 19.434 ( O–Pϭ O), 18.796 ( O–Pϭ O),
18
16
16
18
1
8.778 ( O–Pϭ O), 18.753 ( O–Pϭ O).
General procedures for isotope effect determinations. Removal of the protecting
groups on the 2Ј- and 5Ј-hydroxyl groups was accomplished by dissolving in 30
mL of dilute HCl (pH 1.5) for 45 min at room temperature in the case of 2Ј,5Ј-
tetrahydropyranyl groups and by identical conditions for 30 min for labeled 2Ј,5Ј-(4-
methoxytetrahydropyran-4-yl) groups. NMR experiments monitoring the removal of
the tetrahydropyranyl groups versus hydrolysis showed that under these conditions
deprotection was complete while hydrolysis of the deprotected diester was undetect-
13
15
able by P NMR. For N isotope effects, the natural abundance 2Ј,5Ј-bis(tetrahydro-
pyranyl)uridine-3Ј-p-nitrophenyl phosphate (1a) was used, and for the 18O isotope
effect experiments, remote labeled 2Ј,5Ј-bis(4-methoxytetrahydropyran-4-yl) uridine-
3
Ј-p-nitrophenyl phosphate (1b) was used. It was noted that the four diastereomers
of 1a resulting from the chiral tetrahydropyranyl groups exhibited slightly different
rates of deprotection. There was a possibility that if the diastereomers differed slightly
18
in their isotopic composition, then in the isotopic mixture used for the bridge
O
isotope effects differential deprotection rates might result in slightly different isotopic
mixtures from experiment to experiment. For this reason the achiral protecting groups
used in 1b were used for the remote label experiments.
Deblocking of 1a and 1b was monitored by adding 10-␮L samples to a cuvette
containing 0.4 N NaOH and reading the absorbance at 400 nm. The deblocked
substrate undergoes nearly instantaneous release of p-nitrophenolate under these condi-
tions. After deblocking was complete, the solution was divided into three equal
portions treated under the specific reaction conditions described below.
The isotope effects were calculated using the isotopic ratios of the nitrogen atom
in the nitro group in the product nitrophenol. This ratio was measured for the product
isolated after partial reaction and that in the unreacted substrate following its complete
hydrolysis. The isotope ratio in the product and that in the residual substrate, compared

STUDIES ON A RIBONUCLEASE MODEL
125

126
HENGGE ET AL.
with the isotopic ratio in the starting material, were used for calculations of the isotope
effects as previously described (4). The nitrogen in nitrophenol was converted to
molecular nitrogen for isotopic analysis as previously described (4).
Determination of the isotope effect in the specific base reaction (carbonate buff-
er). A total of 220 mg of either substrate la or substrate 1b was deprotected by
treatment with HCl as described. The resulting uridine-3Ј-p-nitrophenyl phosphate
solution was divided into three 10-mL portions and to each was added 35 mL of ice-
cold water followed by 5 mL of cold 0.5 M carbonate buffer, pH 9.7. Under these
conditions the substrate has a half-life for hydrolysis of approximately 90 s. At times
ranging from 60 to 120 s, reactions were stopped by rapid titration with 5 N sulfuric
acid to give a pH of about 4. The reaction mixtures were extracted with diethyl ether
(3 ϫ 50 mL) to isolate the p-nitrophenol product. After ether was removed by rotary
evaporation, the nitrophenol was taken up in a known volume of water and assayed
by adding an aliquot to 0.1 N NaOH and measuring the absorbance at 400 nm. The
aqueous layer from the original reaction mixture, containing the unreacted substrate,
was titrated to pH 10 with 5 N NaOH and left stand at room temperature for 1 h.
The p-nitrophenol liberated from the residual substrate in this manner was assayed
as above. The aqueous layers were then acidified and extracted with ether to isolate
p-nitrophenol which was purified by vacuum sublimation at 90ЊC prior to isotopic
analysis.
Determination of the isotope effect in the general base reaction (imidazole buffer).
Substrate 1a or 1b was deprotected as described above for the specific base reaction;
then to each 10-mL aliquot of the substrate 40 mL of 0.625 M imidazole buffer (pH
6
.75) was added giving a final buffer concentration of 0.5 M. Under these conditions
at 22ЊC the half-life for hydrolysis was approximately 15 min. Reactions were stopped
at times ranging from 10 to 25 min by titration to pH 3 with 5 N sulfuric acid and
were then extracted with ether (3 ϫ 50 mL). The ether extracts were concentrated
by rotary evaporation, dissolved in 50 mL of water, and assayed for p-nitrophenol
as described above. The solution was titrated to pH 11 and washed with methylene
chloride (50 mL) and then with ether (2 ϫ 40 mL). After acidification to pH ϳ 3,
p-nitrophenol was extracted with ether (3 ϫ 50 mL). These ether layers were dried
over magnesium sulfate and concentrated by rotary evaporation. This back extraction
procedure yielded p-nitrophenol free from contamination by imidazole.
The residual substrate in the aqueous layer of the reaction mixture was titrated to
FIG. 2. At the left are the 31P NMR signals of the 2:1 mixture of the diastereomers of 3Ј-O-[2Ј,5Ј-
bis(tert-butyldiphenylsilyl)uridyl]-O-methyl-O-p-nitrophenyl phosphate (3). To the right of this are the
31
P NMR signals of the cUMP produced in the imidazole-buffered (top) and carbonate-buffered (bottom)
reactions of 5. Prior to methylation the signals for the diastereomers cannot be distinguished; the two
1
8
signals seen are those for the unlabeled and the O-labeled cUMP. At the right are the spectra of the
cUMP products after methylation, corresponding to the four compounds 6a,b and 7a,b in Fig. 3. Each
diastereomer gives a set of three signals consisting of, in order from left to right, a peak for the unlabeled
compound, for the bridge 18O-labeled, and for the nonbridge O-labeled compounds. These are labeled
in the first of the four spectra in the set. The smaller set of signals upfield (in the imidazole spectra) and
downfield (in the carbonate spectra) are ascribed to products from overmethylation of the cUMP.
18

STUDIES ON A RIBONUCLEASE MODEL
127
pH 7, left to stand for 3 h, and then assayed for p-nitrophenol. The p-nitrophenol
thus liberated was then isolated by the same method.
Determination of the isotope effect in the acid hydrolysis reaction. After deprotec-
tion, the substrate solution was made 1 N in HCl by addition of 5 N HCl. Under
these conditions the half-life for hydrolysis was about 12 min. Reactions were effec-
tively stopped by addition of cold 1 N Hepes buffer, pH 8, until the reaction mixtures
had a pH of about 4. The reaction mixtures were extracted with ether (3 ϫ 40 mL).
After evaporation of ether, the p-nitrophenol was dissolved in water and assayed. The
residual substrate solution was titrated to pH 10 by addition of 5 N NaOH, allowed
to stand for Ͼ10 half-lives, and then assayed for p-nitrophenol. The p-nitrophenol
thus liberated from residual substrate was isolated after acidification by ether extraction
and purified by sublimation before isotopic analysis.
Determination of the isotope effect in the ribonuclease A reaction. After deprotec-
tion, 5 mL of the substrate solution was diluted with 75 mL of distilled water and
added to 20 mL of a 500 mM solution of either Bis–Tris buffer at pH 6.2 or of Tris
at pH 7.6. The final buffer concentration was 100 mM, and substrate concentration
was 0.5 mM. A 100-␮L aliquot of a 0.05 mg/mL enzyme solution was added to
initiate the reaction at pH 6.2; 1 mL of a 0.5 mg/mL enzyme solution was used for
the reactions at pH 7.6. The amounts of enzyme were chosen in order to keep the
enzyme-catalyzed reaction at least 10 times faster than the competing solution reaction.
The progress of the reaction was monitored by following the absorbance at 330 nm
of a 50-␮L aliquot added to 950 ␮L of distilled water. When the fraction of reacted
substrate was in the 40 to 60% range, reactions were stopped by acidification with
acid to pH 3, followed by extraction of the p-nitrophenol product with ether (3 ϫ 50
mL). The aqueous layer containing the residual substrate was titrated back to pH 6
and 500 ␮L of the RNase solution was added. This solution was allowed to stand
for Ͼ10 half-lives and then assayed for p-nitrophenol. The ether layers containing
the product p-nitrophenol after partial reaction were evaporated to dryness and the
residue was dissolved in 50 mL of water and assayed for p-nitrophenol. The fraction
of reaction reached when the enzymatic reaction was stopped was determined from
the assay of the initial nitrophenol product and that released from hydrolysis of the
residual substrate. Both samples of p-nitrophenol were isolated by acidification of
the aqueous solutions followed by ether extraction and then purified by sublimation
before isotopic analysis.
Product determination NMR experiments. 2Ј,5Ј-bis(Tetrahydropyranyl) uridine 3Ј-
p-nitrophenyl phosphate (7.5 mg) was dissolved in 600 ␮L of D O and 25 ␮L of 1 N
2
3
1
HCl was added. P NMR was used to monitor the deblocking of the tetrahydropyranyl
groups. When this was complete (approximately 30 min), reactions were initiated by
addition of 0.5 M carbonate buffer (500 ␮L, pH 9.7), imidazole buffer (0.6 N, pH
6
.75, 500 ␮L), or HCl (5.0 N, 100 ␮L). Formation of products was followed by
P NMR.
3
1
RESULTS
The course of the reaction. The courses of the general base, specific base, and
acid hydrolysis reactions were each followed by proton-decoupled 31P NMR. The
2
Ј,5Ј-bis(tetrahydropyranyl) uridine 3Ј-p-nitrophenyl phosphate exhibited a group of

128
HENGGE ET AL.
four signals between Ϫ4.277 and Ϫ4.442 ppm corresponding to four diastereomers
due to the presence the chiral tetrahydropyranyl groups. As the deblocking reaction
progressed, these signals were gradually replaced by a single resonance at Ϫ3.84 ppm.
After addition of imidazole buffer, the resonance of the deblocked substrate was
gradually replaced by a single resonance at 22.14 ppm which did not undergo further
reaction over 45 min. Acidification with 100 ␮L of 5 N HCl resulted in the immediate
replacement of this resonance by a pair of nearly equal resonances at 1.08 and 1.13
ppm. Addition of authentic uridine 3Ј-phosphate resulted in an enhancement of the
more upfield of these two signals.
When carbonate buffer was used to initiate the reaction, the resonance of the
deblocked substrate was gradually replaced by a single resonance at 22.23 ppm. This
signal was unchanged over 2 h, whereupon acidification with 100 ␮L of 5 N HCl
resulted in the immediate replacement this signal by a pair of nearly equal resonances
at 1.19 and 1.24 ppm. Addition of authentic uridine 3Ј-phosphate resulted in an
enhancement of the more upfield of these two signals.
In the acid hydrolysis reaction the single resonance of the deblocked substrate was
gradually replaced by a pair of nearly equal resonances at 1.08 and 1.13 ppm. Addition
of authentic uridine 3Ј-phosphate resulted in an enhancement of the more upfield peak.
Isotope effects. The isotope effects and their standard errors are tabulated in Table
1
for the solution and enzymatic reactions. At least four experiments were conducted
for each set of conditions. Each experiment yields two independent determinations
of the isotope effect, one calculated from the isotope ratios in the product and that
of the starting material and the other from the ratios of the residual substrate and
starting material. These isotope effects agreed in all cases within the standard errors
and were averaged together to give the mean values reported in Table 1. In the
18
experiments with the remote-labeled compound for determination of kbridge, the
15
18
observed isotope effect is that due to both N and O substitution. This observed
15
effect was corrected for the N contribution and for incomplete isotopic incorporation,
as previously described (9), to give the values reported in Table 1.
Stereochemistry. The substrate for the reaction 5 was generated in situ from the
potassium salt of 4 prepared as a 2:1 diastereomeric mixture. Acid-catalyzed deprotec-
tion was accomplished using Dowex 50-X8 in the proton form. A small amount
of cyclization to cUMP and subsequent hydrolysis was observed to occur during
deprotection. When the concentration of the deprotected diastereomeric mixture of
isomers of substrate 5 had reached a maximum, the solution was divided into two
TABLE 1
Isotope Effects for Reactions of Uridine 3Ј-p-Nitrophenyl Phosphate
Reaction
15k
18kbridge
1, Imidazole buffer
1, Carbonate buffer
1, Acid catalysis
1, RNase, pH 6.2
1, RNase, pH 7.6
1.0009 Ϯ 0.0002
1.0001 Ϯ 0.0002
1.0010 Ϯ 0.0002
1.0002 Ϯ 0.0001
1.0005 Ϯ 0.0001
1.0067 Ϯ 0.0009
1.0059 Ϯ 0.0004
1.0063 Ϯ 0.0011

STUDIES ON A RIBONUCLEASE MODEL
129
portions. One of these was treated with imidazole buffer and the other with carbonate
buffer. The cUMP product obtained under both general base and specific base condi-
tions was subsequently methylated, allowing the diastereomeric products to be ana-
3
2
lyzed by P NMR. Prior to methylation of the cyclic UMP, the signals for the
isotopic diastereomers cannot be distinguished. Following methylation, the exo- and
endomethyl esters (6 and 7) (Fig. 2) of cUMP are resolved into separate clusters of
signals. Each cluster consists of signals, from left to right, for the unlabeled compound,
1
8
18
18
with bridging O and with nonbridging O. Unmethylated cUMP with exo- O will
18
produce, after methylation, a mixture of exomethyl ester with bridging O (6a) and
endomethyl ester with nonbridging 18O (7a). Similarly, cUMP with endo- O after
18
18
methylation will produce a mixture of endomethyl ester with bridging O (7b) and
exomethyl ester with nonbridging 18O (6b). The results show essentially identical
stereochemical outcomes for the general base and the specific base-catalyzed reactions.
The approximately 2:1 ratio of diastereomers present in the starting material is essen-
tially unchanged in the products.
DISCUSSION
31
In the P NMR experiments following the products of the general base (imidazole)
and specific base (carbonate) reactions, the signal of the deblocked substrate was
gradually replaced by a more downfield resonance having a chemical shift typical of
5
-membered ring cyclic phosphates. The slight difference in the chemical shift of
this signal between these two sets of conditions was attributed to the difference in
pH of the reactions. This initial product is stable under the conditions of both of the
base-catalyzed reactions, but upon acidification it undergoes ring opening to form a
mixture of 3Ј- and 2Ј-uridine phosphates. The relative intensities of these two signals
in the 31P spectra were similar in the two reactions, with the more downfield peak
being slightly larger. The intermediate cyclic phosphate was not observed in the acid
catalyzed reaction, but its intermediacy is inferred from the fact that the same two
final products are formed, in the same ratio. This result rules out the alternative
reaction of direct acid hydrolysis of the uridine 3Ј-nitrophenyl phosphate, since this
process would form uridine 3Ј-phosphate as the single product. Although isomerization
of nucleotide 3Ј- and 2Ј-phosphates under acidic conditions is known to occur, this
reaction is too slow under the reaction conditions employed to have occurred in the
time period of these experiments. These results indicate that, under all three conditions,
the reaction proceeds via formation of the 2Ј,3Ј-cyclic phosphate, as shown in
Scheme 1.
Interpretation of the isotope effects. Changes in bonding to the p-nitrophenyl leav-
1
8
15
ing group in the transition state are measured by the isotope effects kbridge and k.
The 15k isotope effect monitors how much negative charge had developed on the
18
leaving group in the transition state. The primary isotope effect k
gives informa-
bridge
18
tion about the extent of P–O bond cleavage. As a primary isotope effect, k
has
bridge
contributions from factors other than zero point energy and may not be linearly related
to bond cleavage. In particular, resonance contributions can cause this isotope effect
to exhibit a nonlinear response to transition state structure. In an early transition state,
there is little charge delocalization into the aromatic ring and the nitro group, and
thus the phenolic oxygen atom remains singly bonded to the phenyl ring. In a later

130
HENGGE ET AL.
transition state there will be more charge delocalization and thus partial double bond
character between the phenolic oxygen atom and the ring. This stiffening of the C–O
bond can compensate partially for the loss of P–O bond order and reduce the isotope
1
8
effect. The kbridge isotope effect can, however, distinguish between the considerable
bond cleavage typical of reactions of phosphate monoesters, where this bond is mostly
broken in the transition state (where 18kbridge is in the range 1.02 to 1.03) and more
associative transition states where the degree of bond cleavage is much smaller, as
in phosphodiester reactions (where 18kbridge is Ͻ1.01).
The oxygen (primary) and the nitrogen (secondary) equilibrium isotope effects on
the deprotonation of p-nitrophenol are 18Keq ϭ 1.0153 Ϯ 0.0002 (10) and Keq
15
ϭ
1
.0023 Ϯ 0.0001 (11), respectively. However, deprotonation involves breaking a bond
to a hydrogen atom, not to a phosphoryl group; a phosphoryl group is more electron
withdrawing, as evidenced by the ␤ value of ϩ0.73 for an aryl group in a phospho-
eq
diester (12). This value is very close to those for aryl groups in acetate esters (13).
The positive value reflects electron donation from the bridge oxygen atom to the
phosphoryl group in the ground state, with resultant effects on this bond order. Primary
1
8
kbridge isotope effects in phosphoryl and acyl transfer reactions are as large as 1.03
4,10,14), and this is a better indicator of the maximum for this isotope effect than
(
15
the equilibrium isotope effect for deprotonation. Secondary k isotope effects of
.003 in reactions of the dianion of p-nitrophenyl phosphate, where bond cleavage
1
15
is nearly complete, are also larger than the 1.0023 equilibrium k isotope effect
for deprotonation.
Isotope effects for the alkaline hydrolysis and for the acid hydrolysis reactions of
two other diesters where p-nitrophenol is the leaving group, p-tert-butylphenyl p-
nitrophenyl phosphate (8) and 3,3-dimethylbutyl p-nitrophenyl phosphate (9) have
previously been measured (15) and are reported in Table 2 for comparison with the
values measured in the present work. The prior studies with 8 and 9 were conducted
at higher temperatures, so an estimation of these isotope effects at 25ЊC was made
using the equation ln(KIE at temp x) ϭ (y ЊK/x ЊK) ln (KIE at temp y), which assumes
the isotope effect becomes unity at infinite temperature. The isotope effects increase
by about 20% from this correction.
The three solution reactions studied here differ in some of their mechanistic details.
In the general base reaction of 1, the nucleophile is the 2Ј-hydroxyl group with the
proton partially abstracted in the transition state. Brønsted analysis indicates that the
proton is about 2/3 abstracted by the general base in the transition state (2). In the
TABLE 2
Isotope Effects for Reactions of Other Diesters of p-Nitrophenyl Phosphate (Data from Ref. (15))
Reaction
15k
18kbridge
8
8
9
9
, alkaline hydrolysis
, acid hydrolysis
, alkaline hydrolysis
, acid hydrolysis
1.0012 Ϯ 0.0001
1.0008 Ϯ 0.0001
1.0019 Ϯ 0.0002
1.0011 Ϯ 0.0002
1.0052 Ϯ 0.0008
1.0071 Ϯ 0.0005
1.0072 Ϯ 0.0005
1.0048 Ϯ 0.0004
Note. Corrected for temperature as described in the text.

STUDIES ON A RIBONUCLEASE MODEL
131
specific base reaction in carbonate buffer the nucleophile is the fully ionized alkoxide.
This more powerful nucleophile manifests itself in the faster rate of this reaction
compared to the general base reaction. The acid-catalyzed hydrolyses of diesters
proceed through the nucleophilic attack of water on the protonated, neutral phosphate
ester (16). In comparing the isotope effects under the three conditions it is apparent
that bond cleavage and charge development are, within experimental error, indistin-
guishable in the acid hydrolysis reaction and the general base (imidazole) reaction.
For the acid-catalyzed reaction, the data do not rule out a mechanism with more
extensive bond cleavage to the leaving group than in the basic reactions, but in which
protonation of the leaving group occurs to lower the isotope effects. However, it is
unlikely that acid catalysis would involve protonation of the leaving group in the
transition state, for the following reasons. The acid hydrolysis of phosphate diesters
proceeds via the neutral, phosphoryl protonated species (alkyl nitrophenyl diesters
have a pK of about Ϫ0.5 (3)). The bridging oxygen of the neutral diester will have
a
an extremely low pK in the region of Ϫ9 or lower, by analogy with carboxyl esters.
a
In the transition state, the pK of the bridging oxygen will be somewhere between
a
this value and ϩ7.1, the pK of nitrophenolate. Unless the extent of bond cleavage
a
in the transition state is considerably greater than 50%, the pKa of the bridging oxygen
atom will still be well below the pH of the reaction. In addition, the finding of a
1
5
significant k isotope effect argues against protonation.
Both 18kbridge and k for the acid reaction and the general base-catalyzed reaction
of the uridine substrate are similar to those previously measured for the acid hydrolysis
of phosphodiesters 8 and 9. Thus, the data indicate that the intramolecular nature of
the reaction and the ring strain introduced in the transition state have no measurable
effect on leaving group bond cleavage or on charge development in the transition
state. These parameters are essentially unchanged from reactions of p-nitrophenyl
diesters that do not form a cyclic product.
15
The specific base (carbonate buffer) reaction differs in that the 15k isotope effect
18
is unity within experimental error, and the k
isotope effect is somewhat reduced
bridge
15
from its value in the other reactions. The lack of a k isotope effect implies a lack
of charge development on the leaving group in this reaction, which led us to consider
the possibility that it may have a different rate-limiting step, such as formation of a
phosphorane intermediate. Since no charge would be developed on the nitrophenol
group in formation of a phosphorane, there should be no 15k isotope effect in this
mechanism. Because such a species would be able to pseudorotate before expelling
the leaving group, the stereochemical outcome of the reaction could reveal whether
such an intermediate lies on the reaction pathway of the specific base reaction.
Therefore, with a mixture of diastereomers of uridine 3Ј-p-nitrophenyl phosphate in
known proportion, a concerted reaction would yield diastereomeric products in the
same ratio as that of the starting diester. A reaction proceeding via a phosphorane
intermediate provides the opportunity for epimerization at the phosphorus atom.
Stereochemical experiments. A 2:1 diastereomeric mixture of the substrate 4a
and 4b was prepared as the 2Ј,5Ј-bis(tert-butyldiphenylsilyl) protected compound.
Following deprotection, the compound was subjected to cyclization to cUMP in
31
imidazole buffer and in carbonate buffer. The P NMR (Fig. 2, center) shows the
18
signals for the unlabeled and the O-labeled cUMP; the diastereomers of the labeled

132
HENGGE ET AL.
compound cannot be distinguished. Following methylation using methyl iodide, 31P
NMR (Figure 2, right) can distinguish the diastereomers of the resulting triester.
The stereochemical analysis of the two base-catalyzed reactions shows that both
proceed with the same stereochemical outcome. The results do not determine whether
the reactions proceed with inversion or with retention. However, since the general
base reaction is concerted, the results imply that the specific base one is concerted
15
as well. The most likely explanation for the reduced value for k in the specific base-
catalyzed reaction is that it proceeds with an earlier transition state, with P–O bond
cleavage less advanced and less negative charge is present on the leaving group. This
may be rationalized by the more powerful nucleophile in the specific base-catalyzed
reaction, which is the fully deprotonated 2Ј-alkoxide. In the general base-catalyzed
reaction the 2Ј-hydroxyl group is deprotonated simultaneously with nucleophilic at-
tack, and the nucleophile is essentially neutral in the transition state for the reaction.
Figure 3 shows schematic diagrams of the transition states of the two reactions.
A concerted mechanism is consistent with linear free energy experiments by Wil-
liams and co-workers that indicate that for phosphodiesters with good (i.e., aryl)
leaving groups, phosphoryl transfer is concerted with no intermediate (17). The
Brønsted ␤leaving group value for diester reactions is much smaller than that for monoes-
ters, indicating that reactions of diesters are less dissociative and bond cleavage to
the leaving group in the transition state is less advanced, but that nevertheless leaving
18
group departure is concerted with nucleophilic attack. Similarly, the kbridge isotope
effects in the reactions studied here are all considerably smaller than those found for
reactions of the monoester p-nitrophenyl phosphate.
The ribonuclease reaction. Ribonuclease A catalyzes the cleavage of the P–O(5Ј)
bond of RNA (1). In the generally accepted mechanism, the side chain of His-12 acts
as a base that abstracts a proton from the 2Ј-hydroxyl group, facilitating its attack
on the phosphoryl group. In the same step the side chain of His-119 acts as an acid
to protonate the leaving group (for a review including references to other mechanistic
proposals see Ref. 1). The reported Brønsted dependencies indicate that considerably
less negative charge develops on the leaving group than in the general base-catalyzed
reaction with imidazole buffer (18). The reaction of the substrate 1 with RNase A
has been shown to give an atypical response to mutation of the enzymatic general
acid H119. Specifically, while k /K for H119A is reduced by three to four orders
cat
m
of magnitude with the substrates poly(c) and UpA, with 1 the rate is only slightly
FIG. 3. A diagram of the transition state for the general base (left) and specific base (right) reactions
of 5. In the general base-catalyzed reaction, proton abstraction by imidazole from the nucleophilic 2Ј
hydroxyl group is concerted with phosphoryl transfer. In the specific base-catalyzed reaction, proton
transfer occurs before phosphoryl transfer. The extent of P–O bond cleavage in the transition state is less
in the specific base-catalyzed reaction.

STUDIES ON A RIBONUCLEASE MODEL
133
reduced (19). This is most likely a result of the stability of p-nitrophenolate anion,
which allows it to function as a good leaving group even in the absence of protonation.
The 15k isotope effects for the RNase-catalyzed reaction of 1 at the pH optimum
and at pH 7.6 are smaller than for the imidazole-catalyzed reaction, indicating that
less negative charge develops on the leaving group in the enzymatic reaction in
agreement with the Brønsted studies (18). The isotope effect at pH 6.2 is consistent
with protonation of the leaving group in the transition state. The larger isotope effect
at pH 7.6 may result from a significant amount of reaction occurring from an incorrectly
4
protonated form of the enzyme. Specifically, if H119 is unprotonated this residue
cannot protonate the leaving group in the transition state. The kinetic data from the
H119A mutant indicate that p-nitrophenolate anion is a sufficiently good leaving
group that it can depart as the anion (19), in contrast to the natural substrates for
RNase where this group is a nucleoside. If the rate of catalysis by the H119A mutant
is a fair estimate of the rate of the incorrectly protonated form of the enzyme, then
with the nitrophenyl substrate these rates will be similar. This would permit a signifi-
cant fraction of reaction to proceed via the incorrectly protonated form of the enzymatic
reaction of substrate 1 at pH values above its typical pH optimum.
ACKNOWLEDGMENTS
This work was supported by Grant GM 18938 from the National Institutes of Health to W.W.C. and
GM30327 to M.-D. T.
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4
The 18
kbridge isotope effects were not measured for the ribonuclease reaction. Since this isotope effect
varies very little in the different hydrolysis reactions of 1 in solution, it is unlikely that this isotope effect
would yield additional information about the transition state for the ribonuclease reaction.