Heavy-atom isotope effects on the hydrazinolysis of methyl formate

Article abstract of DOI:10.1021/ja970993+

The carbonyl carbon, carbonyl oxygen, and nitrogen nucleophile isotope effects were measured for the hydrazinolysis of methyl formate at pH 8 and 10. At pH 8, where breakdown of a tetrahedral intermediate to products is rate-determining, the carbonyl carbon isotope effect is k¹²/k¹³ = 1.038, the carbonyl oxygen isotope effect is k¹⁶/k¹⁸ = 1.003, and the nitrogen nucleophile isotope effect is k¹⁴/k¹⁵ = 0.990. The isotope effects at pH 8 are consistent with a late transition state, which greatly resembles the hydrazide product. At pH 10, where formation of a tetrahedral intermediate is rate-determining, the carbonyl carbon isotope effect is k¹²/k¹³ = 1.020, the carbonyl oxygen isotope effect is k¹⁶/k¹⁸ = 1.004, and the nitrogen nucleophile isotope effect is k¹⁴/k¹⁵ = 0.9917. These isotope effects are best rationalized in terms of a concerted general base catalyzed nucleophilic attack of hydrazine on methyl formate as the rate-determining step.

Full text of DOI:10.1021/ja970993+

8838
J. Am. Chem. Soc. 1997, 119, 8838-8842
Heavy-Atom Isotope Effects on the Hydrazinolysis of Methyl
Formate
John F. Marlier,*,† Brandy A. Haptonstall,† Amanda J. Johnson,† and
Katherine A. Sacksteder‡
Contribution from the Department of Chemistry and Biochemistry,
California Polytechnic State UniVersity, San Luis Obispo, California 93407,
and Department of Chemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706
ReceiVed March 28, 1997X
Abstract: The carbonyl carbon, carbonyl oxygen, and nitrogen nucleophile isotope effects were measured for the
hydrazinolysis of methyl formate at pH 8 and 10. At pH 8, where breakdown of a tetrahedral intermediate to products
is rate-determining, the carbonyl carbon isotope effect is k12/k13 ) 1.038, the carbonyl oxygen isotope effect is
k16/k18 ) 1.003, and the nitrogen nucleophile isotope effect is k14/k15 ) 0.990. The isotope effects at pH 8 are
consistent with a late transition state, which greatly resembles the hydrazide product. At pH 10, where formation of
a tetrahedral intermediate is rate-determining, the carbonyl carbon isotope effect is k12/k13 ) 1.020, the carbonyl
oxygen isotope effect is k16/k18 ) 1.004, and the nitrogen nucleophile isotope effect is k14/k15 ) 0.9917. These
isotope effects are best rationalized in terms of a concerted general base catalyzed nucleophilic attack of hydrazine
on methyl formate as the rate-determining step.
Introduction
methoxyl oxygen isotope effect (k16/k18 ) 1.040) and a
considerable carbonyl oxygen isotope effect (k16/k18 ) 1.018).9
Assuming a similar mechanism for the hydrazinolysis of the
two esters, these results imply a slightly earlier transition state
for the methyl benzoate case, where the π bond order is not
completely restored to the carbonyl oxygen in the transition state.
At pH 10, the methoxyl oxygen isotope effect for methyl formate
fell to k16/k18 ) 1.005 and the formyl hydrogen isotope effect
became more inverse (kH/kD ) 0.72).7,8 These results are
consistent with a change in rate-determining step and with a
transition state that is largely tetrahedral. A comparison of the
transition states for methyl formate and methyl benzoate is not
possible at pH 10 because the isotope effects on the hydrazi-
nolysis of methyl benzoate at pH 10 were not reported.
In the present paper we present isotope effects for the carbonyl
carbon, carbonyl oxygen, and nitrogen nucleophile on the
hydrazinolysis of methyl formate. The results of these experi-
ments are used to more fully characterize the bonding for the
transition states at pH 8 and 10.
The reaction of alkyl esters with amines in aqueous solution
is subject to general base catalysis by a second molecule of
amine; a reaction not involving such catalysis occurs to a lesser
extent.1-3 Kinetic investigations of the hydrazinolysis of methyl
formate and other alkyl esters showed a change in rate-
determining step with varying pH.4 These results implied the
existence of tetrahedral addition intermediates. The most widely
accepted mechanism is the one proposed by Jencks,5 which is
given in eq 1. The rate-determining step is believed to change
from breakdown of T- (step 3) at pH 8 to general base catalyzed
formation of T- (step 2) at pH 10.5,6
O–
C
O–
C
+NH2NH2
O
C
k2[B]
k1
k3
H
OCH3 + NH2NH2
H
OCH3
H
OCH3
k–1
k–2[BH]
NHNH2
T±
T–
O
(1)
H
C NHNH2 + HOCH3
Experimental Section
Information about the transition state structures for the
Materials. Methyl formate, hydrazine hydrate, DMSO (anhydrous),
chloramine-T, Dowex 50 W ×8-100 resin, and formic hydrazide were
obtained from Aldrich Chemical Co. Iodine, sublimed, was from
Spectrum Chemical Manufacturing Co. HEPES and CAPS buffers were
from Sigma Chemical Co. All chemicals were of reagent grade or
better and were used without further purification.
Kinetics. The rates of reaction for hydrazinolysis at pH 8 and 10
were determined by following absorbance changes at 242 nm with a
Hitachi model U3210 spectrophotometer with a cell compartment that
was temperature equilibrated at 25 °C.7,8 To correct for the amount of
hydrolysis at pH 10, the total absorbance change at 242 nm was
measured for a reaction containing a large excess of hydrazine (where
no appreciable hydrolysis occurred). The percent of hydrolysis was
calculated from this absorbance change and the changes measured
during the actual isotope effect runs. The percent hydrolysis and the
known isotope effect on alkaline hydrolysis10 were then used to calculate
the isotope effect on hydrazinolysis.
hydrazinolysis of methyl formate and related alkyl esters was
obtained previously from isotope effect studies. For the
hydrazinolysis of methyl formate at pH 8 a large methoxyl
oxygen isotope effect (k16/k18 ) 1.062) together with a small
inverse formyl hydrogen isotope effect (kH/kD ) 0.98) were
consistent with a late transition state, largely resembling the
bonding in the hydrazide product.7,8 At the same pH, the
hydrazinolysis of methyl benzoate gave a slightly smaller
† California Polytechnic State University.
‡ University of Wisconsin.
X Abstract published in AdVance ACS Abstracts, August 15, 1997.
(1) Bannett, J. F.; Davis, G. T. J. Am. Chem. Soc. 1960, 82, 665.
(2) Jencks, W. P.; Carriuolo, J. J. Am. Chem. Soc. 1960, 82, 675.
(3) Bruice, T. C.; Mayahi, M. F. J. Am. Chem. Soc. 1960, 82, 3067.
(4) Blackburn, G. M.; Jencks, W. P. J. Am. Chem. Soc. 1968, 90, 2638.
(5) Satterthwait, A. C.; Jencks, W. P. J. Am. Chem. Soc. 1974, 96, 7018.
(6) Satterthwait, A. C.; Jencks, W. P. J. Am. Chem. Soc. 1974, 96, 7031.
(7) Sawayer, C. B.; Kirsch, J. F. J. Am. Chem. Soc. 1973, 95, 7375.
(8) Bilkadi, Z.; deLorimier, R.; Kirsch, J. F. J. Am. Chem. Soc. 1975,
97, 4317.
(9) O’Leary, M. H.; Marlier, J. F. J. Am. Chem. Soc. 1979, 101, 3300.
(10) Marlier, J. F. J. Am. Chem. Soc. 1993, 115, 5953.
S0002-7863(97)00993-1 CCC: $14.00 © 1997 American Chemical Society

Hydrazinolysis of Methyl Formate
J. Am. Chem. Soc., Vol. 119, No. 38, 1997 8839
Isotope ratios were determined on a Finnigan Delta-E isotope ratio
mass spectrometer. Isotopic compositions are given as δ(13C) for
carbon, δ(18O) for oxygen, and δ(15N) for nitrogen. The δ value
represents a per mil difference in the isotope ratio relative to a tank
standard.
Table 1. Carbonyl Carbon, Carbonyl Oxygen, and Nitrogen
Nucleophile Isotope Effects on the Hydrazinolysis of Methyl
Formate in Water at 25 °Ca,b
carbonyl C
isotope effectc
(k12/k13)
carbonyl O
isotope effect
(k16/k18)
N nucleophile
isotope effectd
(k14/k15)
Carbonyl Carbon and Carbonyl Oxygen Isotope Effect Proce-
dure. The procedure used was essentially that reported in an earlier
paper, but modified to allow analysis of residual substrate.10 The
reaction at pH 8 was initiated by addition of 1 mL of a 0.5 M aqueous
methyl formate solution to 22 mL of a solution containing 0.47-0.57
M total hydrazine at pH 8. The reaction at pH 10 was initiated by
addition of 1 mL of a 0.5 M methyl formate solution to 20 mL of a
solution that contained 0.04 M total hydrazine and 0.4 M total CAPS
buffer in a 100-mL round-bottom flask. An aliquot was withdrawn
for monitoring by the UV assay. At the appropriate fraction of reaction
(the fraction of reaction was varied between 0.46 and 0.73), 2.5 M
NaHSO4 was added to quench the reaction (pH 4). The contents of
the flask were distilled under vacuum, and the unreacted methyl formate
(plus some water) was collected into a 50-mL round-bottom flask
containing an excess of KOH. After complete saponification of the
ester, the flask was equipped with a sidearm containing a stopcock
that was capped at the end with a septum. A 1.0-mL sample of a 1 M
HEPES solution, pH 7, was added to the formate and the contents were
dried at 70 °C under high vacuum for 3 h. Finally, 2 mL of anhydrous
DMSO was added through the sidearm, followed by a solution of 200
mg of I2 in anhydrous DMSO. The reaction was stirred and the
resulting CO2 was collected immediately by vacuum distillation as it
was being formed. The isotopic composition of the starting material
(which is needed to determine the isotope effect) was determined by
quantitative hydrolysis of methyl formate in excess KOH, followed by
oxidation to CO2 in DMSO/I2.
The entire procedure was checked by several controls. A reaction
mixture that contained all reagents except methyl formate failed to
produce detectable amounts of CO2; similar results were obtained when
all the methyl formate was allowed to react with hydrazine. In a
separate experiment, sodium formate was incubated with water enriched
in 18O. This formate was then carried through the entire oxidation
procedure. The δ(18O) value for the resulting CO2 showed no detectable
exchange of the oxygen atoms with the enriched water. When methyl
formate of known isotopic composition was subjected to the entire
isolation procedure, the δ(13C) and δ(18O) remained unchanged.
Nitrogen Nucleophile Isotope Effect Procedure. The reaction was
initiated by adding an aqueous methyl formate solution to an equal
volume of a solution containing hydrazine and either HEPES buffer
(Li+-form) at pH 8 or CAPS buffer (Li+-form) at pH 10. All reaction
mixtures contained a molar excess of hydrazine to methyl formate
(varied from a 2:1 to a 1.25:1 ratio) so that the reaction was limited by
methyl formate. When all the methyl formate had reacted, the reaction
mixture was adjusted to pH 5.0 with 1 M lithium acetate buffer and
the unreacted hydrazine was isolated by ion exchange chromatography
on Dowex 50 resin (Li-form). The product, formic hydrazide, was
first washed off the column with H2O, followed by elution of the
hydrazine with 0.1 M LiOH (6.5-mL fractions). A 1-mL aliquot of
each fraction was titrated with chloramine-T to an iodide end point.11
This allowed determination of the amount of hydrazine in each fraction
and calculation of the fraction of hydrazine consumed in the reaction.
The pooled fractions of hydrazine were placed in a 250-mL round-
bottom flask containing 4 mL of 1.0 M HEPES, pH 7.1, to neutralize
the LiOH. This flask had a sidearm equipped with a stopcock and a
small vessel containing at least a 5-fold molar excess of aqueous
chloramine-T. Atmospheric N2 was removed by several freeze-thaws
and then the chloramine-T was added to the hydrazine solution. The
resulting N2 was collected under vacuum over 4 Å molecular sieves at
liquid N2 temperature, after first passing through one dry ice trap and
two liquid N2 traps. The N2 sample was then analyzed by isotope ratio
mass spectrometry. The concentrations of hydrazine and buffer
(HEPES or CAPS) were varied in some of the runs. No detectable
change in the isotope effect was observed.
pH
8.0 1.038 ( 0.001 (9) 1.003 ( 0.001 (9) 0.990 ( 0.001 (8)
10.0 1.020 ( 0.001 (5) 1.004 ( 0.001 (4) 0.9917 ( 0.0003 (6)
a All isotope effects corrected for percent reaction. b Numbers in
parentheses are the number of determinations. c Corrected for the
amount of alkaline hydrolysis (ref 10). d The isotope effect at pH 8 is
corrected for the equilibrium isotope effect of the pKa of hydrazine
(see text).
Results
The carbonyl carbon and carbonyl oxygen isotope effects for
hydrazinolysis of methyl formate were measured in aqueous
solution at pH 8 and 10 at 25 °C. The unreacted methyl formate
was collected, hydrolyzed to formate ion, and then oxidized to
CO2 by anhydrous DMSO-I2. The resulting CO2 was analyzed
by isotope ratio mass spectrometry. One of the oxygen atoms
of CO2 is derived from the carbonyl oxygen of methyl formate,
the other is from the solvent. The isotopic composition of the
latter oxygen does not change during alkaline hydrolysis because
the solvent is essentially an infinite pool of oxygen atoms. All
hydrolyses were performed by using a single source of 1 M
aqueous KOH that was tightly sealed between reactions. The
control experiments to test the reliability of the procedure are
described in the Experimental Section. The hydrazinolysis at
pH 10 was too rapid at high concentrations of hydrazine (>0.1
M) to allow accurate determination of the fraction of reaction
by the UV assay. Consequently, the concentration had to be
lowered and a significant amount of alkaline hydrolysis oc-
curred. The fraction of hydrolysis was determined for each run,
and the observed carbonyl carbon and carbonyl oxygen isotope
effects were corrected by using the published isotope effects
on alkaline hydrolysis.10 The isotope effects are summarized
in Table 1.
Nitrogen nucleophile isotope effects were determined at pH
8 and 10 under similar conditions to those for the carbonyl
carbon and carbonyl oxygen isotope effects. Both sets of
experiments involved analysis of residual hydrazine. The
unreacted total hydrazine (containing a mixture of hydrazine,
and its conjugate acid) was isolated and oxidized to N2 for
isotopic analysis. The concentration of hydrazine and
buffers (HEPES or CAPS) was varied in several runs, but no
change in the nitrogen isotope effect was detected. Controls
for this procedure are described in the Experimental Section.
The isotope effect at pH 8 had to be corrected for the equilibrium
isotope effect on the pKa of hydrazine. This equilibrium iso-
15
tope effect is technically difficult to measure so the
K
of
eq
1.0167 for protonation of phenylalanine (a primary amine) was
used as an approximate value.12 Because the masses of the
atoms attached to the nitrogen being protonated are similar
15
in both cases, the
K of a primary amine should serve as a
eq
good approximation for that of hydrazine. It should be
noted that substitution of a more electronegative atom (nitrogen)
for a carbon atom of a primary amine might have some
additional small effect on estimation of the 15Keq for hydrazine.
Calculation of the isotope ratio for free hydrazine was ac-
complished by using eq 2, where x1 is the [product]/[reactant]
ratio at equilibrium, y1 is the 15N/14N ratio of the product at
equilibrium, and y2 is the mass ratio of the initial reactant.12
This isotope ratio was subsequently converted to a δ(15N)
value.
As a control, hydrazine of known isotopic composition was subjected
to the isolation procedure. The resulting δ(15N) was unchanged. A
reaction mixture that contained all reagents except hydrazine failed to
yield detectable amounts of N2.
(12) Hermes, J. D.; Weiss, P. M.; Cleland, W. W. Biochemistry 1985,
24, 2959.
(11) Singh, B.; Sing, R. Anal. Chim. Acta 1954, 11, 408.

8840 J. Am. Chem. Soc., Vol. 119, No. 38, 1997
Marlier et al.
15
K
) y2/y1 + x1(y2/y1 - 1)
(2)
eq
This yielded a δ(15N) for free hydrazine of -10.4 at pH 8.0.
The measured δ(15N) in the residual substrate (total hydrazine)
did not change during the course of the reaction, remaining at
0.2. Since the δ(15N) did not change for the total pool of
hydrazine, the δ(15N) for the product must also be 0.2. Thus
the observed isotope effect must be (-10.4 - 0.2) or k14/k15
)
Figure 1. Summary of the isotope effects on the hydrazinolysis of
0.990. These isotope effects are also summarized in Table 1.
methyl formate. All isotope effects are given as klight/kheavy
.
Discussion
and proton removal occurred in a concerted fashion (transition
state structure II), then the carbonyl carbon isotope effect will
be a primary one. The question is as follows: Will a primary
and a secondary isotope effect be different enough to distinguish
between the two possibilities?
Although we are not aware of any direct models in the
literature for this equilibrium isotope effect, an inverse isotope
effect is clearly expected since bonding to the carbon in the
tetrahedral intermediate will be stiffer than that in the ground
state ester. There are at least two examples that allow a
reasonable approximation of the magnitude of this inverse
isotope effect. The first example is the equilibrium formation
of ethyl hydrazine from ethene (eq 3). In this model a C-C
The hydrazinolysis of methyl formate has been shown to
occur by a stepwise mechanism involving one or more
tetrahedral intermediates (eq 1). To more fully characterize the
transition state for this reaction, we have measured the carbonyl
carbon, carbonyl oxygen, and nitrogen nucleophile isotope
effects at pH 8 and 10. These results, as well as the earlier
methoxyl oxygen and formyl hydrogen isotope effects measured
by Kirsch et al.,7,8 are summarized in Figure 1. The isotope
effects are discussed within the framework of this stepwise
mechanism.
Isotope Effects at pH 10. A proton transfer step (step 2, eq
1) is reported to be rate-determining at pH 10; the transition
state for this step is shown as structure I.5,6 Taken together the
reported large inverse formyl hydrogen isotope effect8 and the
small normal methoxyl oxygen isotope effect7 (see Figure 1)
were offered as additional evidence for transition state structure
I. However, a very late (and nearly tetrahedral) transition state
for a concerted, general base-catalyzed formation of T-
(structure II) cannot be rigorously ruled out on the basis of these
reported isotope effects.
NH2NH2 + CH2dCH2 a CH3CH2NHNH2
(3)
double bond (instead of a C-O double bond) is replaced with
a C-N single bond. Although an alkene and a carbonyl group
have different polarities, the masses of the reacting atoms are
13
similar. The data used to estimate this
K
eq
have been
tabulated.14 The reasoning is as follows: The equilibrium
isotope effect for forming ethane from ethene is 13Keq ) 0.998.15
Replacement of a hydrogen on the isotopic carbon atom of
ethane with a heavy atom, such as nitrogen, decreases the value
to 0.983. Addition of a second nitrogen (to make the hydrazine)
will further reduce this equilibrium isotope effect to around
0.979. Since the carbonyl carbon isotope effect on the ionization
of benzoic acid is small (k12/k13 ) 1.001),16 protonation of the
inner nitrogen (effectively the difference between T- and T()
–δ
O–
C
O
O–δ
C
O–δ
C
–δ
OCH3
–δ
OCH3
H
OCH3
H
C
OCH3
H
H
+δ
NHNH2
B–δ
+δ NHNH2
NHNH2
+δ
NHNH2
H
–δ
–δ
B
H
B
H
I
II
III
IV
(a) Carbonyl Carbon Isotope Effects. The carbonyl carbon
isotope effect at pH 10 presented in this study is slightly smaller
(k12/k13 ) 1.020) than most of those reported for the reaction
of alkyl esters with basic nucleophiles (k12/k13 ) 1.03-
1.04).9,10,13 Interpretation of carbonyl carbon isotope effects for
many acyl group transfer reactions is difficult because of the
considerable reaction coordinate motion of this atom and
because carbonyl carbon isotope effects appear to be insensitive
to changes in the structure and the reactivity of esters.13
However, in the present study this carbonyl carbon isotope effect
provides crucial information on the transition state structure.
The small leaving group oxygen isotope effect and the large
inverse formyl hydrogen isotope effect (see Figure 1) argue for
a transition state that is largely sp3 hybridized, with very little
bond reduction to the leaving oxygen. Jencks contends that
the rate-determining step for the aminolysis of esters with poor
leaving groups (like methoxy) is a proton transfer from T( to
a general base. This conclusion can be tested for the hydrazi-
nolysis of methyl formate by the carbonyl carbon isotope effect.
If the rate-determining step is removal of a proton from T(, it
follows that T( must be in equilibrium with methyl formate
and hydrazine. Removal of a proton from the nitrogen of T(
will cause a negligible change in the observed carbonyl carbon
isotope effect. Therefore, the observed carbonyl carbon isotope
effect should arise solely from the equilibrium between T( and
methyl formate. On the other hand, if the nucleophilic addition
is not expected to significantly change the above carbon isotope
13
effect. Therefore,
K
) 0.979 is a rough estimate of the
eq
carbonyl carbon equilibrium isotope effect for formation of T(
(or T-) from methyl formate and hydrazine. The second
example is a theoretical calculation of the equilibrium carbonyl
carbon isotope effect for addition of hydroxide to acetaldehyde
(addition of a heavy atom to a carbonyl).17 This calculated
13
isotope effect is
K
) 0.962 at 25 °C. Both of the above
eq
examples clearly lead to the expectation of a large inverse
carbonyl carbon isotope effect for the equilibrium between
methyl formate and a tetrahedral species (either T- or T() of
between 2 and 4%. By contrast, a primary carbon isotope effect
for a concerted mechanism would be expected to be large and
normal, due to the considerable reaction coordinate motion
experienced by the carbonyl carbon. There are many examples
of this type of kinetic isotope effect in the literature; one
pertinent example is the carbonyl carbon isotope effect on the
alkaline hydrolysis of methyl formate (k12/k13 ) 1.034).10 In
this case carbonyl oxygen exchange and a small leaving group
isotope effect clearly established attack of the nucleophile on
the ester to be rate-determining.7 The present carbonyl carbon
(14) Cleland, W. W. In Methods Enzymol. 1980, 64, 104. Especially
pertinent are the rules in footnote r on page 110.
(15) Hartshorn, S. R.; Shiner, V. J. J. Am. Chem. Soc. 1972, 94, 9002.
(16) Bayles, J. W.; Bron, J.; Paul, S. O. J. Chem. Soc., Faraday Trans.
1 1976, 7, 1546.
(17) Hogg, J. L.; Rodgers, J.; Kovach, I.; Schowen, R. L. J. Am. Chem.
Soc. 1980, 102, 79.
(13) Marlier, J. F.; O’Leary, M. H. J. Am. Chem. Soc. 1990, 112, 5996.

Hydrazinolysis of Methyl Formate
J. Am. Chem. Soc., Vol. 119, No. 38, 1997 8841
isotope effect on the hydrazinolysis of methyl formate at pH
10 is normal and rather large, making it impossible that methyl
formate and T( are in equilibrium, thereby requiring a concerted,
general base mechanism for this reaction. The observation of
general base catalysis1-3 and a large primary isotope effect argue
strongly in favor of a mechanism involving a concerted, general
base catalyzed formation of T- (transition state structure II) as
the rate-determining step. In addition, the large formyl hydrogen
isotope effect8 (Figure 1) indicates this transition state is rather
late and sp3-like.
state (an inverse isotope effect) and will counter the normal
isotope effect for breaking the carbonyl π bond, leading to an
observed effect that is smaller than expected. This stiffening
is more important in methyl formate than in higher molecular
weight esters such as methyl benzoate. Methyl formate does
not have the C-C-C-O torsional mode present in methyl
benzoate, and the H-C-O bending mode of methyl formate is
less 18O sensitive than the C-C-O mode present in methyl
benzoate. Empirical data seem to fit this explanation; the
carbonyl oxygen isotope effects on the alkaline hydrolysis of
methyl benzoate9 (k16/k18 ) 1.0046), p-nitrophenyl acetate18 (k16/
k18 ) 1.0039), and methyl formate10 (k16/k18 ) 0.999) decrease
in the direction predicted. (2) The transition states for all these
reactions may occur either very early during formation of the
tetrahedral intermediate or very late during breakdown of a
tetrahedral species. Both cases would give a transition state
structure containing a large degree of sp2 character (like the
starting ester or amide product) and should yield a small oxygen
isotope effect. This may explain the small isotope effects for
alkaline hydrolysis, but in the present case, the large inverse
formyl hydrogen isotope effect argues for a transition state that
contains considerable sp3 character. (3) The anionic oxygen in
the transition state is more strongly solvated than the ground
state, and this increased hydrogen bonding in the transition state
lowers the observed isotope effect. Several models that support
this conclusion include the vapor pressure isotope effect for
water (1.0091),20 the inverse carbonyl oxygen isotope effect on
Can this conclusion be extended to include the aminolysis
of other esters? The concerted mechanism will occur whenever
T( does not have a finite lifetime. Jencks has postulated that
a concerted mechanism is possible for catalysis by strong bases
of weakly basic amines (giving a T( with a relatively low pKa)
or for esters containing very good leaving groups.5 Because
methyl formate is such a reactive ester (making it necessary to
have a low level of hydrazine to follow the reaction), the present
carbonyl carbon isotope effect experiments at pH 10 employed
a ratio of total buffer (CAPS) to total hydrazine of 10/1. This
makes it likely that CAPS (pKa ) 10.4), rather than hydrazine
(pKa ) 8.3), served as the general base in the reaction.
Therefore, these conditions involve catalysis by a relatively
strong base (CAPS) of the attack on methyl formate by weakly
basic (but highly nucleophilic) hdyrazine. In fact, the pKa of
the nitrogen of T( has been estimated5 to be near 9.6, making
proton transfer from T( to CAPS thermodynamically favorable.
This would not be true when hydrazine is acting as a general
base. The experimental conditions employed by Jencks and
co-workers at or above pH 10 had a ratio of total buffer to total
hydrazine that was 10 to 30 times lower than that used in our
experiments. Our conditions are much closer to those outlined
above where a concerted mechanism becomes possible. There-
fore, at this time it is not possible to rigorously extend the
concerted mechanism to the acetate esters studied by Jencks
without further isotope effect studies.
(b) Carbonyl Oxygen Isotope Effect. The measured car-
bonyl oxygen isotope effect (k16/k18 ) 1.004) is a secondary
kinetic isotope effect because the connection between the carbon
and the oxygen is not severed during the reaction. The largest
measured carbonyl oxygen isotope effects reported in the
literature are those for the hydrazinolysis of methyl benzoate9
at pH 8 (k16/k18 ) 1.018) and the reaction of sulfur anions with
p-nitrophenyl acetate18 (k16/k18 ) 1.012). In contrast, primary
oxygen isotope effects (where an O-C σ bond is completely
severed) can be much larger. For example, the methoxyl oxygen
isotope effect on the hydrazinolysis of methyl formate7 at pH 8
is k16/k18 ) 1.062. This is considerably smaller than the
theoretical maximum of k16/k18 ) 1.19 calculated by Biegeleisen
and Wolfsberg19 for a primary oxygen isotope effect. The
corresponding theoretical maximum for the secondary isotope
effect on breaking a carbonyl π bond is not known, but may be
close to that calculated for the addition of hydroxide to
acetaldehyde (k16/k18 ) 1.03).17
the acid-catalyzed hydrolysis of methyl benzoate (k16/k18
)
0.995),21 and the estimated isotope effect on desolvation of the
carboxyl group (k16/k18 ) 1.01-1.02) during decarboxylations.22
Nitrogen Isotope Effect. Isotope effects on entering nu-
cleophiles, like all primary isotope effects, are composed of two
principal factorssthe temperature-independent factor (TIF) and
the temperature-dependent factor (TDF).23 The TIF is always
normal, whereas the TDF is normal when bonds are being
broken to the isotopic atom in the transition state and inverse
when these bonds are being formed. This usually makes
interpretation of nucleophile isotope effects somewhat complex.
As outlined in the preceding sections, the most likely
transition state for hydrazinolysis at pH 10 is one like that shown
in structure II. Furthermore, this transition state is late and sp3-
like. Although nearly all observed primary kinetic isotope
effects for bond formation are normal (they are dominated by
reaction coordinate motion), calculations suggest that as the
transition state occurs later (i.e. greater bond formation to the
nucleophile) the observed isotope effect will tend to become
smaller.17,24 In the hydrazinolysis reaction the nucleophilic
nitrogen gains a new C-N bond but simultaneously loses a
N-H bond. As a result it is reasonable to expect the primary
nitrogen isotope effect to be a small normal one. The outer, or
nonnucleophilic, nitrogen experiences additional N-N-C bend-
ing and N-N-C-O torsional modes in the transition state
(structure II) and will show an inverse secondary isotope effect
(perhaps as large as 0.5-1.0%). Since both the nucleophilic
and outer nitrogens are analyzed together, this secondary isotope
effect will make the observed isotope effect more inverse.
Three reasons have been given to account for the lesser
magnitude of the secondary carbonyl oxygen isotope effects:10
(1) The breaking of a π bond to oxygen will inherently give
smaller isotope effects than breaking a σ bond. A more detailed
explanation for the hydrazinolysis of methyl formate involves
a qualitative analysis of vibrational modes in the ground state
and transition state for this reaction. Formation of a new C-N
bond adds bending (O-C-N) and torsional (O-C-N-N)
modes in the transition state which are not present in the ground
state. This will serve to stiffen the bonding in the transition
Isotope Effects at pH 8. The reported large methoxyl
oxygen isotope effect7 (Figure 1) indicates considerable C-O
bond breaking in the transition state. The small formyl hydrogen
isotope effect8 (Figure 1) is consistent with a late transition state
(20) Szapiro, S.; Steckel, F. Trans. Faraday Soc. 1967, 63, 883.
(21) Marlier, J. F.; O’Leary, M. H. J. Org. Chem. 1981, 46, 2175.
(22) Headley, G. W.; O’Leary, M. H. J. Am. Chem. Soc. 1990, 112,
1894.
(23) Melander, L. Isotope Effects on Reaction Rates; Ronald Press: New
York, 1960.
(24) Paneth, P.; O’Leary, M. H. J. Am. Chem. Soc. 1991, 113, 1691.
(18) Hengge, A. C.; Hess, R. A. J. Am. Chem. Soc. 1994, 116, 11256.
(19) Bigeleisen, J.; Wolfsberg, M. AdV. Chem. Phys. 1958, 1, 15.

8842 J. Am. Chem. Soc., Vol. 119, No. 38, 1997
Marlier et al.
in which the carbonyl carbon has considerable sp2 hybridization
(like that of the hydrazide product). Thus, at pH 8 the formation
of products (i.e. decomposition of a tetrahedral intermediate)
is clearly rate-determining. The following isotope effects are
rationalized within this framework.
librium isotope effect in two ways. First, the equilibrium isotope
effect for protonation of a primary amine such as phenylalanine
15
15
is
K
eq
) 1.0167 and that for ammonia is
K
eq
) 1.0192.12
By analogy deprotonation of the secondary amine in T( should
slightly increase the equilibrium isotope effect for formation
of T- by about the same 0.2%, giving 15Keq ) 0.986. Second,
the outer nitrogen will make this value even lower by ap-
proximately 0.5-1.0% because of the aforementioned gain of
one new bending and one new torsional mode. Therefore, one
would reasonably expect the equilibrium isotope effect for
formation of T- to be in the range 15Keq ) 0.976-0.981. The
nitrogen kinetic isotope effect on decomposition of T- (transi-
tion state structure III) is a secondary one resulting from the
weakening of one bending mode (N-C-O) and two torsional
modes (N-C-O-C and N-N-C-O) in the transition state
as a result of breaking the bond to the leaving group. This loss
of three vibrational modes will give a normal isotope effect,
which will cause the observed isotope effect to be larger (more
(a) Carbonyl Carbon Isotope Effect. The measured car-
bonyl carbon isotope effect (Figure 1) is similar to that reported
for methyl benzoate at the same pH (k12/k13 ) 1.040).9
Assuming that breakdown of the tetrahedral species T- to
products is fully rate-determining, the observed isotope effect
is composed of an equilibrium isotope effect for formation of
T- and a kinetic isotope effect on its decomposition. If proton
transfer from the nitrogen atom has a negligible effect on this
equilibrium carbon isotope effect, it follows that the equilibrium
isotope effect for formation of T- should be near the 0.979
inverse value calculated earlier for T( (see discussion for pH
10). Consequently, the carbonyl carbon kinetic isotope effect
on the decomposition of T- can be estimated to be on the order
of k12/k13 ) 1.06. If the product forming step is not fully rate-
determining, the estimated kinetic isotope effect on decomposi-
tion of T- will be somewhat smaller.
normal) than the above estimated equilibrium isotope effect of
15
K
eq
) 0.976-0.981. Seen in this light the observed isotope
effect of k14/k15 ) 0.990 is a reasonable result.
The nitrogen isotope effect (as well as the other isotope
effects) is consistent with a second mechanism in which
deprotonation of the inner nitrogen of T( occurs simultaneously
with bond breaking to the leaving group (transition state
structure IV). This is possible because any general base species
present at pH 8 may not be strong enough to remove the proton
from T(. However, as methoxide begins to leave the amide
nitrogen will become more acidic making removal of the proton
more facile. If this is the case, we must estimate the equilibrium
nitrogen isotope effect on the formation of T( (instead of T-)
from hydrazine. This isotope effect will be lower than that
estimated above for the formation of T- (15Keq ) 0.976-0.981)
due to the proton on the inner nitrogen. Once again we can
employ the isotope effect for protonation of the amino group
of phenylalanine as a model.12 Protonation will lower the
(b) Carbonyl Oxygen Isotope Effect. Although there is
strong evidence for a change in the rate-determining step on
going from pH 8 to 10, the carbonyl oxygen isotope effect does
not exhibit a significant change (see Figure 1). Once again,
the stronger solvation of the anionic oxygen in the transition
state as compared to the ground state may partly account for
these similar, small isotope effects. Interestingly, the carbonyl
oxygen isotope effect for hydrazinolysis of methyl benzoate at
pH 8 is significantly larger (k16/k18 ) 1.018)9 than that for methyl
formate. As pointed out earlier, this trend toward larger
carbonyl oxygen isotope effects for methyl benzoate is also true
for other reactions like alkaline hydrolysis, where k16/k18
)
1.0046 for methyl benzoate and k16/k18 ) 0.999 for methyl
formate. The reason for this trend seems to be the additional
bending (O-C-N) and torsional (O-C-N-N) modes present
in the transition state but not in the ground state. As mentioned
previously, methyl benzoate already has similar bending and
torsional modes to the benzene ring making the effect of adding
new ones to the incoming nucleophile proportionally smaller
than in the methyl formate case.
equilibrium isotope effect on formation of T( to approximately
15
K
eq
) 0.959-0.964. If we assume product formation is fully
rate-determining and an equilibrium isotope effect on formation
of T( near
K ) 0.96, the kinetic isotope effect for product
eq
15
formation (including deprotonation) must be approximately k14/
k15 ) 1.03 for this alternative mechanism.
(c) Nitrogen Isotope Effects. At pH 8 the conjugate acid
of hydrazine (pKa 8.3) is the dominant molecular species, and
the equilibrium nitrogen isotope effect on this pKa must be
considered. This equilibrium isotope effect is technically
difficult to measure but should be similar to that for a primary
amine such as phenylalanine (15Keq ) 1.0167).12 Correction
of the observed nitrogen isotope effect for this equilibrium (using
the phenylalanine model) gives an overall isotope effect of k14/
k15 ) 0.990 (see Results section) for formation of the hydrazide
product from free hydrazine.
Summary
The isotope effects presented here complete a study of all
the reacting atoms in the hydrazinolysis of methyl formate and
provide further refinement of the transition state structures.
Strong evidence is offered in support of a concerted, general
base catalyzed formation of a tetrahedral intermediate (T-) as
the rate-determining step at pH 10. This differs from the rate-
determining proton transfer step proposed earlier for other
aliphatic esters.5,6 At pH 8 the isotope effects are consistent
with rate-determining breakdown of an anionic tetrahedral
intermediate (T-) to products, as reported in the literature5,6 or
with a concerted, general base catalyzed decomposition of T(
to products.
If the product-forming step is fully rate-determining, the
observed nitrogen isotope effect would be composed of an
equilibrium isotope effect on tetrahedral intermediate formation
and a kinetic isotope effect on its breakdown. Assuming for
the moment that T- is the only tetrahedral intermediate that
leads directly to products,5 the equilibrium isotope effect for
its formation can be estimated by utilizing the known equilib-
rium isotope effect for formation of phenylalanine (-NH2 form)
from free NH3 and cinnamate (15Keq ) 0.984).12 This model
involves replacement of a hydrogen of ammonia with a
secondary carbon atom from phenylalanine, whereas the forma-
tion of T- involves replacement of a hydrogen of hydrazine
with a carbon containing two heteroatoms (each with nearly
the same mass as carbon). The presence of the outer nitrogen
on hydrazine (versus ammonia) will alter the estimated equi-
Acknowledgment. I am grateful to Professor W. W. Cleland
(GM18938) of the University of WisconsinsMadison for
helpful discussions and for providing summer laboratory space
and supplies. Additionally, I wish to thank Professor Marion
H. O’Leary of Sacramento State University for useful comments
on this work and Isabel Treichel of the University of
WisconsinsMadison for help with mass spectral analysis.
JA970993+