THE JOURNAL OF BIOLO~CAL CHEMISTRY

\“ol. 241, No. 24, Issue of December 25, pp. 5851-5863, 1966
Printed in U.S.A.

The Mechanism of the Condensation of Formaldehyde

with Tetrahydrofolic Acid*
(Received for publication, May 27, 19GG)

ROLAND G. KALLEN~ AND WILLIAM P. JENCKS

From the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts02154

SUMMARY ene-THFi (I), although methylene-THF is not necessarily the

The bell-shaped pH rate profile for the reaction of tetra-
hydrofolic acid with formaldehyde to form 5, lo-methylene
tetrahydrofolic acid has been shown to result from a change
in rate-determining step with changing acidity. In alkaline

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solution, the rate-determining step is the acid-catalyzed dehy-
dration of hydroxymethyl tetrahydrofolic acid and its conju- coo-
gate base. This step is subject to general acid catalysis with
a Bronsted slope, cy, of approximately 0.75. In acid solution, I
attack of tetrahydrofolic acid on formaldehyde is rate-determi- species that is active in eneymic reactions (3-8). A similar
ning. This step is subject to catalysis by general acids, in- reaction occurs with THF and glyoxalate (9). The experi-
cluding the solvated proton, with a Bronsted slope of 0.20. ments reported here were undertaken in an attempt to gain a
At pH values above 4, the reaction is inhibited by thiols, more detailed understanding of the mechanism of this reaction
which combine with formaldehyde in a base-catalyzed reac- and its relationship to other reactions of carbonyl compounds
tion to form hemithioacetals. Depending on the reaction with amines (10). A preliminary account of this work has
conditions, this inhibition can result from a lowering of the appeared (11).
equilibrium concentration of formaldehyde or from a trapping
EXPERIMENTAL PROCEDURE
by thiol anion of unhydrated formaldehyde, as it is formed
from formaldehyde hydrate. The equilibrium constant for Materials and methods were the same as those described in
hemithioacetal formation from formaldehyde and mercapto- the preceding paper. The concentration of mercaptoethanol in
ethanol is approximately 620 M-‘. the THF sample from Nutritional Biochemicals, which was
Secondary amines such as morpholine and imidazole cata- used in some of the early experiments, was not sufficient to
lyze the reaction of tetrahydrofolic acid with formaldehyde by interfere with the kinetic results after dilution I:5000 in the
nucleophilic catalysis by a pathway which must involve the reaction mixtures. THF was diluted daily into deoxygenated
intermediate formation of cationic imines. glass-distilled water which contained 1O-3 to lo-* M ethylene-
\ diaminetetraacetic acid. There was no significant difference in
N+=CHz the kinetic results obtained with the different THF preparations.
Reagent grade formaldehyde was obtained from Fisher in 36.6
to 37.2 s solutions which contained 10 to 11 y0 methanol. This
solution was diluted to a 1.0 M stock solution and was further
diluted to 0.025 M every 1 to 2 days. The formaldehyde con-
The nonensymic condensation of formaldehyde with tetra- centration was determined frequently by iodometric titration
hydrofolic acid occurs rapidly in aqueous solution and exhibits a (12, 13). Amines were redistilled or converted to the hydro-
bell-shaped pH rate profile with a maximum at about pH 4.2 and chlorides and recrystallized; imidazole was recrystallized as the
inflection points at pH 3.2 and 5.2 in the presence of 2-mercap- free base. Reagent grade formic acid, substitut,ed acetic acids,
toethanol (14). The product of this reaction is 5,10-methyl- 2-mercaptoethanol, ethanethiol, mercaptoacetic acid, and in-
organic salts were used without further purificat,ion.
* Contribution 456 from the Graduate Department of Bio- Reaction mixtures for kinetic measurements (3.0 ml) were
chemistry, Brandeis University. Supported by grants from equilibrated in a water bath at 25 f 0.1” and were deoxygenated
the National Science Foundation and the National Institute of by the passage of argon or prepurified nitrogen through the solu-
Child Health and Human Development of the Public Health Serv-
ice (HD-01247). This work was carried out bv R. G. K. durine tion for 5 to 10 min before use. It was shown that no detectable
the ‘tenure of’ a Special Fellowship from the National Cancey loss of formaldehyde occurred from a 0.1 M solution through
Institute of the National Institutes of Health (CA-16,912). which argon was passed for 1 hour. The reaction was started
2 Present address, Department of Biochemistry, University of
Pennsylvania, Philadelphia, Pennsylvania. i The abbreviation used is: THF, tetrahydrofolic acid.

5851
 Reaction of Formaldehyde with Tetrahydrofolic Acid Vol. 241, No. 24

by the addition of THF or formaldehyde from a pipette, and the
solution was mixed with a stream of argon or by inversion.
Spectrophotometric readings were made with a Zeiss PM& II
spectrophotometer equipped with a cell holder maintained
25” after 5 or 10 set and every 23 t,o 5 set thereafter for 5 to
10 half-times, usually with about 15 readings. The end points
were stable, except at alkaline pH at which a slow decomposition
with cuvettes in which the reactants were initially separated by
a partition. After the absorbance of the starting material was
determined, the reactants were mixed and the absorbance was
at followed as a function of time against a blank of p-nitrobenzoic
acid.
RESULTS

of the product was observed after 5 to 10 half-times. At pH 1 The rate of condensation of THF with a large molar excess of
and 7, the spectra of the product agreed with published spectra formaldehyde, measured spectrophotometrically at 290 rnp, is
for enzymically active Ns, NlO-methylene-THF prepared from first order in respect to THF in a given experiment and the
formaldehyde and THF and by the reduction of Ng,Nlo-meth- pseudo first order rate constants were found to increase linearly
enyl-THF with sodium borohydride (3). The experiments were with increasing formaldehyde concentration in the range 0 to
performed at concentrations of formaldehyde and other reagents 0.0025 M at pH 2.33, 3.09, and 4.00 and 0 to 0.00167 M at pH
that were in great excess over THF and pseudo. first order kinetics 5.60 and 7.00, which indicates that the reaction is also first order
were observed unless otherwise noted. The ionic strength was in respect to formaldehyde. In the acid region, and to a lesser
maintained at 1.0 M with potassium chloride. extent in the alkaline region, catalysis by 0.1 M buffers contributes
Pseudo first order rate constants were obtained from the re- significantly to the observed rate. The rate constants in the
lation kobs = 0.693/@ and tt, the half-time of the reaction, was absence of buffer catalysis were obtained by measuring the rates
obtained from linear plots of log (A, - A J against time, in which in duplicate at four to five buffer concentrations at each of a
A, and At are the absorbance at 290 rnp at the end of the reac- series of pH values and extrapolating to zero buffer concentration
tion and at a given time, respectively. The observed changes at each pH. The data obtained with acetate buffers at three

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in absorbance varied with pH from about 0.30 at pH 2 to 4 to pH values are shown in Fig. 1 as an example. In this figure,
about 0.15 at pH 7 and above, with a total absorbance of about the observed pseudo first order rate constants are converted to
1.5 with 5 X 10-S M THF. Readings were made against a second order rate constants for the free base form of THF by
blank cuvette that contained sufficient p-nitrobenzoic acid to give dividing by the formaldehyde concentration and by 01, the frac-
an absorbance of about 1.0 at 290 rnp and required slit widths of tion of THF (pK’ 4.82) present as the free base at each pH value.
0.5 mm or less. The reaction was shown to proceed to comple- The observed pseudo first order rate constants for the reaction
tion by increasing the formaldehyde concentration 4- to 5-fold; of THF with 0.00167 M formaldehyde, extrapolated to zero
the same change in absorbance was observed at all formalde- buffer concentration, exhibit a bell-shaped dependence on pH
hyde concentrations (by extrapolation of the first order plots to both in the presence (lower curve) and the absence (upper curve)
zero time), except in the presence of concentrated formaldehyde of 0.00267 M mercaptoethanol (Fig. 2). The lower curve is
at neutral or alkaline pH in which an “initial burst” of absorb- similar to that obtained by Osborn, Talbert, and Huennekens
ance change was observed. (3) in the presence of approximately the same concentration of
The spectrophotometric measurements of THF in the presence mercaptoethanol, and has a maximum at pH 4.6 with inflection
of concentrated formaldehyde shown in Figs. 6 and 7 were made points at pH 4.0 and 5.2. These reactions were initiated by the
addition of formaldehyde to the reaction mixture. However,
I I if the reaction is carried out in the absence of mercaptoethanol
and the THF is protected from oxidation by argon and ethyl-
enediaminetetraacetic acid, the observed rate constants above
pH 4 are considerably larger and the maximum and inflection
500
points of the pH rate profile occur at higher pH values (uppe?
curve).
The inhibitory effect of mercaptoethanol on the rate is shown
as a function of mercaptoethanol concentration at various pH
T values in Fig. 3. At pH 3.09, mercaptoethanol has little or no
:
-* effect, at pH 4.00 it causes 50% inhibition at 0.02 M, and at
300
'5 pH 5.58 the observed rate constants approach zero with in-
r, creasing mercaptoethanol concentration. This inhibition is
7 attributed to a base-catalyzed reaction of formaldehyde with
'Ei mercaptoethanol to form the unreactive hemithioacetal
T
(12, 14, 15), which decreases the concentration of formaldehyde
.z /
2 100 available for reaction with THF. Similar inhibition was ob-
served with mercaptoacetic acid and ethanethiol, which shows
that the phenomenon is a general one and does not require a
I I I
neighboring hydroxyl group.
0.05 0.10 M At pH 6.57, the inhibition is less if the formaldehyde is pre-
TOTAL BUFFER CONCENTRATION incubated with mercaptoethanol than if the reaction is initiated
by the addition of formaldehyde (Fig. 4). By varying the time
FIG. 1. Effect of acetate buffers on the rate of reaction of form-
aldehyde with THF, as the free base, at ionic strength 1.0 M, of preincubation, it was shown that at a given concentration of
and 25”. The solid lines are calculated for catalysis by acetic thiol a constant degree of inhibition, i.e. an equilibrium, for the
acid (Equation 1, km = 4000 M-~ see-l). reaction of formaldehyde and thiol is reached in less than 2 min
Issue of December 25, 1966 R. G. Kalkn and W. P. Jencks 5853

at this pH. By measuring the amount of the rate decrease which

is caused by the removal of formaldehyde as the hemithioacetal
after a preincubation period of 10 min at this pH at different
thiol concentrations (Fig. 5), it was possible to calculate the
equilibrium constant for hemithioacetal formation. The value

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2.0 4.0 6.0 8.0

FIG. 2. Dependence on pH of the pseudo first order rate con-

stants for the reaction of THF with 0.00167 M formaldehyde, 20 40 60 80
ionic strength 1.0 M and 25” in the presence (X) and absence
Seconds
(0) of 0.00267 M 2-mercaptoethanol. The rate constants are
extrapolated to zero buffer concentration. The pH was main- FIG. 4. Inhibition by mercaptoethanol of the reaction of formal-
tained with hydrochloric acid, formate, acetate, phosphate, dehyde with THF with (O-O) and without (0) a lo-min prior
N-methylmorpholine, and triethylenediamine buffers. The lines incubation of mercaptoethanol and formaldehyde. The reaction
at low (---) and high (-.-) pH are the calculated rates of the conditions were: 0.00167 M formaldehyde, 5 X 10m6M THF, 1.3 X
attack and dehydration steps, respectively, and the upper solid lo-4 M ethylenediaminetetraacetic acid, and 0.03 M phosphate
line is the calculated rate from the steady state rate Equation 8 buffer, pH 6.57, ionic strength 1.0 M, 25”.
(see text and Table IV).

0.15 I I I

0.15 r---- _ 0.10

T
ii

::
2

0.05

0.005 0.010 0.015 M

2- Mercaptoethanol Concentration
0 0.01 0.02 0.03M
FIG. 5. The effect of mercaptoethanol concentration on the
Concentration of 2-Mercaptoethanol
inhibition of the reaction of THF with formaldehyde under condi-
FIG. 3. Effect of mercaptoethanol on the pseudo first order tions in which the formaldehyde and thiol had been allowed
rate constants for the reaction of THF with 0.00167 M formalde- to reach equilibrium by a lo-min incubation before the addition
hyde at ionic strength 1.0 M and 25”. The buffers were 0.033 M of THF. The experimental conditions were the same as in Fig. 4.
formate (A), 0.033 M acetate (0) and 0.067 M acetate (0). The The theoretical curve for an equilibrium constant for hemithio-
reactions were initiated by the addition of formaldehyde. acetal formation of 620 ah1 is shown as the solid line.
5854 Reaction of Formaldehyde with Tetrahydrofolic Acid Vol. 241, No. 24

of KH = [H,C(SR)OH]/[HzC(OH)2] [RSH] = 620 M-’ so ob- I I I I

tained was used to calculate the solid line of Fig. 5, which agrees
satisfactorily with the experimental points.
At pH 8.48, there is a rapid initial increase of the absorbance

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I [ iiCH0 ] (M-‘1
60 /

Seconds FIG. 8. Double reciprocal plot of the pseudo first order rate
constants against formaldehyde concentration for the reaction
FIG. 6. Time dependence of the absorbance change at 296 rnp in of THF with formaldehyde in 0.03 M triethylenediamine buffer,
the reaction of THF with 0.55 M formaldehyde in 0.05 M triethyl- pH 8.50, ionic strength 1.0 M, and 25”. A theoretical curve
enediamine buffer, pH 8.48, ionic strength 1.0 M, 25”. Initial based on the equation
absorbance, 1.763. Readings were taken against a blank of p-
nitrobenzoic acid with a slit width of 0.2 mm. 1 1 2.0 [F]
-=1
k ,,,,a 2.M X 10’ [H+] + 32 X 2.66 X lo7 [H+l [Fl + 2.66 X 107 [H+]
is shown as the solid line.

of THE‘ at 290 rnp in the presence of 0.55 M formaldehyde, which

is too fast to be measured by ordinary techniques, followed by a
relatively slow first order formation of product; the rapid increase
is labeled “Initial Burst” in Fig. 6. At 320 rnp under the same
conditions, the initial burst is in the opposite direction, i.e. to an
absorbance lower than the initial absorbance, and it is followed
by a slower increase to the absorbance of the final product.
Similar experiments at a number of wave lengths made possible a
comparison of the spectra of the starting material, the product,
and the intermediate formed in the initial rapid reaction (Fig. 7).
At low formaldehyde concentrations, the pseudo first order
rate constants for the reaction of THF with formaldehyde in-
crease linearly with the formaldehyde concentration, but at high
formaldehyde concentrations at alkaline pH, under conditions in
which the initial burst is observed, the rates level off and become
independent of formaldehyde concentration. At still higher for-
1 maldehyde concentrations, an inhibition of the rate is observed.
The leveling off is caused by the complete conversion of THF to
the intermediate product which is formed in the initial rapid re-
action, so that a further increase in formaldehyde concentration
cannot cause a further increase in the concentration of the inter-
260 260 300 320 mediate or the rate of its conversion to the final product. The
Wavelength rnp inhibition at very high formaldehyde concentration suggests that
a 2nd molecule of formaldehyde combines with THF to form a
FIG. 7. Spectra of THF (X- - -X), Nr,Nlo-methylene-THF
(O-O), and the hydroxymethyl intermediate of THF (O-.-O) compound which does not react further. The results of a typical
at pH 8.5, constructed from the spectrum of THF and a series of experiment are shown in the form of a reciprocal plot, analogous
experiments similar to that illustrated in Fig. 6. to a Lineweaver-Burk plot, in Fig. 8. The intercept of such a
Issue of December 25, 1966 R. G. Kallen and W. P. Jencks 5855

plot at the ordinate gives the reciprocal of the maximum pseudo
first order rate constant at high formaldehyde concentrations (i.e.
the rate constant for the conversion of the intermediate to prod-
uct), and the intercept at the abscissa gives the negative of the
equilibrium constant for formation of the intermediate. The in-
hibition of the rate at high formaldehyde concentrations
evident in the upward deviation of the experimental points from
the line near the ordinate.
OC
The maximal pseudo first order rate constants at several pH
values, obtained from a series of experiments similar to that il-
lustrated in Fig. 8, are plotted logarithmically as a function of
pH in Fig. 9. The logarithms of the observed rate constants
follow a sigmoid curve, with acid-catalyzed segments at high and
is low pH values connected by a region in which the rate is almost
independent of pH. This behavior suggests that the THF-
formaldehyde intermediate undergoes ionization with a pK,’ of
approximately 10.5 and that both ionic species of the inter-
mediate undergo acid-catalyzed reactions to give the final prod-
uct.
Catalysis of the reaction of THF with formaldehyde by acetate
buffers at several buffer ratios is shown in Fig. 1, with the rate
constants corrected to the non-ionized species of THF (pK,’
4.82; ionization of the carboxyl groups of glutamic acid is neg-
lected). It is evident that the catalysis is proportional to the
acidic species of the buffer, and the experimental points are in
satisfactory agreement with the lines in the figure, which are
calculated from the value 4000 M-’ se6 for the catalytic con-
stant for acetic acid in the rate law

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vc.t = hdTHFl[Fl[HA] (1)
-2.0
in which vcat is the rate of the buffer-citalyzed reaction. How-
ever, this rate law is kinetically indistinguishable from the rate
law
b&md
&at = [THFH+] [F] [A-] = kist [THFH+] [F] [A-] (2)
I I I 1 I I I K’ HA
8.0 9.0 10.0
in which K’THFH+ and Kk, are the ionization constants for
PH THFH+ (pK,’ 4.82) and the catalyzing acid, respectively, and
F is formaldehyde concentration. Since THF is largely in the
FIG. 9. Dependence on pH of the reaction of THF with formal-
dehyde under conditions in which THF is completely converted protonated form under conditions in which catalysis by car-
into the addition intermediate formed in the initial rapid reaction. hoxylic acid buffers is observed, the observed rates of the cata-
The points were obtained from the intercepts of reciprocal plots lyzed reaction in terms of the total THF may increase with
of the data from experiments similar to that shown in Fig. 8. increasing pH, according to this rate law.
Triethylenediamine buffers, 0.03 to 0.10 M, pH 7.98 to 10.20;
sodium carbonate buffers, 0.06 M, pH 10.43 to 10.65; ionic strength The catalytic constants for a number of acids, which were
1.0 M. The solid line is a theoretical line for reaction of the determined in the same manner as that for acetic acid, are sum-
two ionic species of the THF intermediate, based on marized in Table I. The values for the solvated proton and for
water were calculated from the data shown in Fig. 2 and the rate
k* = k’z[H+][l - 011 + k;,[H+][or]
law
in which CYis the fraction of Ns-hydroxymethyl-THF as the anion,
based on a pK’, of 10.5 (see Table IV). v = kJTHF][F][HzO] + k’l[THF][F][H.,:,] (3)

TABLE I
Third order catalytic constants‘ ' Ojf acids for attack of tetrahydrofolic acid on formaldehyde (F) at 86”b
- -
Catalyst PH ( hcentration range No. of experiments Fraction HA PK’~ ~HA (K* 9.x-l)
- _-
M
k&o+. ... 2-4 0.01-0.0001 -1.7 4.8 X lo5
Cyanoacetic acidc. 1.6-3.3 0.034.40 26 0.14.9 2.34 9.1 x 103
Chloroacetic acid.. . . 2.34, 3.18 0.03*.40 18 0.25, 0.5 2.69 8.0 X 103
Formic acid. . .. 3.09-3.65 0.03-0.17 12 0.75-0.5 3.60 5.7 x 103
Glycolic acid. 3.67 0.03-0.40 9 0.5 3.67 5.2 X lo3
Acetic acid. .. . 4.20-4.78 0.017-0.10 57 0.72-0.39 4.60 4.0 x 108
Phosphate monoanion. 5.51-6.38 0.02-0.10 73 0.90-0.55 6.40 2.4 X lo3

-I
N-Methylmorpholine. 3.67-5.71 0.05-0.40 21 1.0 7.80 1.9 x 103
HOH. . .. . . .. . . .. 3.5-6.0 1.0 15.7 1.6
-
o v = kuA [THF][F][HA].
* Ionic strength maintained at 1.0 M by the addition of potassium chloride.
c An additional term is present in the rate law for the reaction of monocationic tetrahydrofolic acid (see “Discussion”), v = k’=A
THFH+][F][HA], in which k’HA = 13 M-~ see-I.
5856 Reaction of Formaldehyde with Tetrahydrofolic Acid Vol. 241, No. 24

which describes the rate of the reaction, exclusive of buffer
catalysis, in the acidic pH region and is shown by the dashed line
at, the left in Fig. 2. In the case of cyanoacetic acid, the strong-
est carboxylic acid examined, the rate constants at acidic pH are
too large to be accounted for entirely by the rate law of Equation
1 and suggest the presence of an additional term
ucat = ~HA[THFI[FI[HA] + ~‘HA[THFH+][FJ[HA]
(4)
The catalytic constants for cyanoacetic acid were evaluated from
a series of experiments carried out at, four different buffer ratios
between 10 and 90% carboxylic acid. Values of 9100 and 13
M+ set+ were obtained for kEIA and k’,,, respectively.
Catalysis by phosphate buffers follows the rate law of Equa-
tion 1 at pH 5.5, 6.1, and 6.4 (o), with the rate of the catalyzed
reaction proportional to the concentration of phosphate mono-
1
0.7
anion (Fig. 10). At pH 6.70 to 6.96 ( l ), the initial rate increase
follows the same rate law &s at the lower pH values, but as the
buffer concentration is increased, the apparent catalytic constant
decreases, and the line shows a tendency to level off. The
pH of phosphate buffers is not constant with increasing buffer
concentration at constant ionic strength in this pH region, but
the change in rate caused by this pH variation is negligible com-
pared to the deviation of the points from the upper straight line.
It will be shown that this leveling off is another reflection of the
mechanism which causes the bell-shaped pH rate profile.
General acid catalysis of the reaction is also observed in the
alkaline pH region. Catalysis by triethylenediamine buffers in
the presence of 0.44 M formaldehyde is proportional to the con-
centration of the conjugate acid of triethylenediamine at pH
8.83 (A) and 8.54 ( l ) (Fig. 11). These experiments were
carried out at high formaldehyde concentrations in order to avoid
a change in the rate caused by the (small) binding of formalde-
hyde to the buffer (16); at these concentrations of formaldehyde,
the rate is independent of formaldehyde concentration and such
binding would not affect the rate. The catalytic constants for
several acids, obtained from similar experiments, are summarized

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1
in Table II.
Secondary amines, such as morpholine and imidazole, are far
0.5 more effective at low concentrations than are other catalysts of
the reaction of THF with formaldehyde. However, as the con-

T
fii
”
:: 0.3
2 i

ii

1 - 0.05
2
I
2
0.1 2

0.1 0.2 M

Phosphate Monoanion Concentration N%H@ Concentration (Ml

FIG. 10. General acid catalysis by phosphate monoanion of the
reaction of THF with 0.00167 M formaldehyde at pH 5.51, 6.10,
6.38 (O), and 6.70 to 6.96 (0) at, ionic strength 1.0 M and 25”.
The curved line is calculated from the steady state rate Equation
9.
FIG. 11. Catalysis by triethylenediamine buffers of the reacti
of THF with 0.44 M formaldehyde at, pH 8.54 (0) and 8.83 (1
ionic strength 1.0 M, and 25”; ko is the rate constant extrar
lated to zero buffer concentration.

TABLE II
Second order rate constants for general acid catalysis of dehydration of hydroxymethyl intermediate of THF at 85””

v = kZHA ( >-CH~~H) (HAI

Catalyst PH Concentration range No. of experiments Fraction HA PK’, kk4 M-1 SIX-’

M
HaO+. . . . . . . . . . . . . . . . . . . .. 7.5-8.5 -1.7 2.7 X lo7
Phosphate monoanion*. . . . . .. 6.7-7.0 0.02-0.40 57 0.314.05 6.40 42
N,iV’-Dimethylpiperazinec. .. 7.60-8.57 0.05-0.50 41 0.50-0.90 8.57 0.32
Triethylenediaminec. . . ... . 8.54-8.83 0.05-0.50 19 0.68-0.78 9.15 0.18

0 Ionic strength maintained at 1.0 M with potassium chloride.

* Obtained from Equation 9 and the data of Fig. 10 (see text).
c Measured at 0.44 M total formaldehyde concentration, for which the reaction rate is independent of the formaldehyde concentration.
Issue of December 25, 1966 R. G. Kallen and W. P. Jencks 5857

centration of secondary amine is increased there is a leveling off TABLE III

and then an inhibition of the rate at high amine concentrations, Apparent second order rate constants for condensation of formalde-
as shown for morpholine in Fig. 12. The catalyzed reaction be- hyde and free base fom of tetrahydrofolic acid as function of
comes faster as the pH is decreased. The rate of the secondary imidazole concentrationa
amine-catalyzed reaction was found to be directly proportional
to the formaldehyde concentration
tions: 0 to 0.0033 M formaldehyde
under the following condi-
at pH 7.84 for 0.0033 and
Total imidazole
I- pH 5.0Zc pH
kl[rb
5.51d pH 5.19'

0.017 M morpholine; 0 to 0.01 M formaldehyde at pH 8.49 for 0.033 M

M morpholine; 0 to 0.0063 M formaldehyde at pH 6.29 and 0 to 0 85.0
0.008 M formaldehyde at pH 6.86 for 0.067 M imidazole. The 0.00167 96.3
second order rate constants for the maximal velocities, k’n = 0.0033 112 115 177
kobs (max)/[F] in which k&s (max) is obtained from the intercept 0.0067 119 127 180
of extrapolations of the linear portions of plots of l/kobs against 0.0133 156
l/[amine], are plotted logarithmically against pH in the inset 0.0167 129 206
of Fig. 12. The data in Fig. 12 suggest that the mechanism of 0.033 208 218 256
0.067 366 302 308
catalysis involves an acid-catalyzed reaction of an intermediate
0.099 368
which accumulates at high concentrations of secondary amine.
0.130 693 449 368
Catalysis of the reaction by imidazole was studied in less 0.340 822
detail than that by morpholine, but it is apparent from the data 0.400 462
shown in Table III that the imidazole-catalyzed reaction k’~ (max), 3860 816 465

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is also catalyzed by acid and shows saturation with increasing
imidazole concentration. Under conditions of saturation in a The reaction was started after 10 min of prior incubation of
imidazole and formaldehyde; formaldehyde, 2.1 X lo+ M, ionic
r strength 1.0 M, 25".
b k’~ (M-I see-r) = k,b,/[F]~u, where 01 is the fraction of total
THF as the free base.
c Sodium acetate, 0.02 M.
d Potassium phosphate, 0.02 M.
e Potassium phosphate, 0.05 M.
/ Extrapolated value, from a plot of l/k'N against l/[imidazole].

0.200

0.100

20 40 60
0.0025 Seconds
FIG. 13. The induction period in the reaction of THF with
0.0033 M formaldehyde catalyzed by 0.038 M morpholine in the
presence of 0.0167 M triethylenediamine buffer, pH 8.02, at ionic
0.01 0.02 0.03 &32 0.136 strength 1.0 M and 25”. O----O, no preincubation; A----A, 10
min preincubation of morpholine and formaldehyde.
Total Morpholine Concentration (M)
FIG. 12. Morpholine catalysis of the reaction of THF with respect to the catalyst and at a given formaldehyde concentration
4.7 X 10-’ M formaldehyde at different pH values in 0.05 M phos-
phate or triethylenediamine buffers at ionic strength 1.0 M and and pH, imidazole is about IO-fold less effective than morpholine
25”. The Zower curve was calculated from data obtained with as a catalyst. N-Methylpiperazine was also found to be an
0.00167 M formaldehyde; the rate constants are linear in respect effective catalyst and to show saturation with respect to amine
to formaldehyde concentration under these conditions. The mor- concentration at pH 5.38.
pholine and formaldehyde were allowed to reach equilibrium dur-
In the presence of secondary amine catalysts there is an in-
ing a IO-min incubation prior to the addition of THF. Inset:
Dependence on pH of the logarithm of the maximum second order duction period if the reaction is started by the addition of formal-
rate constant, k’~ = k&s (max)/[F]. dehyde, as shown for the reaction of morpholine at pH 8.02 in
5858 Reaction of Formaldehyde with Tetrahydrofolic Acid Vol. 241, No. 24

Fig. 13 ( 0). The induction period is abolished by brief pre- conditions, the THF competes with thiol for the limited amount
incubation of the amine and formaldehyde before addition of of unhydrated formaldehyde and, since the reactive thiol is
THF (Fig. 13, A). This evidence also suggests that an inter- present in much larger concentrations than that of THF, the
mediate is formed from the amine and formaldehyde in this re- THF reaction is inhibited. U n d er equilibrium conditions, the
action. smaller inhibition is caused only by the decrease in the equilib-
rium concentration of unhydrated formaldehyde, because at
DISCUSSION
equilibrium the rate of formation of hemithioacetal is equal to
Hemithioiacetal Formation from Formaldehyde and ThiolsThe its rate of breakdown to unhydrated formaldehyde, so that the
inhibition by thiols of the condensation of THF with formalde- formation of unhydrated formaldehyde is no longer rate-
hyde at neutral and alkaline nH is undoubtedlv
1
caused bv the determining in the THF reaction.
well known reaction of thiols with formaldehyde and other The fact- that inhibition of the THF-formaldehyde reaction
carbonyl compounds (12, 14, 15) to form the hemithioacetal by thiols can occur by the trapping of free formaldehyde as it is
(Equation 5). formed demonstrates that it is free formaldehyde and not formal-

H’ ‘O-H
H\ ,o-H ~HOH . P
(5)
dclwH’ &r H/c\H

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5, IO-methylene-THF -I- HOH

The fact that inhibition of the reaction of formaldehyde with dehyde hydrate that is the reactive species in the reaction with
THF is not observed in acid solution indicates that hemithio- THF.
acetal formation is base-catalyzed; i.e. the thiol anion is the re- The equilibrium constant of 620 M-~ for hemithioacetal forma-
active species. It was shown by Ratner and Clarke that the tion from formaldehyde and mercaptoethanol at pH 6.57,
reaction of N-acetylcysteine with formaldehyde is subject to estimated from the inhibition of the THF-formaldehyde react.ion
base catalysis (15). under conditions in which it was shown that equilibrium had
Inhibition by thiols of the reaction of THF with formaldehyde been reached in the hemithioacetal reaction, is similar to that of
occurs by two different mechanisms, depending on the experi- approximately 500 M-’ for the addition of glutathione
mental conditions. If the thiol and formaldehyde are allowed to methylglyoxal, which is also hydrated in water (20), and is
to react with each other to reach equilibrium before THF is somewhat larger than the values of 18 to 136 M-’ for the addition
added to the reaction mixture, the inhibition is simply the result of various thiols to pyridoxal phosphate (21, 22).
of the decreased concentration of formaldehyde at equilibrium The fact that the inhibition by mercaptoethanol is dependent
which is caused by hemithioacetal formation. However, if the on pH accounts for the difference in the positions of the ascending
reaction is initiated by the addition of formaldehyde, the in- and descending limbs of the pH rate profile in the presence and
hibition is greater, at a given thiol concentration (Fig. 4), and a absence of mercaptoethanol (Fig. 2) and means that the inflec-
different mechanism of inhibition must occur. This type of tion points of these curves in the presence of mercaptoethanol
inhibition is attributed to a competition between THF and thiol cannot be assigned to dissociation constants of THF (cf. Refer-
for the limited amount of free, unhydrated formaldehyde which is ence 3). Preliminary evidence has been reported for the ex-
made available by the dehydration of formaldehyde hydrate. istence of an enzyme which catalyzes the reaction of THF with
The equilibrium constant for formaldehyde hydration, KH = formaldehyde in the presence of 0.01 M mercaptoethanol at pH
[H2C(OH)2]/[HCHO][HOH], is 103 to lo4 and indicates that 7.5 (23). This concentration of mercaptoethanol would be
formaldehyde is more than 99.9% hydrated in aqueous solution expected to reduce the rate of reaction of formaldehyde with
(17, 18). The rate of dehydration of formaldehyde hydrate to THF to a small fraction of the uninhibited value and raises the
free formaldehyde has been determined (19) and is more than possibility, among several others, that this enzyme activity in-
adequate to supply unhydrated formaldehyde for the reaction volved a facilitation of the liberation of free formaldehyde from
of THF with formaldehyde at the concentrations of these com- the hemithioacetal, rather than of the condensation of formalde-
pounds which were used in these experiments in the absence of hyde with THF.
competing nucleophilic reagents. That the dehydration of Significance of Bell-shaped pH Rate ProjZe for Reaction of THF
formaldehyde hydrate is ordinarily not rate-determining is also and Formaldehyh-The midpoints of the ascending and descend-
shown by the fact that the reactions are first order in respect to ing limbs of the pH rate profile for the reaction of THF with for-
THF. maldehyde do not correspond to dissociation constants of
However, if formaldehyde is added to a solution which contains THF (24), either in the presence or in the absence of mercap-
an excess of thiol at a sufficiently high pH, the unhydrated for- toethanol. Although the midpoint of the acidic limb of the
maldehyde will react with thiol to form hemithioacetal as fast as curve in the absence of mercaptoethanol is close to the pK’,
it is formed by the relatively slow dehydration of formaldehyde value for ionization of the NS position of THF, there is no ioniza-
hydrate, thereby lowering the concentration of unhydrated tion in either THF or (hydrated) formaldehyde near the mid-
formaldehyde available for the reaction with THF. Under these point of the alkaline limb, at about pH 7. The bell-shaped pH
Issue of December 25, 1966 R. G. Kallen and W. P. Jencks 5859

rate profile must be, therefore, an expression of the kinetics of the dine part of THF (4, 27). At still higher formaldehyde concen-
reaction rather than of the ionization constants of the reactants. trations, inhibition of the THF reaction is observed, probably
Such behavior has been observed in a number of carbonyl group because of addition of formaldehyde to the Nlo position to form
reactions (10,25) and at least one enzymic reaction (26) and may the unreactive Ng,N1O-dihydroxymethyl-THF (IV). The equi-
be most simply described as a change in the rate-determining
step of the reaction with changing pH, such that attack of the
P” P”
nucleophilic reagent on the carbonyl group is rate-determining
in one pH region, while dehydration of the resulting addition FH2 YH*
-N N-
product is rate-determining in another pH region. These steps 6 w 10
are shown for the reaction of THF with formaldehyde in Equa- IV
tion 6. It is probable that the intramolecular attack of an
amine on the cationic imine in the last step of the reaction is librium constant for the addition of the second mole of formalde-
very fast.,2 so that the pH rate profile may be interpreted in terms hyde is estimated to be approximately 2.0 Mu’ from the kinetic

Step 1 II step 2 III

of rate-determining attack of the basic N5 nitrogen atom of THF data, which is similar to the value of 2.4 ~-1 for the addition of

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in one pH region (Step 1) and rate-determining dehydration of the formaldehyde to NB-formyl-THF (4, 27).
carbinolamine intermediate (II) to a cationic imine (III) in At the acid and alkaline extremes of the pH rate profile, one
another pH region (Step 2). or the other of the two steps is almost completely rate determin-
The dependence on pH of the rates provides evidence for a ing and the observed rate may be accounted for by the rate law
change in rate-determining step, but does not show which step for these steps. At alkaline pH, where acid-catalyzed dehydra-
is rate-determining in each pH region. Analogy with other con- tion is rate-determining, the rate law is
densations of carbonyl groups with nitrogen compounds suggests
that attack of amine on the carbonyl group is rate-determining 21 = k; [Int] [H+] = F [THF] [F] [H+] (7)
1
at acidic pH, while dehydration of the carbinolamine inter-
mediate is rate-determining at alkaline pH (10). The dehydra- in which Int is the hydroxymethyl-THF adduct II. The calcu-
tion step is acid-catalyzed, which accounts for the increasing rate lated line for this rate law, based on the measured equilibrium
with decreasing pH in this region, but as this rate becomes very constant for addition product formation, and the rate constant
fast with increasing acidity the rate of amine attack can no for its dehydration, which are summarized in Table IV, is shown
longer keep up with dehydration and amine attack becomes rate- as the dot-dashed line on the right side of Fig. 2. The fact that
determining. The rate of the attack step increases with in- the observed rates do not drop to zero with increasing acidity
creasing pH in the region in which the amine is protonated, be- at pH values below the maximum means that there is an acid-
cause the free base form of the amine is the reactive species. catalyzed as well as an uncatalyzed addition of the free base
It was possible to obtain direct evidence that this assignment of form of THF to formaldehyde, so that the rate law in this region
the rate-determining steps is correct also for the reaction of THF is that of Equation 3. The dashed line on the left side of Fig. 2
with formaldehyde. At alkaline pH values there is a very rapid is based on this equation and the rate constants summarized in
reaction of THF with formaldehyde to form an adduct, which Table IV and accounts satisfactorily for the observed rates in
must be the carbinolamine addition product (Step 1, Equation 6) the acidic pH region.
followed by a slow further reaction to give the final product At intermediate regions of pH, both steps are partially rate-
(Step 2, Equation 6, Fig. 6). Ko such intermediate would be determining and the rate can be described only by the use of a
observed if attack of the amine on formaldehyde were rate- steady state rate equation with the form of Equation 8, which is
determining. Further evidence for the formation of such an based on the mechanism of Equation 6.
adduct is the leveling off of the rate with increasing formaldehyde
concentration at alkaline pH (Fig. 8). When THF is completely k ohs = k; [H+l (kl + k; [X3+1)[HCHOI
k’ohs = - (8)
converted to the hydroxymethyl (carbinolamine) addition a k-1 + k:l [H+l + k; [H+l
product, a further addition of formaldehyde cannot result in a
The calculated rate from this equation and the rate constants
further rate increase and the rate of dehydration of the inter-
given in Table IV is shown as the solid line in Fig. 2. This line
mediate may be observed directly. The equilibrium constant
shows satisfactory agreement with the observed rate constants
for the formation of this adduct, obtained from a series of recipro-
over the entire range of pH, within the experimental error of up
cal plots of these data similar to that shown in Fig. 8, is 32 f 6
~-1, which is in the range of the values of 18 to 42 M-’ reported to 20% for reactions with half-times of 3 to 5 sec.
Further evidence for a change in rate-determining step comes
for the addition of formaldehyde to 2-amino-6-methyl-5,
from the dependence of the rate on buffer concentration at a
6,7,8-tet,rahydro-4-pteridinone, a model for the tetrahydropteri-
constant pH. At pH 5.51, 6.10, and 6.38, the reaction is cata-
2 Evidence that supports this assumption is given in the last lyzed by phosphate buffers and the rate is directly proportional
section. to the concentration of the phosphate monoanion. However, at
5860 Reaction of Formaldehyde with Tetrahydrofolic Acid Vol. 241, No. 24

TABLE IV may be brought about by a change in buffer concentration, as well

Apparent rate and equilibrium constants for reaction of tetrahy- as by a change in pH (28).
drojolic acid and formaldehyde at 66”, ionic strength 1 .O M, The steady state rate Equation 9 may be derived from the
water activity 1 .O scheme of Equation 6 by including the rate constants for buffer
k
-=ohs (kl + k; [H+l + k;’ [HAD (k; [H+l + k;’ [HAI) [HCHOI cgj
cz k-1 + k, [H’] + k:l [HA] + k; [H+l + k;’ [HA]
3.2 X lo4 M--l’

catalysis k”JHA], k”-JHA] and k”,[HA] for the first and second
steps. The lower solid line of Fig. 10 was calculated from Equa-
tion 9 and the rate constants shown in Table IV and shows
32 M-l satisfactory agreement with the experimental data.
General Acid Catalysis of Attack Step-The Brijnsted slope, (Y,
is approximately 0.20, for a logarithmic plot of the catalytic
constants of a series of general acid catalysts against pK’, for
catalysis of the attack of the NS position of THF on formal-
103
dehyde (Fig. 14). The solvated proton and water show deviations
of about one order of magnitude from the line drawn through
the points for carboxylic acids. This value of Q!for the attack of
kl u = k, [THFI [FI 90 M-l set-1 the Nb position of THF (pK’, 4.8) is similar to the values of
0.25 for general acid catalysis of the attack of semicarbazide

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k; u = k; [THFl[Fl [H+l 4.8 X 106 M* see-l (pK’, 3.7) and aniline (pK’, 4.6) on aldehydes and suggests that
k;’ v = k;’ [THFl IF] [H,PO,-1 2400 M* see-l the catalysis occurs by a similar mechanism in these reactions
(28, 29). There is evidences which supports the mechanism
k-1 v = k-1 [>,,,,,OH] 2.8 se6 shown in transition state V for these reactions (10, 28, 30), but
Mechanism VI, which has the same stoichiometric composition
and is kinetically indistinguishable, must also be considered. In
k:, v = k:l [-&H.OH]IH+I 1.5 x 104 M-’ se0 addition to the analogy to other, similar reactions, the following
evidence provides support for Mechanism V for the reaction of
THF with formaldehyde.
Icl; u = kl; [ >,cHQoH] [H2Po,-l 75 M-l see-1
H-\N . ..&+)...&..A A-..+..& ..a b,+oH
/ I I I
kj v = k; [=,,,,,OH],H+l 2.7 X 107 M-I see-1 V VI
For the reaction catalyzed by the solvated proton, the most
k;’ v = k;’ [ >,,,oH] [H2P04-l 42 M-~ se0 probable pathway for the formation of transition state VI in-
volves the protonation of free formaldehyde in a prior equilibrium
kk v = ka [Al [H+l 1.3 x 109 M-l se0 step, followed by the removal of a proton by water, acting as a
general base catalyst, from the attacking THF. From the lowest
H
HYH
Q

“‘leference 5’ A = F = formaldehyde hydrate

(THF anion)

pH 6.70 to 6.96, in the region in which the change in rate-deter-

mining step occurs, the rate shows the same proportionality to
phosphate monoanion concentration at low phosphate concentra-
tion, but shows a tendency to level off at higher buffer concen-
trations (Fig. 10). The step which is subject to catalysis by
phosphate monoanion at low pH is the attack step and this is
the step which shows catalysis at low buffer concentrations at
neutral pH; however, as the buffer concentration is increased in
this pH region the attack step becomes faster and the dehydra-
tion step, which is less subject to buffer catalysis, becomes FIQ. 14. Briinsted plot for general acid catalysis of the attack
of THF on formaldehyde at 25”, ionic strength 1.0 M. The line
partly rate-determining. Similar behavior is observed in semi- has been drawn through the points for carboxylic acid catalysts
carbazone formation, in which a change in rate-determining step and has a slope, 01 = 0.20.
Issue of December 25, 1966 R. G. Kahn and W. P. Jencks 5861

estimated value for the equilibrium constant for formaldehyde change in rate-determining step occurs at lower pH and the reac-
hydration, KH = [H&(OH)Q[H&O][H20] = lo3 (17) and tion of the hydroxymethyl-THF anion becomes significant at
the assumption that the b&city of free formaldehyde is as large higher pH. The limited data which could be obtained for this
as that which has been estimated for acetone by spectrophoto- catalysis (Table II) indicate that it has a Brtjnsted coefficient,
metric titration (pK’, for protonated acetone = -7.2 (32)) a a, of approximately 0.75 and the absence of detectable catalysis
value of 7.4 X lOI M-I see-i may be calculated for the rate of the dehydration step by water is in accord with such a high
constant of the solvated proton catalyzed reaction by this mech- value of (Y. This large value of ar also reflects the experimental
anism. This is more than four orders of magnitude larger than fact that catalysis by the solvated proton is dominant, so that
the value of 1.4 X 10” M-’ se@ for the fastest known diffusion- catalysis by weaker acids is of comparatively little importance.
controlled reaction, the reaction of hydroxide ion with the sol- The difference between this value and the value (Y = 0.20 for
vated proton (32), which effectively rules out this mechanism. general acid catalysis at low pH values is further evidence for a
Reactions which involve other general acids, HA, would require difference in the nature of the rate-determining step at alkaline
involvement of their conjugate bases A- according to Mech- and acid pH.
anism VI. These bases would be expected to be more effective The probable mechanism of this step, shown in transition state,
catalysts than water, so that the rates for such reactions would VII, is similar to that for related reactions (10).
have to be even faster than that for the solvated proton if Mech-
\ s+ H 6+ 6-
anism VI held. These considerations favor Mechanism V for
all general catalysts. N - C& . . . () . . . H . . . A
Cyanoacetic acid, a relatively strong carboxylic acid, catalyzes /
the attack on formaldehyde of THFH+ as well as THF (Equa- VII

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tion 4). The reactive species of THFH+ in this reaction is not In this case there is no kinetic ambiguity, because the alternative
established, but it is probable that it is one of the forms of mechanism, which involves pre-equilibrium protonation of the
THFHf formed in a microscopic ionization, with the N5 position leaving hydroxyl group and general base catalysis by removal of
free and the proton on the Nr or Nro positions; rough estimates a proton from the nitrogen atom, concomitantly with the expul-
of the expected microscopic ionization constants suggest that sion of water, is not possible for THF, because THF has no proton
this is a reasonable assumption. In any case, the ratio of the on the nitrogen atom of the hydroxymethylated secondary amine.
rate constants for the cyanoacetic acid-catalyzed reactions of The suggestion by Swain et al. that catalysis may involve solva-
THF and THFH+, kHA/k’nA = 700. A similar ratio is expected tion of the partial positive charge on the nitrogen atom by a
for the water-catalyzed reaction of THF and THFH+ with catalyzing anionic base in the transition state (33) does not ap-
formaldehyde. Such reactivity ratios show only a very small sen- pear to be tenable in this case, because catalysis is observed with
sitivity to the strength of the catalyzing acid; for example, the re- both neutral and anionic catalysts; this point is considered in
activity ratio for p-nitrobenzaldehyde to p-hydroxybenzaldehyde greater detail elsewhere (34, 35).
with semicarbazide changes less than a-fold with a change of 1017 Nucleophilic Catalysis bySecondaryAmine-The evidence that
in the strength of the catalyzing acid (28). This gives an ex- catalysis of the reaction of THF with formaldehyde by secondary
pected value of 0.13 M+ set-i for the water-catalyzed reaction of amines, such as morpholine and imidazole, is nucleophilic cataly-
THFH+. This is much smaller than the value of 7.3 ~-1 set+ sis is as follows. (a) Secondary amines are far more effective
based upon the observed rate of the proton-catalyzed reaction, catalysts than general acid-base catalysts and show effective
but expressed as the kinetically indistinguishable water-catalyzed catalysis in the 0.01 M concentration range. (b) As the concen-
reaction of THFH+. Thus, the latter mechanism appears to tration of amine catalysts is increased, the rate levels off and
make only a small contribution to the observed rate at low pH finally decreases at high concentrations of amine. This suggests
and the observed reaction may be interpreted as true general the formation and accumulation of an intermediate at high con-
acid catalysis by the solvated proton of the attack of the free centration. (c) An induction period in the amine-catalyzed reac-
base form of THF. tion is eliminated if the amine and formaldehyde are allowed to
Dehydration Step-The rate of the dehydration step can be react with each other before the addition of THE‘.
measured directly under conditions in which THF has been This evidence and the fact that the rate of the amine-catalyzed
largely converted to the hydroxymethyl adduct in an initial fast reaction increases with increasing acidity support the mechanism
reaction; the specific rate of the dehydration step is obtained shown in Equation 10 for catalysis by secondary amines. The
from extrapolation to infinite formaldehyde concentration, at secondary amine catalyst (with the circled nitrogen atom) and
which all of the THF is hydroxymethylated, by the use of re- \
ciprocal plots (Fig. 8). The dependence on pH of the dehy- formaldehyde react to form a hydroxymethylamine, NCHzOH,
dration step indicates that it is acid-catalyzed and that both /
hydroxymethyl-THF and the conjugate base of hydroxymethyl- which undergoes dehydration to give the reactive cationic imine
THF react at significant rates (Figs. 2 and 9). The pK’, of \+
hydroxymethyl-THF, which may be estimated from Fig. 9, is intermediate, N=CHz. This intermediate reacts with the Na
10.5, the same as the pK’, of the THF itself; the hydroxymethyl /
group would not be expected to have a large effect on the loss of nitrogen atom of THF to give the methylenediamine intermedi-
a proton from the N3 position (24). The solid line in the figure ate, VIII, which undergoes acid-catalyzed loss of the catalyzing
is a calculated line based on this pK value. amine to give the same cationic imine as in the uncatalyzed
The dehydration step is subject to general acid catalysis, but pathway, III, which rapidly undergoes ring closure to give 5, lo-
this can be measured only between pH 7.5 and 8.5, because the methylene-THF (I). Inhibition of the reaction at high concen-
5862 Reaction of Formaldehyde with Tetrahydrofolic Acid Vol. 241, No. 42

H\c/o-H
H’ ‘O-H

(10)
‘X,
i, CH,
x.‘..(
Y, H&$ ‘“N
“..,, /\/\
“._.,
“...
‘X.,
...__
.-..._
VIII

-.....,_ E
II
.‘......_.._._..___._....
.‘.

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H\ /H FAST
fi F ’ ,‘q
;NsuNr ,NstiNm + Hm

THF
III I

trations of amine is caused by the formation of the unreactive the rate constants for the morpholine-catalyzed reaction as a
\ / function of total morpholine concentration and the concentra-
methylenediamine, NCHzN from formaldehyde and 2 tions of hydroxymethylmorpholine (-) and methylenedi-
/ \ morpholine (- - -), calculated from the equilibrium constants for
molecules of secondary amine (Step G). the formation of these species. It is evident that, within experi-
The determination of the equilibrium constants for the forma- mental error, the rate of the catalyzed reaction follows the con-
tion of the hydroxymethylamine and methylenediamine adducts centration of hydroxymethylmorpholine and not that of methyl-
of morpholine (16) permits a comparison of the rate of the cat- enedimorpholine. In particular, the decrease in the rate at high
alyzed reaction with the concentrations of these species, which morpholine concentrations occurs as the concentration of hy-
provides further support for this scheme. In Fig. 15 are plotted droxymethylmorpholine begins to decrease, because of methyl-
enedimorpholine formation, and as the concentration of methyl-
enedimorpholine continues to increase. Hydroxymethylamines
0’02=
r--+-l and methylenediamines have both been suggested as intermedi-
ates in a number of other reactions which involve formaldehyde

ICI
. l 1.25
. .
.
and amines (36-39).
. The induction period which is abolished by prior incubation of
.
/W’ formaldehyde and amine is attributed to the time-dependent
formation of hydroxymethylamine (Equation 10, Steps A and B).
0.63
The rate constant of 90 M-I set-l for the reaction of the NC
nitrogen atom of THF with formaldehyde suggests that the reac-
e-4
tion of relatively concentrated solutions of the considerably more
_-- --
__--
_#--
basic morpholine would be even faster, so that the rate-deter-
.02
mining step for hydroxymethylamine formation would be the
0.01 .03 :13 .I4
dehydration of formaldehyde hydrate to the reactive free formal-
Total Morpholine Concentration (MI
dehyde. The rate constant for this dehydration has been re-
FIG. 15. Dependence on total morpholine concentration of the ported to be 0.27 min+ (19), which is of the correct magnitude
pseudo first order rate constants for morpholine catalysis of the to account for the observed induction period. As in the case of
reaction of THF with 0.00167 M formaldehyde in 0.05 M triethylene- thiols, morpholine will compete with the much more dilute and
diamine buffers, pH 8.49, ionic strength 1.0 M and 25”. The solid
line shows the calculated concentration of hydroxymethylmor- less reactive THF for the free formaldehyde as it is formed, so
pholine, NE, and the dashed line that of methylenedimorpholine, that the reaction with THF will be inhibited until enough of the
ND (ordinates on right). The value of ko, for the small contri- reactive formaldehyde-amine intermediate is formed to allow the
bution of the noncatalyzed reaction, was calculated from kobs catalyzed reaction pathway to become significant.
at zero morpholine concentration multiplied by the calculated The above evidence, the fact that the rate of the catalyzed
fraction of formaldehyde in the free, unbound form. The mor-
pholine and formaldehyde were allowed to react for 10 min prior reaction is proportional to the proton concentration under condi-
to the addition of THF. tions in which all of the formaldehyde is converted to hydroxy-
Issue of December 25, 1966 R. G. Kallen and W. P. Jencks 5863

methylmorpholine (Fig. 12, inset), and the fact that the rate is OSBORN, M. J., TALBERT, P. T., .IND HUENNEKENS, F. M.,
J. Am. Chem: Sot., 82, 4921 (1960).
first order in respect to THF permit the conclusion that the
BLAKLEY. R. L.. Biochem. J.. 72. 707 (1959).
transition state for the catalyzed reaction contains the elements BLAKLEY; R. L:, Biochem. J., 74, 71(196b); J. Biol. Chem.,
of formaldehyde, THF, 1 mole of morpholine and a proton, under 238, 2113 (1963).
conditions in which the induction period has been abolished by KISLIUK, R. L., J. Biol. Chem., 227, 805 (1957).
prior incubation of amine with formaldehyde. The rate-deter- JAENICKE, L., in A. V. S. DE REUCH AND M. O’CONNOR (Edi-
tors), The mechanism of action of water-soluble vitamins,
mining step in the scheme of Equation 10, therefore, can be either Ciba Symposium Study Group No. 11, Little, Brown and
D, the reaction of the cationic imine with THF, or E, the acid- Company, Boston, 1961, p. 38.
catalyzed expulsion of morpholine from the intermediate VIII. 8. HUENNEKENS, F. M., WHITELEY, H. R., AND OSBORN, M. J.,
It has not yet been possible to distinguish between these two possi- J. Cell. Comp. Physiol., 54 (suppl. l), 109 (1959).
bilities. 9. Ho, P. P. K., SCRIMGEOUR, K. G., AND HUENNEKENS, F. M.,
J. Am. Chem. Sot., 82, 5957 (1960).
The rapid rate of the catalyzed reaction, incidentally, shows 10. JENCKS, W. P., Progr. Phys. Org. Chem., 2,63 (1964).
that the ring closure in the final step of the reaction (Step F) is 11. KALLEN. R. G.. AND JEXCKS. W. P.. Federation Proc.. 24, 541
not rate-determining in either the catalyzed or uncatalyzed reac- (1965) :
tion. It is not rate-determining in the uncatalyzed reaction be- 12. WALKER, J. F., Formaldehyde, Ed. 3, American Chemical
Society Monograph Series, Reinhold Publishing Corpora-
cause the catalyzed reaction proceeds at a much fast,er rate and
tion, New York, 1964.
involves the same intermediate. It is not rate-determining in 13. ROMIJN, G., 2. Anal. Chem., 36, 18 (1877).
the catalyzed reaction because this step does not involve the 14. POSNER. T.. Chem. Ber.. 36. 296 (1903).
catalyst and the kinetic evidence shows that a molecule of 15. RATNER’, S.; AND CLARKE, h. T.; J. Am. Chem. Sot., 69, 200
(1937).
catalyst is present in the transition state.
16. KALLEN, R. G., AND JEXCKS, W. P., J. Biol. Chem., 241, 5864
Mechanisms can also be written for both the catalyzed and

Downloaded from http://www.jbc.org/ by guest on July 16, 2018

(1966).
uncatalyzed reaction pathways in which an Sn2 displacement 17. GRUEN, L. C., AND MCTIGUE, P. T., J. Chem. Sot., 5217 (1963).
occurs on a saturated carbon atom (IX and X). Similar mecha- 18. BIEBER, R., AND TR~~MPLER, G., Belv. Chim. Acta, 30, 1860
(1947).
19. LE HI~NAFF, P., Compt. Rend., 266, 1752 (1963).
20. CLIFFE, E. E., AND WALEY, S. G., Biochem. J., 79, 475 (1961).
21. BERGEL, F., AND HARRAP, K. R., J. Chem. Sot., 4051 (1961).
22. MACKAY, D., Arch. Biochem. Biophys., 99, 93 (1962).
23. OSBORN,.M. j., VERCAMER, E. N.,‘TALBERT, P. T., AND HUEN-
NEKENS. F. M.. J. Am. Chem. Sot., 79. 6565 (1957).
24. KALLEN, k. G., AND JENCKS, W. P., j. Biol. Chem.,‘241, 5845
nisms have been proposed for the Mannich reaction and related
(1966).
reactions (37, 38, 40, 41). We reject such mechanisms on the 25. JENCKS, W. P., J. Sm. Chem. Sot., 81, 475 (1959).
ground that almost all reactions of carbonyl compounds or their 26. BENDER, M. L., CLEMENT, G. E., KEZDY, F. J., AND HECK,
derivatives that have been studied in detail occur by an elimina- H. D’A., J. Am. Chem. Sot., 86, 3680 (1964).
27. BLAKLEY. R. L.. Nature. 182. 1719 (1958).
tion-addition pathway rather than by a direct SN2 displacement
28. CORDES, E. H.,‘AND JE~CKS; W. P:, J.‘Am. Chem. Sac., 84,
(10). In particular, the condensation of acetone, cyanide, and a 4319 (1962).
secondary amine (42) and the amine-catalyzed reaction of alde- 29. CORDES, E. H., AND JENCKS, W. P., J. Am. Chem. Sot., 84,
hydes with hydroxylamine and semicarbazide (43) occur at rates 832 (1962).
that are independent of the concentration of the final carbonyl 30. SWAIN, C. G., AXD WOROSZ, J. C., Tetrahedron Letters, 36,
3199 (1965).
group acceptor. This shows that the formation of an unsat- 31. CAMPBELL. H. J., AND ED~.~RDS. , J. T., Can. J. Chem.. 38, 2109
urated imine intermediate, rather than an SN2 displacement on a (1960). ’ ’
saturated intermediate, must be the rate-determining step in 32. EIGEN, M., Angew. Chem. (Intern. Ed. Engl.), 3, 1 (1964).
these reactions. 33. SWAIN. C. G.. KUHN, D. A., AND SCHOWEN. R. L.. J. Am.
Chem. Sot., 87, 1553 (1965):
The foregoing supports the hypothesis (7, 8) that in some en-
34. DO AMARAL, L., SANDSTROM, W. A., AND CORDES, E. H., J.
symic reactions of formaldehyde and THF, cationic imine inter- Am. Chem. Sot., 88, 2225 (1966).
mediates (e.g. III), rather than methylene-THF itself, are the 35. REIMANN, J., AND JENCKS, W. P., J. Am. Chem. Sot., 88, 3973
reactive species. The fact that the Ng position of THF is a (1966).
secondary amine ensures the formation of such an intermediate 36. WAGNER, E. C., J. Org. Chem., 19, 1862 (1954).
37. FERNANDEZ, J. E., AND FOWLER, J. S., J. Org. Chem., 29, 402
rather than the less reactive, neutral imine which would be (1964) .
formed from a primary amine. Secondary amine catalysts such 38. BURCKHALTER, J. H., WELLS, J. N., AND MAYER, W. J., Tetra-
as imidazole, in the active sites of enzymes may also facilitate hedron Letters, 21, 1353 (1964).
such reactions. 39. HELLMAN, H., AND OPITZ, G., Chem. Ber., 89, 81 (1956).
Imidazole, although less efficient than morpholine, is an effec- 40. ALEXANDER, E. R., AND UNDERHILL, E. J., J. Am. Chem. Sot.,
71, 4014 (1949).
tive nucleophilic catalyst. The aromatic ring of imidazole would 41. CUMMINGS, T. F., AND SHELTON, J. R., J. Org. Chem., 26, 419
be expected to destabilize a cationic imine formed from this com- (1960).
pound and imidazole was found to be ineffective as a nucleophilic 42. STEWART, T. D., .*ZYD LI, C.-H., J. Am. Chem. Sot., 60, 2782
catalyst for the formation of pyridoxal phosphate semicarba- (1938).
43. CORDES, E. H., .~ND JENCKS, W. P., J. Am. Chem. Sot., 84,
zone (44).
826 (1962).
REFERENCES 44. JENCKS, W. P. AND CORDES, E., in E. E. SNELL, P. M. FASELLA,
A. E. BRAUNSTEIN, AND A. ROSSI FANELLI (Editors), Chemi-
1. KISLIUK, R. L., Federation Proc., 16, 289 (1956). cal and biological aspects of pyridoxal catalysis, Pergamon
2. JAENICKE, L., Federation Proc., 16, 281 (1956). Press, New York, 1963, p. 57.
 The Mechanism of the Condensation of Formaldehyde with Tetrahydrofolic
Acid
Roland G. Kallen and William P. Jencks
J. Biol. Chem. 1966, 241:5851-5863.

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