Dissection of Nucleophilic and General Base Roles for the Reaction of Phosphate with p-Nitrophenyl Thiolacetate, p-Nitrophenyl Thiolformate, and Phenyl Thiolacetate

Article abstract of DOI:10.1021/jo970904b

Phosphate buffers are well-known to catalyze the decomposition of various active acyl compounds. This study was undertaken to determine the extent to which it acts as a nucleophile and general base toward some activated esters and thiolesters. Thus, the hydrolyses of p-nitrophenyl acetate (3a), p-nitrophenyl thiolacetate (3b), phenyl acetate (4a), phenyl thiolacetate (4b), and p-nitrophenyl thiolformate (5) have been studied in aqueous phosphate, μ = 1.0 (K₂SO₄). Both phosphate monoanion and dianion are reactive toward the thiolesters 3b, 4b, and 5. For 3b, reaction of the dianion exhibits a solvent kinetic isotope effect (SKIE) of 1.00 ± 0.11 while that for the monoanion is 2.13 plusmn; 1.1. For the reaction of phosphate dianion with 5, the SKIE is 0.8 ± 0.2 and that for the monoanion at pH 3.05 is roughly 1.5. Phosphate dianion reacts with each thiolacetate and its oxygen analogue at comparable rates: the reactivity ratio of the formyl to acetyl thiolesters, 5:3b, toward phosphate dianion is 685. ¹H NMR analysis of the 3b hydrolysis mixtures in H₂O and D₂O containing phosphate shows the transient formation, and subsequent hydrolysis, of acetyl phosphate. Analysis of the kinetics of these processes indicates that in H₂O at pH = 8.5, phosphate dianion functions as both a nucleophile and general base toward 3b, the nucleophilic role comprising 80-93% of the reaction. In D₂O, the process is entirely nucleophilic. For the reaction of phosphate dianion with 4b, the ¹H NMR analysis indicates that the nucleophilic role comprises 40-50% of the reaction, the general base role being 50-60%. The reaction of phosphate dianion with 5 is entirely nucleophilic, while the monoanion reacts as a general base. The data are interpreted in terms of standard carbonyl addition/elimination mechanisms in which the ability of the attacking phosphate di- or monoanion to displace a given leaving group is tied to the pK_a of the conjugate acids of the nucleophile and leaving groups.

Full text of DOI:10.1021/jo970904b

J . Org. Chem. 1997, 62, 7351-7357
7351
Dissection of Nu cleop h ilic a n d Gen er a l Ba se Roles for th e
Rea ction of P h osp h a te w ith p-Nitr op h en yl Th iola ceta te,
p-Nitr op h en yl Th iolfor m a te, a n d P h en yl Th iola ceta te
Manjinder S. Gill, Alexei A. Neverov, and R. S. Brown*
Department of Chemistry, Queen’s University, Kingston, Ontario, Canada, K7L 3N6
Received May 20, 1997X
Phosphate buffers are well-known to catalyze the decomposition of various active acyl compounds.
This study was undertaken to determine the extent to which it acts as a nucleophile and general
base toward some activated esters and thiolesters. Thus, the hydrolyses of p-nitrophenyl acetate
(3a ), p-nitrophenyl thiolacetate (3b), phenyl acetate (4a ), phenyl thiolacetate (4b), and p-nitrophenyl
thiolformate (5) have been studied in aqueous phosphate, µ ) 1.0 (K2SO4). Both phosphate
monoanion and dianion are reactive toward the thiolesters 3b, 4b, and 5. For 3b, reaction of the
dianion exhibits a solvent kinetic isotope effect (SKIE) of 1.00 ( 0.11 while that for the monoanion
is 2.13 ( 1.1. For the reaction of phosphate dianion with 5, the SKIE is 0.8 ( 0.2 and that for the
monoanion at pH 3.05 is roughly 1.5. Phosphate dianion reacts with each thiolacetate and its
oxygen analogue at comparable rates: the reactivity ratio of the formyl to acetyl thiolesters, 5:3b,
toward phosphate dianion is 685. 1H NMR analysis of the 3b hydrolysis mixtures in H2O and D2O
containing phosphate shows the transient formation, and subsequent hydrolysis, of acetyl phosphate.
Analysis of the kinetics of these processes indicates that in H2O at pH ) 8.5, phosphate dianion
functions as both a nucleophile and general base toward 3b, the nucleophilic role comprising 80-
93% of the reaction. In D2O, the process is entirely nucleophilic. For the reaction of phosphate
1
dianion with 4b, the H NMR analysis indicates that the nucleophilic role comprises 40-50% of
the reaction, the general base role being 50-60%. The reaction of phosphate dianion with 5 is
entirely nucleophilic, while the monoanion reacts as a general base. The data are interpreted in
terms of standard carbonyl addition/elimination mechanisms in which the ability of the attacking
phosphate di- or monoanion to displace a given leaving group is tied to the pKa of the conjugate
acids of the nucleophile and leaving groups.
Sch em e 1
In tr od u ction
In an earlier report we have described the catalysis of
acyl transfer between certain formamides and water by
an imidazole thiol.1 This reaction proceeds as in Scheme
1 and was proposed as a mimic of the chemistry believed
to occur in the active site of the cysteine proteases. As
part of this program we were interested in the partition-
ing of the formyl thiolester intermediate (2) between
attack of water and amine nucleophiles. Surprisingly,
when 2 was placed in an aqueous phosphate buffer, an
immediate quantitative formation of formyl phosphate2,3
occurred (confirmed by 1H and 31P NMR). The apparent
facility of the acyl transfer from a thiolester to phos-
phate seemed an unusual process and worthy of further
study.
phoimidazolide-activated nucleotides,7 and acyl imida-
zoles8 by phosphate has been reported numerous times.
The mode of buffer catalysis has been discussed4-8,9 in
terms of general acid/base and nucleophilic mechanisms,
but often the exact process is unclear unless one can
actually observe an intermediate such as the one formed
during nucleophilic catalysis.10 To the best of our knowl-
edge, the first unambiguous demonstration of a nucleo-
philic role for phosphate reacting with an activated ester
was provided in 1984 by O’Connor and Wallace.6 In
that study, the hydrolysis of p-nitrophenyl acetate (pNPA)
in the presence of phosphate was monitored by both
UV/vis spectrophotometry and 1H NMR, the latter
showing the transient appearance of acetyl phosphate.
Phosphate is a commonly used buffer for controlling
the pH around neutrality, but it is often not a kinetically
innocent species. Catalytic enhancement of the rate of
hydrolysis of such species as activated esters,4-6 phos-
X Abstract published in Advance ACS Abstracts, September 15, 1997.
(1) Keillor, J . W.; Neverov, A. A.; Brown, R. S. J . Am. Chem. Soc.
1994, 116, 4669.
(2) J ahansouz, H.; Mertes, K. B.; Mertes, M.; P.; Himes, R. H. Bioorg.
Chem. 1989, 117, 207.
(3) Neverov, A. A.; Brown. R. S., unpublished observations.
(4) (a) Bruice, T. C.; Lapinski, R. J . Am. Chem. Soc. 1958, 80, 2265.
(b) J encks, W. P.; Carriuolo, J . J . Am. Chem. Soc. 1960, 82, 1778. (c)
Shames, S. L.; Byers, L. D. J . Am. Chem. Soc. 1981, 103, 6170 and
references therein. (d) Koehler, K.; Skora, R.; Cordes, E. H. J . Am.
Chem. Soc. 1966, 88, 3577. (e) Fedor, L. R.; Bruice, T. C. J . Am. Chem.
Soc. 1965, 87, 4138. (f) Bruice, T. C.; Bruno, J . J .; Chou, W. J . Am.
Chem. Soc. 1963, 85, 1659.
(7) Kanavarioti, A.; Rosenbach, M. T. J . Org. Chem. 1991, 56, 1513.
(8) J encks, W. P.; Carriuolo, J . J . Biol. Chem. 1959, 234, 1272, 1280.
(9) J encks, W. P. Catalysis in Chemistry and Enzymology;
McGraw-Hill: New York, 1969; pp 24-242.
(5) J ohnson, S. L. Adv. Phys. Org. Chem. 1967, 5, 237.
(6) O’Connor, C.; Wallace, R. G. Aust. J . Chem. 1984, 37, 2559.
(10) Gold, V.; Oakenfull, D. G.; Riley, T. J . Chem. Soc. B 1968, 515.
S0022-3263(97)00904-3 CCC: $14.00 © 1997 American Chemical Society

7352 J . Org. Chem., Vol. 62, No. 21, 1997
Gill et al.
1 (K2SO4)) were made from purified, deoxygenated water and
were further degassed by bubbling argon through them for
0.5-1 h before using. The rate of hydrolysis of thiol esters
was followed by observing the change in absorbance at 284
nm (decrease) for 3b; 263 nm (increase) for 4b; 275 nm
(decrease) for 3a ; 270 nm (increase) for 4a ; and 280 nm
(decrease) or 412 nm (increase) for 5. In a typical kinetic
experiment, 3-3.5 mL of deoxygenated buffer solution in a
quartz cell were equilibrated at 37.5 °C or 25 °C for 20-30
min in the spectrophotometer cell holder, after which the run
was initiated by injection of 5-7 µL of a stock solution of thiol
ester in dry CH3CN. The concentration of substrates varied
from 6 to 8 × 10-5 M. Reactions were followed to at least 5
half-times, and the pseudo-first-order rate constants (kobs) were
obtained for each run by nonlinear least-squares (NLLSQ)
fitting of the abs vs time data to a standard exponential model
(At ) A∞ + (A0 - A∞) exp(-kobst)). The final pH of the cell was
measured after each run. For initial rates the reaction was
followed up to 10% completion, and the final absorbance was
obtained from the observed absorbance at that particular
wavelength when equivalent amounts of the reaction products
were added to the same buffer solution. For the reactions
conducted in D2O, the solution pD values were determined as
pD ) pH + 0.4.19a
(C) P r od u ct Stu d ies. The products produced during the
hydrolysis of the esters in the presence of phosphate were
determined by 1H NMR spectroscopy. A buffer solution of a
particular pH or pD (0.5 mL) in an NMR tube was equilibrated
to 37.5 °C or 25 °C for 0.5 h. The reaction was started by
adding a stock solution of (2-3) × 10-4 M of 3b or 4b in 5-10
µL of CD3CN or (2-3.3) × 10-3 M of thiolformate (5) in 12-20
µL of CD3CN, and the spectra were recorded at various times.
The rate constants for the hydrolysis of acetyl phosphate and
formyl phosphate, (0.3-4) × 10-2 M, were also determined
under similar conditions. The hydrolysis of formyl phosphate
was conducted using two sample preparations. An authentic
sample containing formic acid and acetic acid was used
directly, or 5 was allowed to react with aqueous phosphate
buffer (0.1 M) at pH ∼8 for 1 min, after which the pH of the
reaction mixture was quickly reduced to pH 3.05 with subse-
quent hydrolysis of the formyl phosphate transient being
monitored by 1HMR spectroscopy. In the trapping experi-
ments, an aqueous pH 3.05 solution containing hydroxylamine
(7.91 × 10-3 M) was allowed to react with 5 (3.73 × 10-3 M)
at ambient temperature, and the spectrum was recorded after
30 min. This procedure was repeated with pH 3.05 aqueous
solutions containing phosphate buffer (0.01 and 1.0 M). The
fractions of formamide and formic acid were calculated from
the integration of singlets for the respective formyl protons.
Subsequently, Byers has suggested, on the basis of
detailed kinetic studies (including solvent kinetic isotope
effects and activation parameters), that phosphonates4c
and molybdate11 are good nucleophiles toward both pNPA
and p-nitrophenyl thiolacetate (pNPTA).
As pointed out by O’Connor and Wallace,6 the reaction
of activated esters with phosphate may serve as a model
for a biological process wherein the enzyme phospho-
transacetylase converts an acyl phosphate and CoA-SH
into various acyl CoA esters,12 eq 1. Given the analogy
of the transfer from the formyl thiolester (2) to phosphate,
and the rapidity of that reaction, we undertook a more
complete study of this process to compare the reactivities
of oxygen and thiol esters 3 and 4 toward phosphate at
37.5 °C, as well as the hydrolysis of p-nitrophenyl
thiolformate (5) in aqueous phosphate buffer at 25 °C.
The following indicates that (1) phosphate dianion is
indeed a nucleophile toward all species with similar
preference for oxygen and thiol esters; (2) phosphate
monoanion acts as a general base; and (3) there is a
competition between the nucleophilic and general base
roles that depends upon the pKa of the conjugate acids
of the attacking nucleophile and leaving group.
Exp er im en ta l Section
(a ) Ma ter ia ls a n d Gen er a l Meth od s. p-Nitrophenyl
thiolacetate (3b),13 phenyl thiolacetate (4b),14 and p-nitrophen-
yl acetate (3a )4b,15 were prepared according to the literature
procedures. Phenyl acetate (4a ) (Aldrich) was used as sup-
plied. p-Nitrophenyl thiolformate (5) was prepared according
to the procedure of Bax et al.16 from the reaction of acetic
formic anhydride17 with p-nitrobenzenethiol and a catalytic
amount of pyridine. Potassium dihydrogen orthophosphate
and dipotassium hydrogen orthophosphate were analytical
grade (BDH) and were used as supplied. Acetonitrile was
distilled from P2O5. Acetyl phosphate was prepared according
to the procedure of J encks et al.18 Formyl phosphate was
obtained as a solid mixture containing formic acid and acetic
acid as impurities by the reaction of aqueous K2HPO4 with
excess acetic formic anhydride at pH ∼7 in an ice bath for 45
min, followed by precipitation by addition of cold acetone.
(B) Kin etics. The kinetics of the reactions were followed
Resu lts a n d Discu ssion
The following will be broken into two main parts. In
the first we will describe the information that is gained
simply by observing, using UV/vis spectroscopy, the rate
of production of the hydrolysis products of the various
esters in the presence of varying [phosphate]. These
experiments will show the spontaneous rate of reaction,
and the dependence of the rate on [phosphate], but they
will not tell whether the phosphate acts as a nucleophile
1
by UV/vis spectrophotometry and H NMR using instruments
and procedures previously described.1 Buffer solutions (µ )
1
or a general base. This can be obtained using H NMR,
the analysis of which will comprise the second part of
the discussion.
(i) UV/Vis Kin et ics. (a ) Wa t er a n d Hyd r oxid e
(11) (a) Wikjord, B. R.; Byers, L. D. J . Am. Chem. Soc. 1992, 114,
5553. (b) Wikjord, B. R.; Byers, L. D. J . Org. Chem. 1992, 57, 6814.
(12) Lehninger, A. L.; Nelson, D. L.; Cox, M. M. Principles of
Biochemistry; Worth Publishers: New York, 1972; p 100.
(13) (a) Douglas, K. T.; Yaggi, N. F.; Mervis, C. M. J Chem. Soc.,
Perkin Trans. 2 1981, 171. (b) Dessoliw, M.; Lalio-Dirard, M.; Vilkas,
M. Bull. Soc. Chim. Fr. 1970, 2573.
(14) (a) Demaria, P.; Fini, A.; Hall, F. M. J . Chem. Soc., Perkin
Trans. 2. 1973, 196. (b) Street, J . P.; Brown, R. S. J . Am. Chem. Soc.
1985, 107, 6084.
Rea ction s. The pseudo-first-order rate constants (kobs
)
for the hydrolysis of esters 3a ,b and 4a ,b were deter-
mined at a given pH (pD) at 37.5 °C as a function of
[phosphate]total. The primary data (Tables 1S-5S, Sup-
porting Information), when plotted (not shown), give
straight lines, the slopes and intercepts of which are the
(15) Chattaway, F. D. J . Am. Chem. Soc. 1931, 2495.
(16) Bax, P. C.; Stevens, W. Recl. Trav. Chim. Pays-Bas. 1970, 89
(3), 265.
(17) Krimen, L. I. Organic Syntheses; Wiley: New York, 1973;
Collect. Vol. V, p 8.
(18) Herschlag, D.; J encks, W. P. J . Am. Chem. Soc. 1986, 108, 7938.
(19) (a) Covington, A. K.; Paabo, M.; Robinson, R. A.; Bates, R. G.
Anal. Chem. 1968, 40, 700. (b) Lide, D. R. CRC Handbook of Chemistry
and Physics, 76th ed; CRC Press: Baco Raton, 1995; pp 8-57.

General Base Roles for Phosphate Reactions
J . Org. Chem., Vol. 62, No. 21, 1997 7353
Ta ble 1. Ra te Con sta n ts for th e Hyd r olysis of
p-Nitr op h en yl Th iola ceta te (3b) in Aqu eou s P h osp h a te
Bu ffer a t 37.5 °Ca
Ta ble 4. Ra te Con sta n ts for th e Hyd r olysis of
p-Nitr op h en yl Aceta te (3a ) in P h osp h a te Bu ffer (H2O) a t
37.5 °Ca
b
c
b
c
pH
k0 (s-1
)
kcat (M-1 s-1
)
pH
k0 (s-1
)
kcat (M-1 s-1
)
3.05d
4.05d
5.0d
5.7
6.1
6.5
7.0
7.5
8.5
9.0
(5.75 ( 0.46) × 10-6
(5.42 ( 0.01) × 10-6
(5.26 ( 0.45) × 10-6
(6.74 ( 0.1) × 10-6
(9.58 ( 1.79) × 10-6
(1.08 ( 0.73) × 10-5
(1.88 ( 0.46) × 10-5
(2.95 ( 0.68) × 10-5
(1.65 ( 0.001) × 10-4
(4.00 ( 0.25) × 10-4
(1.07 ( 0.09) × 10-3
(3.84 ( 0.20) × 10-3
(1.18 ( 0.06) × 10-2
(3.38 ( 0.05) × 10-2
(5.92 ( 2.1) × 10-6
(9.72 ( 0.01) × 10-6
(2.64 ( 0.21) × 10-5
(9.77 ( 0.05) × 10-5
(1.98 ( 0.08) × 10-4
(2.76 ( 0.34) × 10-4
(5.10 ( 0.21) × 10-4
(6.43 ( 0.31) × 10-4
(6.90 ( 0.001) × 10-4
(8.53 ( 1.17) × 10-4
(1.26 ( 0.40) × 10-3
6.5
7.5
8.5
9.0
9.5
(2.00 ( 0.06) × 10-5
(2.84 ( 0.76) × 10-5
(2.17 ( 0.14) × 10-4
(4.91 ( 0.52) × 10-4
(1.20 ( 0.15) × 10-3
(2.31 ( 0.03) × 10-4
(6.39 ( 0.36) × 10-4
(6.02 ( 0.66) × 10-4
(1.69 ( 0.24) × 10-3
(3.39 ( 0.69) × 10-3
µ ) 1 (K2SO4). b From the intercept of linear regression of plots
a
of pseudo-first-order rate constant for hydrolysis of 3a ; errors are
from standard deviations of the plots. c Second-order rate constant
for the buffer catalysis from the slope of plots of pseudo-first-order
rate constant for hydrolysis of 3a vs [phosphate]total; errors are
from standard deviations of the plots.
9.5
10.0
10.5
11.0
Ta ble 5. Ra te Con sta n ts for th e Hyd r olysis of P h en yl
Aceta te (4a ) in P h osp h a te Bu ffer (H2O) a t 37.5 °Ca
a
b
µ ) 1 (K2SO4). From the intercept of linear regression of
plots of pseudo-first-order rate constant for hydrolysis of 3b; errors
are from standard deviations of the plots. c Second-order rate
constant for the buffer catalysis from the slope of plots of pseudo-
first order rate constant for hydrolysis of 3b vs [phosphate]total
errors are from standard deviations of the plots. Rate constants
from initial rates of hydrolysis reactions.
b
c
pH
k0 (s-1
)
kcat (M-1 s-1
)
7.80
8.75
9.60
(1.76 ( 0.09) × 10-5
(7.24 ( 0.29) × 10-5
(4.01 ( 0.09) × 10-4
(9.21 ( 0.21) × 10-3
(4.06 ( 0.26) × 10-5
(3.87 ( 0.19) × 10-5
(3.41 ( 0.18) × 10-5
;
d
11.0
8.5d
8.5e
8.5f
(3.11 ( 0.38) × 10-5
(3.25 ( 0.27) × 10-5
(3.70 ( 0.25) × 10-5
Ta ble 2. Ra te Con sta n ts for th e Hyd r olysis of
p-Nitr op h en yl Th iola ceta te (3b) in P h osp h a te Bu ffer in
D2O a t 37.5 °Ca
b
aµ ) 1 (K2SO4). From the intercept of linear regression of plots
of pseudo-first-order rate constant for hydrolysis of 4a ; errors are
from standard deviations of the plots. c Second-order rate constant
for the buffer catalysis from the slope of plots of pseudo-first order
rate constant for hydrolysis of 4a vs [phosphate]total; errors are
b
c
pD
k0 (s-1
)
kcat (M-1 s-1
)
2.70d
2.70d
4.55d
4.70d
6.0d
(2.07 ( 0.38) × 10-6
(2.71 ( 0.25) × 10-6
(3.88 ( 0.55) × 10-6
4.27 × 10-6
(4.05 ( 1.73) × 10-6
(3.02 ( 2.50) × 10-6
(2.45 ( 0.03) × 10-5
(1.77 ( 0.61) × 10-4
(2.84 ( 0.21) × 10-4
(5.82 ( 0.68) × 10-4
(6.30 ( 0.29) × 10-4
(1.02 ( 0.07) × 10-3
from standard deviations of the plots. µ ) 2.5 (K2SO4). e As in
d
d, except µ ) 2.5 (KCl). f As in d, except [4a ] ) 0.8 mM.
ko ) kL O + kLO-[-OL]
(2)
6.7
7.1
2
7.50
8.50
9.60
10.6
11.0
(7.75 ( 3.9) × 10-6
(7.47 ( 0.62) × 10-5
(2.58 ( 0.16) × 10-4
(2.20 ( 0.10) × 10-3
(7.90 ( 0.20) × 10-3
D2O
pKw
) 14.54 at 37.5 °C,19b fits of the ko data in Tables
-
1 and 2 to eq 2 gave rate constants kL O and kLO of (7.13
2
( 0.79) × 10-6 s-1 and (17.1 ( 1.83) M-1 s-1 in H2O; in
D2O the values were (3.29 ( 0.75) × 10-6 s-1 and (29.6
( 7.0) M-1 s-1
. Therefore, the water reaction has a
aµ ) 1 (K2SO4). b From the intercept of linear regression of plots
of pseudo-first-order rate constant for hydrolysis of 3b; errors are
from standard deviations of the plots. c Second-order rate constant
for the buffer catalysis from the slope of plots of pseudo-first-order
rate constant for hydrolysis of 3b vs [phosphate]total; errors are
computed solvent deuterium kinetic isotope effect (SKIE)
of kH / kD O) 2.2 ( 0.9, while that for the lyoxide term
2O
2
-
-
is kHO /kDO ) 0.6 ( 0.3. These are in the typical range
for hydrolyses of activated oxygen and thiol esters which
show normal values for the water reaction20 and unit or
slightly inverse SKIE’s for the acyl transfer to oxyanions
including LO- 21 consistent with transition states 6 and
7, respectively.
d
from standard deviations of the plots. Rate constants from initial
rates of hydrolysis reactions.
Ta ble 3. Ra te Con sta n ts for th e Hyd r olysis of P h en yl
Th iola ceta te (4b) in P h osp h a te Bu ffer (H2O) a t 37.5 °Ca
b
c
pH
k0 (s-1
)
kcat (M-1 s-1
)
6.5
8.5
(1.62 ( 0.77) × 10-6
(5.94 ( 0.15) × 10-5
(5.31 ( 0.31) × 10-5
(4.73 ( 0.83) × 10-5
(2.24 ( 0.01) × 10-4
(7.88 ( 0.4) × 10-3
8.5d
8.5e
9.5
(1.01 ( 0.05) × 10-4
(8.29 ( 1.1) × 10-5
11.0
a
b
µ ) 1 (K2SO4). Fom the intercept of linear regression of plots
of pseudo-first-order rate constant for hydrolysis of 4b; errors are
from standard deviations of the plots. c Second-order rate constant
for the buffer catalysis from the slope of plots of pseudo-first-order
rate constant for hydrolysis of 4b vs [phosphate]total; errors are
The rate constants for the alkaline hydrolysis of 3a and
4a ,b were calculated from fitting the data in Tables 3-5
d
from standard deviations of the plots. µ ) 2.5 (K2SO4), duplicate
runs, mean values of kobs and standard deviations. e As in d, except
[4b] ) 1 mM.
-
to eq 2; the respective kHO values at 37.5 °C are 23.9 (
4.5, 5.6 ( 1.4, and 6.26 ( 2.8 M-1 s-1. Although there
second-order rate constant for the phosphate reaction
(kcat) and spontaneous hydrolysis rate constant (ko),
respectively. These are given in Tables 1-5.
(20) (a) Alvarez, F. J .; Schowen, R. L. In Isotopes in Organic
Chemistry; Buncel, E., Lee, C. C., Eds.; Elsevier: Amsterdam, 1987,
pp 1-60 and references therein. (b) Hegazi, M.; Mata-Segreda, J . F.;
Schowen, R. L. J . Org. Chem. 1980, 45, 307.
(21) (a) Kellogg, B. A.; Tse, J . E.; Brown, R. S. J . Am. Chem. Soc.,
1995, 117, 1731. (b) Kellogg, B. A.; Brown, R. S.; McDonald, R. S. J .
Org. Chem. 1994, 59, 4652. (c) Pohl, E. R.; Hupe, D. J . J . Am. Chem.
Soc. 1980, 102, 2763 and references therein.
The spontaneous rate of hydrolysis of 3b in H2O and
D2O followed the rate expression given in eq 2. Using
values for [OH-] and [OD-] based on pKw
and
) 13.614
H2
O

7354 J . Org. Chem., Vol. 62, No. 21, 1997
Gill et al.
Ta ble 6. Ra te Con sta n ts for th e Hyd r olysis of
p-Nitr op h en yl Th iolfor m a te (5) in P h osp h a te Bu ffer
(H2O) a t 25 °Ca
b
c
pH
k0 (s-1
)
kcat (M-1 s-1
)
1.00
1.45
2.05
3.05
4.05
4.6
(7.56 ( 0.09) × 10-4
(7.15 ( 0.12) × 10-4
(6.94 ( 0.18) × 10-4
(7.00 ( 0.13) × 10-4
(7.64 ( 0.70) × 10-4
(6.02 ( 0.47) × 10-4
(6.26 ( 0.14) × 10-4
(2.67 ( 0.40) × 10-4
(2.80 ( 0.57) × 10-4
(6.04 ( 0.83) × 10-4
(1.21 ( 0.06) × 10-3
(3.29 ( 0.32) × 10-3
(4.61 ( 0.89) × 10-3
(5.51 ( 0.22) × 10-2
(6.29 ( 0.09) × 10-2
(1.57 ( 0.06) × 10-1
(1.22 ( 0.04) × 10-1
(1.76 ( 0.06) × 10-1
(3.84 ( 0.15) × 10-1
(4.38 ( 0.16) × 10-1
(5.16 ( 0.20) × 10-1
5.7
6.1
6.5
7.1
7.5
8.1
(1.36 ( 0.14) × 10-3
(1.96 ( 0.33) × 10-3
(2.48 ( 0.34) × 10-3
F igu r e 1. A plot of the second-order rate constants for the
reaction of 3b in H2O, phosphate buffer, as a function of pH,
T ) 37.5 °C, µ ) 1 (K2SO4). The points are experimental, and
the line is calculated from the NLLSQ fitting of the data to
µ ) 1 (K2SO4). b From the intercept of linear regression of plots
a
obs
2
equation kcat ) kM + kD(1 + [H+]/Ka ).
of pseudo-first-order rate constant for hydrolysis of 5; errors are
from standard deviations of the plots. c Second-order rate constant
for the buffer catalysis from the slope of plots of pseudo-first order
rate constant for hydrolysis of 5 vs [phosphate]total; errors are from
standard deviations of the plots.
7.16 ( 0.05) for the D2O reaction. The reported value of
the second pKa of phosphoric acid in H2O at 25 °C and µ
) 1.0 (NaCl) is 6.36,23a while the value at µ ) 0 is 7.2.23b
The former value, assuming the same temperature
dependence as at µ ) 0, should drop by only 0.01 unit23b
at 37.5 °C and therefore agrees well with the best-fit
value of 6.52 we determined here in H2O. It is known
that the second pKa value for phosphate in D2O is 0.64
unit higher than in H2O, the reported value of 7.1623c
agreeing exceptionally well with the best-fit value. The
SKIE for the reaction of 3b with the phosphate dianion,
F igu r e 2. A plot of the second-order rate constants for the
reaction of 3b in D2O, phosphate buffer, as a function of pD,
T ) 37.5 °C, µ ) 1 M (K2SO4). The points are experimental
and the lines is calculated from the NLLSQ fitting of the data
kD O)/kD(D O), is 1.00 ( 0.11, while that for the reaction
(H2
2
of the monoanion, is kM O)/kM(D O), is slightly higher at
(H2
2
obs
2
to equation kcat ) kM + kD(1 + [H+]/Ka ).
2.13 ( 1.1. The unit value for the reaction of the dianion
can be compared with those for attack of molybdate2- and
the dianions of phosphonic acids on 3a ,b for which values
in the range of 1.03 ( 0.1 4c,11 are reported. Nucleophilic
mechanisms have been proposed for the latter reactions.
The values for the monoanion and dianion computed in
our study appear different from each other and may
suggest nucleophilic dianions, and general base monoan-
ions, but the appreciable errors in the best-fit values for
the monoanion rate constants limits our ability to draw
meaningful mechanistic conclusions about this reaction.
While we have not investigated the reaction of phos-
phate with pNPA (3a ) in as much detail as with 3b, 4a ,b,
the data given in Table 4 indicate that in region between
pH 7.5 and 8.5 where the dianion is the dominant species,
kcat ) (6.2 ( 0.4) × 10-4 M-1 s-1. This value is very close
to that obtained for dianion attack on 3b (6.68 × 10-4
M-1 s-1), which indicates that there is little difference in
the attack of this anion on the p-nitro S- or O-ester.
From the limited data for phenyl acetate (4a ) and phenyl
thiolacetate (4b) given in Tables 3 and 5, the ratio for
are substantial errors in the fit values, these data
indicate that the analogous S- and O-esters are of
comparable susceptibility to hydroxide attack; this can
be compared with a reported reactivity ratio toward OH-
of about 1/2 reported for 3a /3b at 25 °C.22
(b) P h osp h a te Rea ction s. Plots of the second order
rate constants for the phosphate catalysis (log kcat vs pH,
data of Tables 1 and 2) for the reaction of 3b in H2O and
D2O are shown in Figures 1 and 2. The lines through
the experimental points were calculated by NLLSQ
fitting of the data to eq 3 where
obs
2
kcat ) kM + kD(1 + [H+]/Ka )
(3)
kM and kD, are the rate constants for mono- and dianion
attack on 3b, and Ka2 is the second dissociation constant
of H3PO4. The so-derived constants are; kM ) (6.31 (
0.56) × 10-6 M-1 s-1, kD ) (6.68 ( 0.47) × 10-4 M-1 s-1
,
and Ka ) (3.04 ( 0.38) × 10-7 (pKa ) 6.52 ( 0.05) in
H2O. For the data in D2O the fitting procedure consisted
of two parts. The high pH domain was fit to eq 4 to
2
2
4b
their phosphate reaction at pH 8.5 is roughly kcat4a / kcat
) (3.3 × 10-5 M-1 s-1/9 × 10-5 M-1 s-1) ) 1/3.
2
determine kD and Ka . These constants were then “fixed”
(c) p-Nitr op h en yl Th iolfor m a te. A fit of the Table
6 data for the hydrolysis of p-nitrophenyl thiolformate
to derive the value for kM from a fit of all the data to eq
3. The computed rate and equilibrium constants
(5) in H2O at 25 °C to eq 2 gives kH ) (7.09 ( 0.54) ×
2O
obs
2
kcat ) kD(1 + [H+]/Ka )
(4)
(23) (a) Ilyasova, A. K.; Saulebekova, M. S. Zh. Neorg. Chim. 1983,
28, 553; Chem. Abstr. 1983, 98, 167945x. (b) Perrin, D. D. In
Dissociation Constants of Inorganic Acids and Bases in Aqueous
Solution; IUPAC, Pages Bros. Ltd.: Norwich, 1969. (c) Salomaa, P.;
Hakala, R.; Vasala, S.; Aalto, T. Acta Chem. Scand. 1969, 23, 2116.
(d) CRC Handbook of Chemistry and Physics, 50th ed; CRC Press:
Boca Raton, 1969-70; p D117.
are kM ) (2.95 ( 0.83) × 10-6 M-1 s-1, kD ) (6.69 ( 0.23)
× 10-4 M-1 s-1, and Ka ) (6.84 ( 0.74) × 10-8 (pKa
)
2
2
(22) Hupe, D. J .; J encks, W. P. J . Am. Chem. Soc. 1977, 99, 451.

General Base Roles for Phosphate Reactions
J . Org. Chem., Vol. 62, No. 21, 1997 7355
Ta ble 7. Ra te Con sta n ts for th e Hyd r olysis of
p-Nitr op h en yl Th iolfor m a te (5) in P h osp h a te Bu ffer in
D2O a t 25 °Ca
b
c
pD
k0 (s-1
)
chdkcat (M-1 s-1
)
3.05
3.50
4.05
8.15
(2.09 ( 0.01) × 10-4
(2.47 ( 0.1) × 10-4
(2.05 ( 0.6) × 10-4
(4.29 ( 0.03) × 10-4
(6.0 ( 0.46) × 10-4
(7.30 ( 2.8) × 10-4
(5.55 ( 1.00) × 10-1
µ ) 1 (K2SO4). b From the intercept of linear regression of plots
a
of pseudo-first-order rate constant for hydrolysis of 5; errors are
from standard deviations of the plots. c Second-order rate constant
for the buffer catalysis from the slope of plots of pseudo-first-order
rate constant for hydrolysis of 5 vs [phosphate]total; errors are from
standard deviations of the plots.
F igu r e 4. Plots of the fractions of 3b (open circles), acetic
acid (open squares), and acetyl phosphate (open triangles) as
observed by 1H NMR in H2O in pH 8.5, 0.1 M phosphate buffer
at 38 °C. The lines are calculated from NLLSQ fit to the
data: for 3b from first-order exponential decay; for acetyl
phosphate from eq 7 and for acetic acid from eq 8.
F igu r e 3. A plot of the second-order rate constants for the
reaction of 5 in H2O, phosphate buffer, as a function of pH, T
) 25 °C, µ ) 1 (K2SO4). The points are experimental and the
line is calculated from the NLLSQ fitting of the data to
obs
1
2
equation kcat ) kM(1 + [H+]/Ka ) + kD(1 + [H+]/Ka ).
10-4 s-1 and kHO ) (7.35 ( 1.6) × 103 M-1 s-1. Water
-
F igu r e 5. Plots of fractions of 3b (open circles), acetic acid
and hydroxide attack on the formyl ester are roughly 100-
fold and 350-fold faster than on the corresponding acetyl
ester 3b as expected on the basis of steric grounds. These
values can be compared with the findings of Pohl, Wu,
and Hupe24 who reported that 5 reacts roughly 1000-fold
faster with anions than does 3b regardless of whether
the nucleophile is an oxyanion or thiol anion. A limited
number of kinetic runs for the hydrolysis of 5 in the
presence of phosphate in D2O were conducted, the val-
ues for the rate constants being presented in Table 7.
Between pD 3.05 and 4.05 (the pD independent region),
(filled squares), and acetyl phosphate (open triangles), as
1
observed by H NMR in D2O, pD 8.5, 0.1 M phosphate buffer
at 38 °C. The lines are calculated from NLLSQ fit to the
data: for 3b from first order exponential decay; for acetyl
phosphate from eq 7 and for acetic acid from eq 8.
kD value in water ((4.58 ( 0.41) × 10-1 M-1 s-1), with
the limiting value found for kcat in D2O ((5.5 ( 1.0) ×
10-1 M-1 s-1, Table 7) gives a computed value for kD
kD
/
H2
O
D2O
) 0.8 ( 0.2 for the SKIE of the dianion attack on
5. These values support a nucleophilic role for the
dianion and either a general base or nucleophilic role for
the monoanion in assisting the hydrolysis of 5. Further
delineation of these roles is possible using NMR analysis
of the reaction media as described below.
the average value of the water reaction in D2O is kD O
)
2O
2
(2.2 ( 0.2) × 10-4 s-1. The SKIE for this process is kH
/
kD O ) 3.2 ( 0.6, consistent with the general process
2
described above (transition state 6) where two or more
waters are involved in the attack on the carbonyl.20
Shown in Figure 3 is a plot of the log kcat vs pH profile
for the reaction of phosphate with 5 at 25 °C (data of
Table 6). The line through the data is the best fit to eq
5 where kM and kD are rate constants for attack of the
1
(ii) H NMR k in etics. (a ) Th iolester s 3b a n d 4b.
The changes in the 1H NMR intensities of the signals due
to unreacted 3b (singlet at δ 2.4), acetyl phosphate
(doublet at δ 2.0), and the hydrolysis product, acetic acid
(singlet at δ 1.83) were monitored in 38 °C H2O, 0.1 M
phosphate, pH 8.5 as a function of time. The same
determination was conducted in D2O solution at pD )
8.5. Plots of the fractions of the reactant and products
vs time are shown in Figures 4 and 5, from which it can
be seen that there is a substantial buildup of the
transient intermediate, acetyl phosphate: this buildup
is more prominent in D2O than in H2O.
1
2
mono- and dianion, and Ka and Ka are the first and
second dissociation constants for the ionization of H3PO4.
obs
1
2
kcat ) kM(1 + [H+]/Ka ) + kD(1 + [H+]/Ka ) (5)
Using the previously computed dissociation constant
2
(pKa ) 6.52) as a fixed value gave kM ) (9.27 ( 2.01) ×
10-4 M-1 s-1, kD ) (4.58 ( 0.41) × 10-1 M-1 s-1 and Ka
1
Shown in Scheme 2 is the minimum process required
to explain the data. In that scheme there are two roles
for the phosphate, namely as a nucleophile (knuc) to give
acetyl phosphate (8), or as a general base (kgb) to assist
in the direct formation of acetic acid. Spontaneous
hydrolysis of the thiolester also can occur (ko) to yield
acetic acid directly.
) (2.55 ( 1.06) × 10-2, pKa ) 1.60 ( 0.15. Comparing
1
the kcat value at pD 3.50 with the water value at pH )
3.05 gives a kcatH O/kcatD O of (9.3 × 10-4 M-1 s-1/6.0 × 10-4
2
2
M-1 s-1) ≈ 1.5. Comparison of the best-fit value of the
(24) Pohl, E. R.; Wu, D.; Hupe, D. J . J . Am. Chem. Soc. 1980, 102,
2759.

7356 J . Org. Chem., Vol. 62, No. 21, 1997
Gill et al.
rate constant kD ) (6.69 ( 0.23) × 10-4 M-1 s-1, obtained
by UV/vis kinetics. Given the inherent differences in the
methods and possibility of experimental error, we do not
view these rate constants as being different, particularly
Sch em e 2
1
at the 95% confidence level. Importantly, the H NMR
experiment indicates that in D2O the phosphate reaction
is entirely nucleophilic while, as above, in H2O roughly
7-20% of the phosphate reaction goes via a general base
route. This would be the expected effect since the general
base route should have a significant SKIE while the
nucleophilic route should show a much smaller one.
The reaction of phenyl thiolacetate (4b), monitored by
1H NMR in a 38 °C, pH 8.5 aqueous solution of 1.0 M
phosphate, also showed signals assignable to acetyl
phosphate, but these were not as prominent as in the
reaction of 3b. Fitting of the data using eqs 6-8 and
rate constants given in Table 8 shows that knuc contrib-
utes 40-50% to the reaction, kgb contributing the rest.
This suggests that the nucleophilic reaction depends upon
the relative pKa of the thiol leaving group (pKa p-
nitrothiophenol ) 4.50, pKa thiophenol ) 6.43):27 the
higher the pKa of the leaving group, the less prone it is
to nucleophilic substitution by phosphate. This is a
common finding for other carbonyl addition/elimination
processes such as the reaction of acetate with aryl
acetates where the nucleophilic reaction gives way to a
general base one as the pKa of the leaving aryloxy group
increases.10
The disappearance of 3b follows an exponential first-
order decay. Reversal of the acyl phosphate (8) is
expected to be negligible as acetyl phosphate reacts with
thiols very slowly if at all in aqueous solution.25a,b In fact
this was confirmed under the present conditions by
following the hydrolysis of authentic acetyl phosphate by
1H NMR in phosphate buffer (1.0 M) of pH 8.5 in the
presence of a large excess of thiophenol. Even though
phenyl thiolate should be more nucleophilic than p-
nitrophenyl thiolate, no signal assignable to 4b was
observed. The rate constant (knuc) representing the
nucleophilic attack of phosphate on 3b was calculated
from standard equations for consecutive pseudo-first-
order reactions.26
1
(b) Th iolester 5. The H NMR spectrum of 5 (δ 8.22
d[8]/dt ) knuc[3b] - kac[8]
(6)
(s)) in aqueous phosphate buffer (0.01 M) of pH 8.1 after
15 min showed signals due to formyl phosphate (8, >80%,
δ 8.41 (d, J ) 3.7 Hz)) and <20% of formic acid (9, δ 8.33
(s)): formyl phosphate further hydrolyses at a slower
rate. Hydrolysis of 5 in pH 3.05 aqueous phosphate
buffers (0.1 and 1.0 M), followed by recording the 1H
NMR spectrum after 15 min, showed no signal assignable
to formyl phosphate. This does not necessarily rule out
the formation of formyl phosphate as a transient since
independent experiments showed that an authentic
sample hydrolyzes quickly to formic acid under the pH 3
reaction conditions.
Integration of eq 6 using initial [3b] ) 1 and [8] ) 0 gives
eq 7:
[8] ) knuc/(kac - knuc(e-k nuc t - e-kt))
(7)
The fraction of acetic acid (9) was calculated from eq 8:
[9] ) 1 - [3b] - [8]
(8)
The rate constant for the hydrolysis of acetyl phosphate
(kac) was determined in a separate experiment under
similar conditions by following the decomposition of
authentic material using 1H NMR. This kac was then
used as a constant for the fitting of the data for the
fraction of acetyl phosphate to eq 7 to evaluate the rate
constant knuc. The computed rate constants are listed in
Table 8. After supplying the rate constants for disap-
pearance of 3b and 8, a reasonably good fit to the data
for the fraction of acetic acid (9) was obtained from eq 8,
the lines through the data in Figure 4 being those
calculated on the basis of these constants. On the
premise that the kcat (entirely due to the dianion (kD) at
pH 8.5) determined for phosphate in the UV/vis kinetics
consists of both a general base and nucleophilic processes,
and the 1H NMR experiment provides knuc, one can
ultimately compute kgb ) kcat - knuc. From the numbers
given in Table 8, the nucleophilic process contributes 80-
93% of the overall reaction, the general base process
contributing 7-20%.
In an attempt to detect the formation of a transient
intermediate of formyl phosphate at pH 3, a trapping
experiment (Scheme 3) was conducted. J ahansouz et al.2
have determined that the strong nucleophile, hydroxy-
lamine, reacts rapidly with formyl phosphate and have
used this reaction to quantify the amount of 8 (R ) H)
remaining in solution at various times during hydrolysis.
1
Therefore, the hydrolysis of 5 was studied by H NMR
in a pH 3.05 aqueous solution of hydroxylamine (pKa
HONH3 ) 6.03)23d in the presence and absence of
+
phosphate. Without phosphate, the observed products
were formic acid (9) and formyl hydroxamic acid (HC(O)N-
HOH, 10) in a 1:2 ratio. In an aqueous phosphate buffer
(0.1 M) of pH 3.05, the ratio was, within experimental
error, the same. However, in a 1.0 M phosphate buffer,
pH 3.05, formic acid and the hydroxamic acid were found
to be produced in a 1.3:1 ratio. Using the rate constants
given in Table 6, and assuming an exclusive nucleophilic
role for the H2PO4-, a computed ratio of 1:4 for formate:
10 at 1.0 M phosphate is determined. Conversely, if the
exclusive role is general base, then the formate:10 ratio
should be 3:2, this value being experimentally indistin-
guishable from the 1.3:1 ratio observed.28 These findings
indicate that the dominant, and perhaps exclusive, role
A similar experiment was conducted in D2O, [phos-
phate]total ) 0.1 M, pD ) 8.5. NLLSQ fitting of the D2O
D2O
data to eq 7 gave knuc
) (7.19 ( 0.06) × 10-4 M-1 s-1
,
a value that is only slightly higher than the second-order
(25) (a) Sabato, G. D.; J encks, W. P. J . Am. Chem. Soc. 1961, 83,
4393. (b) Moore, S. A.; J encks, W. P. J . Biol. Chem. 1982, 257, 10874.
(26) Frost, A. A.; Pearson, R. G. In Kinetics and Mechanisms; Wiley
International: New York, 1961; pp 161-166.
(27) J encks, W. P.; Salvesen, K. J . Am. Chem. Soc. 1971, 93, 4433.

General Base Roles for Phosphate Reactions
J . Org. Chem., Vol. 62, No. 21, 1997 7357
Ta ble 8. Ra te Con sta n ts for th e Hyd r olysis of p-Nitr op h en yl Th iola ceta te (3b)a a n d P h en yl Th iola ceta te (4b)b in
P h osp h a te Bu ffer a t T ) 37.5 °C
c
d
e
f
g
h
thiolacetatee
pH/pD
kobs (s-1
)
kobs (s-1
)
k0 (s-1
)
kcat (M-1 s-1
)
kac (s-1
)
knuc (M-1 s-1
)
3b
pH ) 8.5
2.34 × 10-4
1.36 × 10-4
(2.26 ( 0.11)
× 10-4
(1.65 ( 0.01)
× 10-4
(6.68 ( 0.47)
× 10-4
(1.08 ( 0.06)
× 10-4
(5.79 ( 0.03)
× 10-4
3b
4b
pD ) 8.5
pH ) 8.5
(1.28 ( 0.03)
× 10-4
(7.47 ( 0.62)
× 10-5
(6.69 ( 0.23)
× 10-4
(8.26 ( 1.05)
× 10-5
(7.19 ( 0.06)
× 10-4
(1.30 ( 0.11)
× 10-4
(1.10 ( 0.09)
× 10-4
(4.73 ( 0.83)
× 10-5
(9 ( 1) × 10-5
(8.87 ( 0.51)
× 10-5
(4.05 ( 0.06)
× 10-5
[Phosphate]total ) 0.1 M. [Phosphate]total ) 1.0 M. c From UV/vis kinetics, single run for 3b and four runs for 4b. From 1H NMR
kinetics, single run. e From UV/vis kinetics, from the intercept of the linear regression of plot of kobs vs [phosphate]total, errors from standard
deviations. f From UV/vis kinetics, from the fits of the kcat data for 3b to eq 3. Note that at pH 8.5, kcat ) kD. Value of kcat for 4b average
a
b
d
of two values in Table 3. From 1H NMR kinetics as the slope of the linear regression of the plot of ln of fraction of unhydrolyzed acetyl
g
h
phosphate vs time, errors from standard deviations. From NLLSQ fitting of the data for the fraction of acetyl phosphate (8) to eq 7,
using [phosphate] ) 0.1 M for 3b and [phosphate] ) 1 M for 4b.
Sch em e 3
4b proceeds in much the same way in terms of reaction
rate and mechanism. In two cases where we have data,
phosphate monoanion behaves as a general base in
promoting the hydrolyses of 3b and 5.
All the above observations are, after the fact, reconciled
by well-established principles of carbonyl addition/
elimination reactions, namely that nucleophiles cannot
displace substantially poorer leaving groups from the
addition intermediates.29 The available data suggest that
displacements of phosphate dianion on esters with leav-
ing thiol anions or oxyanions having pKa’s for their
conjugate acids of about 7 or less can occur readily. At
lower pH, when the catalytic agent becomes the phos-
phate monoanion, the mode of catalysis for these esters
changes to general base. This change in the mode of
catalysis indicates that the nucleophilic addition to the
carbonyl has a larger Brønsted coefficient than does the
general base-promoted reaction, a finding that is also
consistent with well-established principles for reactions
at ester carbonyl groups. For example, nucleophilic
addition of a variety of oxyanion and amine nucleophiles
to activated esters, such as p-NPA, have Brønsted
â-values of 0.66-0.92.30 The reactions of a series of
activated esters such as the ethyl chloroacetates and
various anhydrides with neutral oxygen nucleophiles
(water, alcohols) are subject to general base catalysis, but
here the Brønsted â-values are in the range of 0.47.31
for the phosphate monoanion at pH 3.05 is general base,
while that for the dianion at pH 8.5 is nucleophilic.
Con clu sion s
Despite the fact that phosphate is a commonly used
buffer and catalyzes numerous acyl transfer reactions,
the number of cases where it has been unambiguously
shown to be a nucleophile are surprisingly few. In the
above study we have shown that p-nitrophenyl thiolac-
etate (3b), and p-nitrophenyl thiolformate (5) react with
phosphate dianion in the neutral pH domain predomi-
nantly by a nucleophilic route. The study also shows that
phosphate dianion behaves as both a nucleophile and
general base toward phenyl thiolacetate (4b), the ratio
of the two roles being roughly 1:1 at 37.5 °C. O’Connor
and Wallace6 have suggested that the nucleophilic to
general base ratio for phosphate dianion reacting with
p-nitrophenyl acetate (3a ) at 37.5 °C is about 11:1 in D2O,
but could not refine the ratio further since the salt effect
of phosphate on the reaction was uncertain. Under
comparable conditions the reaction of phosphate dianion
with the oxygen/thiolester pairs of 3a or 3b and 4a or
Ack n ow led gm en t. The authors gratefully acknowl-
edge the financial support of the Natural Sciences and
Engineering Research Council of Canada and Queen’s
University. In addition they are grateful to the review-
ers of the manuscript for their helpful comments.
Su p p or tin g In for m a tion Ava ila ble: Tables of rate con-
stants for the hydrolysis of esters 3a ,b and 4a ,b in H2O, T )
37.5 °C, at various pH values as a function of [phosphate]total
(5 pages). This material is contained in libraries on microfiche,
immediately follows this article in the microfilm version of the
journal, and can be ordered from the ACS; see any current
masthead page for ordering information.
(28) The calculation is as follows: at the concentration of hydroxy-
lamine and substrate 5 used (7.9 × 10-3 and 3.76 × 10-3 M), 1 part of
formic acid and 2 parts of hydroxamic acid are produced at pH 3.05.
From the rate constants given in Table 6, at pH 3.05, 1 M phosphate,
no hydroxylamine, about 2/3 of the reaction goes via the phosphate
process. If all the reaction of phosphate is general base, then the
amount of formic acid is 3 parts, and the amount of hydroxamic acid
is 2 parts. If all the reaction is nucleophilic and the formyl phosphate
reacts quantitatively with hydroxyl amine, then the amount of formic
acid is 1 part and the amount of hydroxamic acid is 4 parts.
J O970904B
(29) J encks, W. P. In Catalysis in Chemistry and Enzymology;
McGraw-Hill: New York, 1969; pp 463-554.
(30) Bruice, T. C.; Benkovic, S. J . In Bioorganic Mechanisms;
Benjamin, Inc.: New York, 1966; Vol. I, p 43.
(31) J encks, W. P.; Carrioulo, J . J . Am. Chem. Soc. 1961, 83, 1743.