Determination of the rates of formation and hydrolysis of the schiff bases formed by pyridoxal 5′-phosphate with L-tryptophan and its methyl and n-butyl esters

Article abstract of DOI:10.1246/bcsj.75.2471

The apparent rate constants of the formation (k₁) and hydrolysis (k₂) of the Schiff bases formed by pyridoxal 5′-phosphate with L-tryptophan and their methyl and n-butyl esters at a variable pH, 25 °C, and an ionic strength of 0.1 M were determined, along with the equilibrium constant (K_pH). The individual rate constants of formation and hydrolysis of the Schiff bases of systems corresponding to different chemical species present in the medium as a function of its acidity were also determined, as were the pK values for the Schiff bases. The influence of the α-carboxyl group on the formation and hydrolysis constants of the Schiff bases, and also on their pK values, is demonstrated.

Full text of DOI:10.1246/bcsj.75.2471

c. Jpn.
© 2002 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 75, 2471–2476 (2002) 2471
e Chemical
ciety of Ja-
n
e Chemical
ciety of Ja-
n
Determination of the Rates of Formation and Hydrolysis of the Schiff
Bases Formed by Pyridoxal 5ꢀ-Phosphate with L-Tryptophan and Its
Methyl and n-Butyl Esters
Kinetics of PLP with Esters of l-Tryptophan
G. R. Echevarría Gorostidi et al.
Gerardo. R. Echevarría Gorostidi,* José G. Santos,† Andrea Basagoitia,† and
Francisco García Blanco††
02
71
76
SJA8
09-2673
016
Department of Physical Chemistry, University of Alcalá, E-28871 Alcalá de Henares, Spain
†Faculty of Chemistry, Pontificia Universidad Católica de Chile, Casilla 306, Santiago 22, Chile
††Department of Physical Chemistry, Faculty of Pharmacy, Complutensian University, E-28040 Madrid, Spain
02
02
(Received January 21, 2002)
The apparent rate constants of the formation (k1) and hydrolysis (k2) of the Schiff bases formed by pyridoxal 5ꢀ-
phosphate with L-tryptophan and their methyl and n-butyl esters at a variable pH, 25 °C, and an ionic strength of 0.1 M
were determined, along with the equilibrium constant (KpH). The individual rate constants of formation and hydrolysis of
the Schiff bases of systems corresponding to different chemical species present in the medium as a function of its acidity
were also determined, as were the pK values for the Schiff bases. The influence of the α-carboxyl group on the forma-
tion and hydrolysis constants of the Schiff bases, and also on their pK values, is demonstrated.
One form of vitamin B6 is Pyridoxal 5ꢀ-phosphate (PLP).
Material and Methods
This molecule plays an important part as a coenzyme in many
reactions, such as transaminations, transiminations, dealdola-
L-Tryptophan (TRP), L-tryptophan methyl ester (MTRP), and
tions, and eliminations, which contribute to amino acid metab-
olism.1–3 PLP participates in the formation of a carbinolamine
(α-hydroxy amine) intermediate through a reaction of its car-
bonyl with the ε-amino moiety of the L-lysine residue in the
peptide chain.1,4 This intermediate decomposes to yield a
Schiff base and one molecule of water in an acid-catalyzed
process.4,5
In almost all PLP-dependent enzymes, the first step of the
process mentioned above is a transimination reaction, which
involved a transformation of the PLP-lysine imine into a PLP-
substrate imine.6 This means that the covalent bond in the
Schiff base should be cleaved during the catalytic cycle. Thus,
a new base is formed by the reaction of the coenzyme with the
substrate (commonly an amino acid).1
The literature abounds with references to Schiff bases
formed by PLP and various compounds bearing amino groups,
such as amines, amino acids, and polypeptides.7 However,
very few kinetic studies of the reactions of PLP with amino ac-
ids as a function of the pH have so far been conducted.8
In this work, the stability and the kinetics of the formation
and hydrolysis of the Schiff bases of pyridoxal 5ꢀ-phosphate
with L-tryptophan and their methyl and n-butyl esters (viz. the
PLP–TRP, PLP–MTRP, and PLP–BTRP systems) were deter-
mined, and the results were compared with those previously
obtained for other amino acids.8,9 In order to shed additional
light on the interactions of the α-carboxyl group, the results
were examined in terms of the individual rate constants for the
species involved in the process (see Scheme 1).
L-tryptophan n-butyl ester (BTRP) were purchased from Sigma
Chemical Co. Pyridoxal 5ꢀ-phosphate (PLP) and all other chemi-
cals used were of reagent-grade and purchased from Merck.
Acetate, phosphate, and carbonate buffers were used over ap-
propriate pH ranges. The buffer concentration used was typically
0.02 M and the ionic strength was maintained at 0.1 M by adding
appropriate amounts of KCl to the medium.
The hydrolysis reaction of MTRP and BTRP was monitored by
measuring the presence of TRP by HPLC in a Star chromatogra-
phy workstation Varian 4.51 equipped with a Merck-Hitachi
LaChrom L-6250 pump and a Merck-Hitachi LaChrom L-7420 UV
detector. Conditions: LC-18-DB (25 cm, 5 mm) Supelco column,
eluant methanol–water 50:50, pH = 6.5, and isocratic mode
0.5 mL min−1. For BTRP in the whole studied pH range and for
MTRP at pH < 8, the hydrolysis was less than 1% after the time
necessary for the preheating and more than 10 half-lives of the re-
actions. Nevertheless, at pH > 8 the hydrolysis of MTRP was
more important (e.g. at pH = 10, 3% of TRP was found in 10 min-
utes).
PLP solutions were made in appropriate buffers and stored in
the dark. Their exact concentrations were determined by dilution
with 0.1 M HCl,10 and were found to be in the region of 2 × 10−5
M. TRP solutions spanning the concentration range from 5 × 10−4
to 2 × 10−2 M were also prepared, on a daily basis, by diluting ap-
propriate amounts of stock solutions in the corresponding buffer.
In order to prevent the hydrolysis of MTRP and BTRP, the solu-
tions were prepared inmediatly prior to use in the corresponding
buffer, spannning the same concentration range as that of the TRP
solutions.

2472 Bull. Chem. Soc. Jpn., 75, No. 11 (2002)
Kinetics of PLP with Esters of L-Tryptophan
Kinetic measurements were made at a variable pH by using a
Hewlett-Packard 8453 diode array spectrophotometer and a
Uvikon 941-Plus spectrophotometer furnished with thermostated
cells of 1-cm light path. In each case, the reaction was started by
adding a known volume of PLP buffered solution to prethermo-
stated TRP, MTRP (pH < 8), and BTRP solutions at (25 0.05)
°C. For the reaction of PLP with MTRP at pH > 8, where the hy-
drolysis of MTRP is important, freshly prepared PLP and MTRP
stock solutions at pH = 7 and 25 °C were added to a preheated so-
lution of the buffer at the desired pH.
The presence of TRP in the reactions was checked at the end of
the reactions, and was never greater than 3% (in most cases <
1%). Those reactions showing TRP > 3% at the end of the reac-
tion were disregarded.
The difference between the initial and final pH in the reaction
cell never exceeded 0.03 units. pH measurements were made by
using a Crison pH-meter equipped with a Metrohm EA120 elec-
trode that was previously calibrated with aqueous buffers at
25.0 °C.
variation of the logarithmic overall rate constants of formation
(k1) and hydrolysis (k2), and the equilibrium constant (KpH), for
the Schiff bases of PLP–TRP, PLP–MTRP, and PLP–BTRP
systems as a function of the pH.
Reaction measurements could only be made up to pH 10.5
because more alkaline media resulted in secondary imine in-
tramolecular cyclization by an attack of the indole group on
the azomethine carbon, thereby hindering the reaction (as pre-
viously observed in that between PLP and histidine12,13) and
precluding an examination beyond this alkalinity level.
In PLP–MTRP and PLP–BTRP systems, k1 increased with
increasing pH in acid and neutral media, and peaked at a pH of
about 7.0; however, for the PLP–TRP system, k1 increased
with increasing pH, but no peak was observed. Below pH 9, k1
were greater for the ester systems than for the PLP–TRP sys-
The overall reaction between an aldehyde and an amine can be
schematized as follows:
k1
R1 – CHO + NH2 – R2
R1 – CHwN – R2 + H2O,
k2
(1)
where k1 and k2 are the overall rate constants of formation and hy-
drolysis, respectively, of the Schiff base. The rate Eq. 1, taking
into account the Beer–Lambert law and that the kinetics of both
the forward and the reverse reactions are second-order, is Eq. 2
with the application of Eq. 3, where a and b are the initial alde-
hyde and amine concentration, respectively:
A∞ − A0
A∞ − A
ab−xxe
ln
= −ln
+ kobst,
xe2
(2)
(3)
1/ 2
2
kobs
=
k + k
(
a + b
)
− 4abk12
.
[
{
]
}
2
1
Fig. 1. Plot of log k1 vs pH for the PLP–TRP (ꢀ), PLP–
MTRP (ꢁ), and PLP–BTRP (ꢂ) systems. Curves calculat-
ed using Eq. 7 and data from Table 1.
Here, A0, A, and A are the absorbances at times 0, t, and ∞, re-
∞
spectively; k1 and k2 are the overall formation and hydrolysis rate
constants for the Schiff base.
The kobs values were obtained from the slopes of the plots of
ln(A −A) versus time, since ab >> xxe. The k1 and k2 values
∞
were calculated from the kobs values obtained at a given pH for dif-
ferent (a + b) values.11 Their ratio coincides with the equilibrium
constant (KpH = k1/k2). Nucleophilic rate constants (kN) were ob-
tained from the slopes of linear plots of kobs vs the free amine con-
centration, or by dividing the k1 values into the corresponding free
amine mole fractions.
The deprotonation equilibrium constant (KN) values for TRP,
MTRP, and BTRP were determined potentiometrically by titration
with 0.1 M NaOH of 0.01 M solutions of the corresponding amine
and 0.01 M HCl, using a Radiometer autotitrator equipped with a
PHM-62 pH-meter, an ABU-11 autoburette, a TTT-60 titrator, a
REA-160 recorder, a TTA-60 thermostatic support, a G-2040
glass electrode, and a K-4040 calomel electrode. The experimen-
tal conditions used were the same as those for the kinetic measure-
ments. The pKN values for TRP, MTRP, and BTRP thus found
were 9.2, 7.6, and 8.0, respectively.
Fig. 2. Plot of log k2 vs pH for the PLP–TRP (ꢀ), PLP–
MTRP (ꢁ), and PLP–BTRP (ꢂ) systems. Curves calculat-
ed using Eq. 8 and data from Table 1.
Results
Figures 1–3 show the experimental results in the form of the

G. R. Echevarría Gorostidi et al.
Bull. Chem. Soc. Jpn., 75, No. 11 (2002) 2473
ference between the two systems was the presence of a carbox-
yl (or carboxylate) group or an ester function in the carbon α,
it must have been the origin of the observed differential behav-
ior.
Figure 3 is consistent with the results for other similar sys-
tems. As can be seen, KpH increases with increasing pH up to a
value in between that of pKN for the amine and the value of the
pK3P for PLP (see Scheme 1);14 the experimental maximum
lies at about 8.7 for the PLP–TRP system and 7.8, and 8.0 for
the PLP–MTRP and PLP–BTRP systems, respectively (the
pKN values for TRP, MTRP, and BTRP are 9.2, 7.6, and 8.0, re-
spectively, and the pK3P for the PLP15 is 8.2).
The apparently increased reactivity of MTRP and BTRP
with PLP relative to TRP with PLP below pH 9 (see Fig. 1)
arises from the differential values of pKN for the amino group
in the three compounds. One way of comparing their reactivi-
ty is by making the formation rate constants for the Schiff
bases independent of pK for the amino group. Figure 4 shows
the variation of the nucleophilic rate constant, kN, with pH; as
can be seen, TRP is more reactive than MTRP and BTRP
throughout the pH range studied, but in the 6 to 8 pH range is
very symilar.
Fig. 3. Plot of log KpH vs pH for the PLP–TRP (ꢀ), PLP–
MTRP (ꢁ), and PLP–BTRP (ꢂ) systems. Curves calculat-
ed using Eq. 9 and data from Table 1.
tem (Fig. 1).
As shown in Scheme 1, the overall rate constants of forma-
tion and the hydrolysis of the Schiff bases can be described in
On the other hand, k2 was greater for the ester systems
throughout the pH range studied (Fig. 2). Because the sole dif-
Scheme 1.

2474 Bull. Chem. Soc. Jpn., 75, No. 11 (2002)
Kinetics of PLP with Esters of L-Tryptophan
kOH + k22 ⋅a/ K3B + k21 ⋅a2 /(K3B ⋅ K2B) + k20 ⋅a3 /(K3B ⋅ K2B ⋅ K1B
)
,
k2
=
(8)
(9)
1 + a/ K3B + a2 /(K3B ⋅ K2B) + a3 /(K3B ⋅ K2B ⋅ K1B
)
(1 + a/ K3B + a2 /(K3B ⋅ K2B) + a3 /(K3B ⋅ K2B ⋅ K1B))KM
(1 + a/ KN )(1 + a/ K3P + a2 /(K3P ⋅ K2P ) + a3 /(K3P ⋅ K2P ⋅ K1P ))
,
KpH
=
3
2
where kOH = k2 + kOH (KW/K3B), a = 10−pH and KW is the
ionic product of H2O.
The experimental values of k1, k2, and KpH were fitted simul-
taneously to Eqs. 7–9 by a nonlinear regression method involv-
ing minimization of the functions U1, U2, and UpH:
U1 =
(logk1, e − logk1, t )2,
(logk2, e − logk2, t )2,
(logKpH, e − logKpH, t )2,
(10)
(11)
(12)
∑
∑
U2
=
Fig. 4. Plot of log kN vs pH for the PLP–TRP (ꢀ), PLP–
MTRP (ꢁ), and PLP–BTRP (ꢂ) systems. Curves calculat-
ed using theoretical values of k1.
UpH
=
∑
terms of the individual constants for the different chemical
species present in the medium at each pH.
where subscripts e and t denote the experimental and theoreti-
cal data, respectively.
The values of the pK of PLP are found in ref. 15 and those
for TRP, MTRP, and BTRP were determined in this work (see
Material and Methods).
i
i
Thus, k1 and k2 (with i = 0, 1, 2, or 3) are the individual
rate constants of formation of the Schiff bases and of their hy-
drolysis by H2O, and k2OH is the rate constant of hydrolysis of
species B2 (a Schiff base with a net charge of −2) by OH−
ions. Pi (with i = 0, 1, 2, or 3) denotes the different chemical
species of PLP, and pK3P, pK2P, and pK1P the different pK val-
ues that relate them. Bi (with i = 0, 1, 2, or 3) are the different
chemical species of the Schiff bases, and pK3B, pK2B, and pK1B
are the pK values that relate them. Finally, KN is the deproto-
Table 1 gives the individual rate constants of the formation
i
i
(k1 ) and hydrolysis (k2 , kOH), as well as the pK values obtained
in the fitting of k1, k2, and KpH to Eqs. 7, 8, and 9. For compar-
isons, the values for the Schiff bases of PLP with other amino
acids are also listed.8,9
At this point it is interesting to notice that the reaction
mechanism is through a carbinolamine intermediate, as in Eq.
13 (R1–CHO is Pi (with i = 0, 1, 2, or 3) and R1–CHwN–R2 is
+
nation equilibrium constant of the –NH3 group in TRP,
MTRP, or BTRP.
i
In Scheme 1 the hydrolysis reactions of the forms Bi (i = 0,
1) for OH− have been omitted because of the very small con-
centration of OH− at the pH at which the concentration of Bi (i
= 0, 1) is important.
Bi (with i = 0, 1, 2, or 3)). The k1 rate constants in Scheme 1
involve the formation of carbinolamine rate constants (kci) and
those from the intermediate carbinolamine to form the Schiff
base by water elimination (kdi) and to return to reactants by
amine elimination (k−ci):
The rate of formation of the Schiff base is given by
3
kci
v1 =
[
RNH2
]
k1i
[
P
i
]
= k1
[
RNH2 ]T[PLP
]
,
(4)
∑
T
R1 – CHO + NH2 – R2
R1 – CH(OH) – NH – R2
i = 0
k−ci
where T denotes the concentration of all the species.
The hydrolysis of the Schiff base conforms to Eq. 5:
kdi
,
(13)
R1 – CHwN – R2 + H2O
3
k−di
2
v2 = kOH
The equilibrium constant (KpH) is given by
KpH = [Schiff Base]T/([RNH2]T[PLP]T).
[
B2
]
[
OH−
+
k2i
[
Bi
]
= k Schiff Base .
[
]
]
2
∑
T
i = 0
(5)
If Kci = kci/k−ci is defined as the equilibrium formation con-
stant for carbinolamine, provided that this is formed and split
fairly rapidly, and is completely shifted to the reactants, then
k1 = Kcikdi and k2 = k−di. This is in accord with the linear
plots kobs vs [amine] obtained for the three amine-group bearer
at different pH values.
i
i
(6)
Taking into account the equilibria in Scheme 1 and the fact
that the equilibrium formation constant for the Schiff base at
very high pH values is given by KM = k1 /k2 , Eqs. 4–6 can be
Discussion
3
3
i
readily transformed into
As can be seen from Table 1, the k1 values for the reactions
of PLP with different amino acids are quite similar; conse-
quently, the side chains in the amino acids play no significant
role in the formation of Schiff bases with PLP, as has been de-
k13 + k12 ⋅a/ K3P + k11 ⋅a2 /(K3P ⋅ K2P ) + k10 ⋅a3 /(K3P ⋅ K2P ⋅ K1P
(1 + a/ KN )(1 + a/ K3P + a2 /(K3P ⋅ K2P ) + a3 /(K3P ⋅ K2P ⋅ K1P ))
)
k1
=
,
(7)

G. R. Echevarría Gorostidi et al.
Bull. Chem. Soc. Jpn., 75, No. 11 (2002) 2475
Table 1. Best Kinetic Constant Values Obtained in the Fitting of Experimental k1 (L mol−1 min−1), k2 (min−1), and KpH (L mol−1
Values to Scheme 1, and Those Corresponding to the PLP–GLY,9 PLP–LEU,9 PLP–ILE,9 PLP–PHE,8 and PLP–ALA8 Systemsa)
)
PLP–TRP
PLP–MTRP
PLP–BTRP
PLP–GLY
PLP–LEU
PLP–ILE
PLP–PHE
PLP–ALA
0
1
2
3
log k1
log k1
log k1
log k1
pK1P
pK2P
pK3P
log k2
log k2
log k2
6.83 0.07
5.15 0.03
3.14 0.07
1.96 0.08
3.46b)
5.77 0.09
4.94 0.03
2.64 0.10
1.80 0.05
3.46b)
6.19 0.07
5.11 0.03
2.26 0.10
1.76 0.06
3.46b)
7.30
5.52
3.79
2.50
3.58
7.16
5.05
3.36
2.25
3.61
7.31
5.20
3.54
2.34
3.61
—
4.75
2.96
2.23
—
5.95
8.14
—
—
5.01
3.23
2.39
—
5.95
8.14
—
—
—
—
—
6.02b)
6.02b)
6.02b)
5.90
8.33
5.90
8.34
5.86
8.33
8.22b)
8.22b)
8.22b)
0
1
2
−0.42 0.10
0.33 0.05
−1.16 0.07
0.01 0.17
6.04 0.35
6.33 0.30
10.58 0.30
2.10 0.28
0.66 0.12
0.69 0.04
−0.70 0.09
−0.34 0.07
4.34 0.20
6.72 0.13
9.66 0.22
2.10 0.18
0.67 0.09
0.65 0.04
−1.08 0.25
0.32 0.08
4.54 0.14
7.09 0.11
10.1 0.60
1.48 0.60
0.584
0.676
−0.550
1.160
5.46
6.36
11.35
1.33
0.170
0.080
−0.900
1.080
5.68
6.65
11.61
1.19
−0.010
−0.170
−1.000
1.020
5.67
6.66
11.57
1.31
—
0.477
0.64
—
6.55
12.3
—
log kOH
pK1B
pK2B
pK3B
—
11.78
—
log KM
a) GLY, LEU, ILE, PHE and ALA denote glycine, L-leucine, L-isoleucine, L-phenylalanine, and L-alanine, respectively. b) From
Ref. 15.
Scheme 2.
i
scribed.8 A comparison of the k1 values obtained for the PLP–
MTRP and PLP–BTRP systems reveals the absence of signifi-
cant differences (see Table 1); therefore, the methyl or n-butyl
group size is not important in Schiff-base formation.
bear a deprotonated carboxyl group (the reported pK for the
carboxyl group in tryptophan is17 2.4). If this is an explanation
to the observed behavior, the Brönsted must be curved up in
the acidic media; nevertheless, the plot is linear with α = 0.77
(Fig. 5), as in the other α-amino acids9 α = 0.74–0.78, pre-
cluding a catalytic effect due to the carboxylic group. On the
other hand, the PLP–MTRP and PLP–BTRP show non-linear
Brönsted plots (Fig. 5), showing that the ester groups inhibed
the Schiff-base formation, probably due to a steric inhibition.
The maxima in the k1 curves (Fig. 1) are a result of the pKN
of the amino group (the mole fraction of free amine increases
i
A comparison of the k1 values obtained for PLP–TRP and
ester systems (Table 1) reveals the presence of a strong differ-
ences in k10 and k1 . This effect is also apparent by comparing
2
the kN curves of Fig. 4, which exhibit appreciable differences
in acid and alkaline media, but virtually none in neutral media.
The rate-determining step in the formation of a Schiff base
is known to be the dehydration of an intermediate carbinola-
mine formed by an attack of the amine on the carbonyl
group.4,5 It is also known that, with PLP, the dehydration is
subject to intramolecular acid catalysis, and that the phenol
group at C-3 on the pyridine ring plays an especially promi-
nent role in acid media.16 The dehydration of carbinolamines
of these systems in an acid medium (structures ꢁ and ꢂ in
Scheme 2) is catalyzed by the phenol group; in addition, struc-
ture I, which corresponds to the PLP–TRP system, can release
a water molecule by the interaction of the proton in the carbox-
yl group with the OH group in the carbinolamine. This is im-
possible in the ester and in those chemical species of TRP that
2
with increasingpH) and of the sequence k10 > k11 > k1 (the re-
activity decreases with increasing pH). The net balance between
these two opposing effects leads to the maximum in Fig. 1.
Figure 2 reveals an increased stability of the Schiff base of
TRP relative to that of esters, consistent with the values of the
i
individual k2 constants in Table 1. The presence of non-polar
groups in the vicinity of the hydrolysis site reportedly protects
the imine bond from hydrolysis,9 as found, for example, in
comparing the Schiff bases of PLP with L-leucine (LEU) and
L-isoleucine (ILE), and those of the same substrate with gly-
cine (GLY) and LEU (Table 1). This effect has also been ob-

2476 Bull. Chem. Soc. Jpn., 75, No. 11 (2002)
Kinetics of PLP with Esters of L-Tryptophan
and Programa para la Cooperación con Iberoamérica.
References
1
E. E. Snell, “Pyridoxal Phosphate: History and
Nomenclature,” in “Vitamin B-6 Pyridoxal Phosphate. Chemical,
Biochemical and Medical Aspects. Part A,” ed by D. Dolphin, R.
Poulson, and O. Avramovic, J. Wiley & Sons, New York (1986),
pp. 1–12.
2
V. C. Emery and M. Akhtar, “Pyridoxal Phosphate
Dependent Enzymes,” in “Enzyme Mechanisms,” ed by M. I. Page
and A. Williams, The Royal Society of Chemistry, London
(1989), pp. 345–389.
3
A. E. Braunstein, “Transamination and Transaminases,” in
“Transaminases,” ed by P. Christen and D.E. Metzler, J. Wiley &
Sons, New York (1985), pp. 2–19.
4
W. P. Jencks, in “Catalysis in Chemistry, and
Enzymology,” McGraw-Hill, New York (1969), pp. 490–496.
S. Rosemberg, S. M. Silver, J. M. Sayer, and W. P. Jencks,
J. Am. Chem. Soc., 96, 7986 (1974).
R. B. Silverman, in “The Organic Chemistry of Drug
Fig. 5. Brönsted plot for the PLP–TRP (ꢃ), PLP–MTRP
(ꢁ), and PLP–BTRP (ꢂ) systems.
5
6
served in the Schiff bases of PLP with poly L-lysines,18 where
the polypeptide chain provides a less polar environment and
leads to bases of increased stability. Accordingly, the methyl
or n-butyl group in the ester should result in less readily hydro-
lyzed bases. However, the behavior of the Schiff bases studied
here leads to the opposite conclusion. Thus, the presence of
the carbonyl group or the carboxylate anion stabilizes the
Schiff base in relation to the alkyl group in the ester.
Design and Drug Action,” Academic Press Inc., San Diego (1992),
pp. 116–125.
7
a) G. R. Echevarría Gorostidi, A. Basagoitia, E. Pizarro, R.
Goldsmitd, J. G. Santos, and F. García Blanco, Helv. Chim. Acta,
81, 837 (1998), and refs. therein. b) G. R. Echevarría Gorostidi,
M. P. Martín Pérez, J. G. Santos, and F. García Blanco, Helv.
Chim. Acta, 82, 769 (1999).
8
E. Gout, M. Zador, and C. G. Beguin, Nouv. J. Chim., 8,
In the same way, compared to other described models,7–9 the
curves log k2 vs pH of Fig. 2 show a minimum in accordance
243 (1984).
9
M. A. Vázquez, F. Muñoz, J. Donoso, and F. García
2
Blanco, Int. J. Chem. Kinet., 22, 905 (1990).
10 E. A. Peterson and H. A. Sober, J. Am. Chem. Soc., 76, 169
(1954).
with the fact that k2 is the minor of the individual hydrolysis
rate constants (see Table 1).
Table 1 also gives the pK values for the various forms of the
Schiff bases derived from the different amino acids; as can be
seen, there are no marked differences in this respect. However,
a comparison of the pK values for the Schiff bases of the PLP–
TRP and PLP–MTRP or PLP–BTRP systems reveals signifi-
cant differences in pK1B and pK3B, which are smaller in the es-
ter. It should be noted that the differences are quite close to
that in the pKN for the amino group in TRP relative to that in
MTRP or BTRP. The difference in pK3B is quite normal, be-
cause it involves an imine nitrogen; on the other hand, the fact
that the effect is passed onto pK1B suggests that this corre-
sponds to the pyridine nitrogen. The effect can be transmitted
to this atom, but not to the phosphate group, to which pK2B can
be assigned. This result allows one to unequivocally assign the
protonation sequence of Scheme 1.
11 M. A. Garcia del Vado, J. Donoso, F. Muñoz, G.
Echevarría, and F. García Blanco, J. Chem. Soc., Perkin Trans. 2,
1987, 445.
12 T. C. Bruice and A. Lombardo, J. Am. Chem. Soc., 91,
3009 (1969).
13 M. Coll, J. Frau, J. Donoso, F. Muñoz, and F. García
Blanco, An. Quim., 87, 179 (1991).
14 C. M. Metzler, A. Cahill, and D. E. Metzler, J. Am. Chem.
Soc., 102, 6075 (1980).
15 G. Echevarría, M. A. García del Vado, F. García Blanco, M.
Menéndez, and J. Laynez, J. Solution Chem., 15, 151 (1986).
16 J. M. Sánchez-Ruiz, J. M. Rodríguez Pulido, J. Llor, and
M. Cortijo, J. Chem. Soc., Perkin Trans. 2, 1982, 1425.
17 C. R. Cantor and P. R. Schimmel, in “Biophysical
Chemistry,” W. H. Freeman & Company, New York (1980), p. 49.
18 M. A. García del Vado, G. R. Echevarría, F. García Blanco,
J. G. Santos, M. Blázquez, J. M. Sevilla, and M. Domínguez, J.
Mol. Catal., 68, 379 (1991).
This work was funded by Spain’s DGI (Project BQ2000-
0646), FONDECYT (Chile, Projects 1990551 and 2990006)