Acid-Catalyzed Hydrolysis of Formamide
carbonyl-O and carbonyl-C KIEs (13δ ) -6.3) was determined
previously.6 Isotope ratios for carbon, nitrogen, and oxygen in
heavy-atom isotope effects were measured on an isotope ratio mass
spectrometer. The m/z ) 46/45 and 47/45 of formamide for the
measurement of the formyl-H KIE and carbonyl-O exchange were
determined on a GC-MS with an XT-1 nonpolar column. Proton-
decoupled 15N NMR spectra (40 MHz) of formamide in water and
in HCl were taken at 300 K; the D2O lock solvent resided in a
coaxial insert. The spectral width was 20.3 kHz in 32 K data points
and 128 scans. The acquisition time was 0.40 s, and the relaxation
delay was 3 s. Formamide (0.4 N) samples were prepared by the
addition of 15N formamide (8.1 uL, 0.2 mol) to H2O and to seven
different concentrations of HCl (aq; 0.2, 5, 1, 3, 6, 9, and 12 N).
15N NMR spectra were taken immediately (<2 min) after the
addition and mixing.
Nessler’s reagent) in fractions 1-6. Enough 10 M NaOH was added
to the pooled fractions until the concentration was 1 M, and the
hydrolysis to formate was allowed to proceed overnight. A 0.156-g
sample of MES (H+ form) was added to this hydrolysis mixture,
followed by the addition of 2.0 M HCl until the pH was near 6.0.
The neutralized solution was then reduced to a total volume of about
2 mL by rotary evaporation and placed in a 100-mL round-bottom
flask equipped with two stopcocks. One stopcock was on a sidearm
that was capped with a septum. The second stopcock was for
attachment to the high vacuum line. The solution was dried under
high vacuum at 70 °C overnight. While under vacuum, 2 mL of
anhydrous DMSO containing 250 mg of I2 was added via a syringe
through the sidearm to the dried formate, and the resulting CO2
was collected into a liquid nitrogen trap, as previously described.6
Isotopic analysis gave the δ for both the oxygen atom and the
carbon atom. There are two oxygen atoms in formate, one derived
from the solvent and one from the nucleophile. The observed per
mil isotopic abundance, 18δ(obs), is related to that for the carbonyl,
Determination of the Fraction of Reaction. The fraction of
reaction for the formyl-H KIE was determined via a formate
dehydrogenase assay. (a) Typically, a 50-µL aliquot of the quenched
reaction mixture was added to 2000 µL of 0.10 M phosphate buffer
at pH 7.6. A 50-µL aliquot of this diluted solution was added to a
solution composed of 100 µL of 15 mM NAD+ and 650 µL of the
same phosphate buffer in a 1 mL quartz glass cuvette. The reaction
was initiated by the addition of 200 µL of a formate dehydrogenase
solution (∼50 U/mL in the phosphate buffer), and the increase in
absorbance at 340 nm was determined. Formamide was not reactive
under these conditions. (b) To determine total formate plus
formamide present at quench, a 50-µL aliquot of the quenched
reaction mixture was mixed with 250 µL of 1.0 M NaOH and
allowed to react for 1 h. A 150-µL aliquot of this reaction mixture
was then added to 100 µL of 1.0 M HCl and 2000 µL of the
phosphate buffer. A 50-µL aliquot was then assayed via formate
dehydrogenase, as described above. The fraction of the reaction
was the µmol of formate from (a) divided by the total µmol of
formate plus formamide from (b).
18δ(CO), and the nucleophile, 18δ(NU), as shown in eq 8. The
18
magnitude of
δ
does not change during the course of the
(NU)
reaction, because the nucleophile (water, 56 M) is in huge excess
over formamide. Thus 18
δ
(CO) can be calculated from eq 9. Because
18
the actual magnitude of
δ
is not known, only an apparent
(NU)
18
18
δ
can be calculated from eq 10. This apparent
δ
is
(CO)
(CO)
displaced from the true value by the same constant (eq 9), where
C ) (0.5)(18δ(NU))/0.5. The value of the constant is not important
because the magnitude of the isotope effect depends only on the
18
difference between the apparent
δ
for the low and high
(CO)
conversion samples. As a result, the apparent 18
in the KIE calculations.
δ(CO) values are used
18
δ
) (0.5)18δ(CO) + (0.5)(18δ(NU)
)
(8)
(obs)
18
δ
) [18δ(obs) - (0.5)(18δ(NU))]/0.5 ) 18δ(obs)/0.5 - C (9)
The fraction of the reaction for the carbonyl-C, carbonyl-O, and
leaving-N KIEs was determined using Nessler’s reagent. (a) For
the carbonyl-C and carbonyl-O KIEs, a 40-µL aliquot of each
fraction obtained from the ion-exchange column containing only
unreacted formamide (see below) was added to 160 µL of 1.0 M
NaOH and reacted for 1 h. Next, a 100-µL aliquot of the above
was added to 800 µL of water and 100 µL of Nessler’s reagent.
The absorbance at 425 nm was then determined for each fraction,
and the total number of µmol of NH3 was calculated from a standard
curve. (b) For the leaving-N KIE, a 25-µL aliquot of fractions from
the ion-exchange column that contained only NH3 produced during
hydrolysis was added to 875 µL of water and 100 µL of Nessler’s
reagent. (c) The total number of µmol of nitrogen in each reaction
mixture (NH3 plus unreacted formamide) was determined by
alkaline hydrolysis of an aliquot obtained at quench but prior to
addition to the ion-exchange column, followed by analysis with
Nessler’s reagent. For the carbonyl-O and carbonyl-C KIEs, the
fraction of reaction was the µmol of NH3 from (a) divided by the
total µmol of NH3 from (c). For the leaving-N KIE, the fraction of
reaction was the ratio of the µmol of NH3 from (b) divided by the
total µmol of NH3 from (c) subtracted from 1.
(CO)
18
apparent
δ
)
18δ(obs)/0.5
(10)
(CO)
Nitrogen Isotope-Effect Procedure. The acidic hydrolysis and
quenching was carried out as described above. The quenched
reaction mixture was applied to a column composed of 7 mL of
strong cation-exchange resin (in the Na+ form). The column was
eluted first with water (8 fraction, 4 mL each), followed by 1 M
NaCl (8 fractions, 8 mL each). The early fractions contained
unreacted formamide; the later ones contained the product, am-
monium ion. The unreacted formamide was hydrolyzed in 1 M
NaOH for 2 h, then neutralized with H2SO4. This solution was then
made basic with NaOH and steam distilled into 0.1 M H2SO4. The
volume of the steam-distilled solution was reduced to about 2 mL
by rotary evaporation and oxidized with NaOBr to N2. The N2 was
analyzed by isotope ratio mass spectrometry. The ammonium ion
isolated by ion exchange chromatography was treated in a similar
manner, except no alkaline hydrolysis was needed.
Formyl-Hydrogen Isotope-Effect and PIX Procedures. The
formyl-H KIE and PIX were measured by using a 1:1 mixture of
1-d-formamide to 1-h-formamide. The acidic hydrolysis and
quenching was carried out as described above. The unreacted
formamide was isolated by repeated extraction with ethyl acetate.9
The ethyl acetate solution was dried over anhydrous sodium
carbonate and filtered, and the ethyl acetate was removed by rotary
evaporation. The isolated formamide was then dissolved in methanol
(as a carrier), and the appropriate ratio (m/z ) 46/45 or 47/45) was
directly measured by GC-MS without prior oxidation.
Carbonyl-Carbon and Carbonyl-Oxygen Isotope-Effect Pro-
cedures. A solution containing 500 µL of water and 16 µL of
formamide (400 µmol) was incubated at 25 °C. Then a 500-µL
aliquot of a 2.0 M HCl solution was added with stirring. At a
designated time, the reaction was quenched with 1.0 M NaOH. The
resulting pH was between 5 and 7. Control experiments show that
there was negligible further reaction under these conditions. A 1.2-
mL aliquot of the quenched solution was applied to a mixed bed
resin containing 5 mL of strong cation-exchange resin (Na+ form)
plus 10 mL of strong anion-exchange resin (acetate form). The
column was eluted first with water (8 fractions, 4 mL each).
Formamide was shown to elute (via hydrolysis, followed by
Determination of the 18δ for Water. A small sample of CO2
(<100 µmol) was added to an evacuated round-bottom flask
J. Org. Chem, Vol. 71, No. 10, 2006 3835