Effect of pH on the reactivity of dipeptides and α-amino acids in the N-acylation

Article abstract of DOI:10.1134/S1070363212110126

The reaction kinetics of Gly, L-α-Ala, Gly-Gly, L-α-Ala-L- α-Ala and β-Ala-β-Ala with picryl benzoate in water (40 wt %)-2-propanol was investigated. At pH = 4-8 the rate constants of N-acylation of the anionic form of dipeptides are less than those of the corresponding amino acid anions, in agreement with their basicity, whereas the relative effective rate constants of reactions depend on pH: in acidic, neutral and slightly alkaline media the k_ef values are higher for the dipeptides, and in a strongly alkaline medium, for the amino acids. These differences are due to the changes in the concentrations of reactive forms of amino acids and dipeptides in the system at varying the medium pH.

Full text of DOI:10.1134/S1070363212110126

ISSN 1070-3632, Russian Journal of General Chemistry, 2012, Vol. 82, No. 11, pp. 1810–1814. © Pleiades Publishing, Ltd., 2012.
Original Russian Text © L.V. Kuritsyn, N.V. Kalinina, L.B. Kochetova, 2012, published in Zhurnal Obshchei Khimii, 2012, Vol. 82, No. 11, pp. 1820–1824.
Effect of pH on the Reactivity of Dipeptides
and α-Amino Acids in the N-Acylation
L. V. Kuritsyn, N. V. Kalinina, and L. B. Kochetova
Ivanovo State University, ul. Ermaka 39, Ivanovo, 153025 Russia
e-mail: kochetova_lb@mail.ru
Received September 13, 2011
Abstract—The reaction kinetics of Gly, L-α-Ala, Gly-Gly, L-α-Ala-L-α-Ala and β-Ala-β-Ala with picryl
benzoate in water (40 wt %)–2-propanol was investigated. At pH = 4–8 the rate constants of N-acylation of the
anionic form of dipeptides are less than those of the corresponding amino acid anions, in agreement with their
basicity, whereas the relative effective rate constants of reactions depend on pH: in acidic, neutral and slightly
alkaline media the kef values are higher for the dipeptides, and in a strongly alkaline medium, for the amino
acids. These differences are due to the changes in the concentrations of reactive forms of amino acids and
dipeptides in the system at varying the medium pH.
DOI: 10.1134/S1070363212110126
Over the years we have studied the kinetics of N-
acylation of α-amino acid with the derivatives of
aromatic carboxylic acids in aqueous-organic solvents
[1–10]. In continuation of these studies we aimed to
study the kinetics of reactions Gly (I), Gly–Gly (II), L-
α-Ala (Ia), L-α-Ala–L-α-Ala (IIa) and β-Ala–β-Ala
(IIb) with picryl benzoate in the solvent water (40 wt %) –
2-propanol.
compound concentration over the ester. The observed
first-order reaction rate constant kobs was calculated by
the method of Guggenheim. The second order reaction
rate constants k– were calculated according to Eq. (2):
k– = kobs/k–
(2)
The equations of the relationship between the rate
constants kobs and k– of the α-amino acid reactions with
esters are presented in [2–4].
The studied reaction proceeds according to Eq. (1):
The kobs and k– values of the studied reactions, as
well as the activation energy and entropy E– and ΔS≠,
respectively, of the picryl benzoate reaction with
anionic forms IIa and IIb, calculated from the
temperature dependence of the rate constants k–, are
presented in Table 1. The data in Table 1 indicate the
relationship of basicity of the compounds studied to
their reactivity in the N-acylation, which is manifested
in the decrease in the acylation rate constant k– of
dipeptides in comparison with the corresponding
amino acids. A similar pattern is observed in the
interaction of α-amino acids and dipeptides with
benzoyl chloride in binary mixtures of water with
dioxane [1, 3, 6]. However, the basicity of the amino
group of dipeptides is not the only factor governing
their reactivity in the N-acylation.
С6Н5СOOС6Н2(NO2)3 + NH2RСОО–
k–
→ С6Н5CONHRСОО– + HOС6Н2(NO2)3,
(1)
where R is the substituent in compounds I, II, Ia–IIb;
k– is the rate constant of the N-acylation of α-amino
acid (dipeptide) anion.
The reaction rate was monitored spectrophoto-
metrically (λ = 400 nm). The conditions of kinetic
experiments were chosen in order to provide only one
reactive form of α-amino acid or dipeptide in solution:
the anionic form. We have previously shown that at the
concentration ratio of zwitter-ions and anions of α-
amino acid c±/c– in the range of 4–10 the conditions
are provided under which the rate of hydrolysis of the
ester can be neglected [2–4]. Therefore, a known amount
of alkali was added to the amino acid (dipeptide)
solution. The reaction was performed in the first-order
conditions with 102–103 times excess of amino
The properties of α-amino acids and dipeptides in
solution largely depend on their acid–base interactions
with the components of the medium. The protonation
1810

EFFECT OF pH ON THE REACTIVITY OF DIPEPTIDES
1811
Table 1. Kinetic parameters of N-acylation of anionic forms of amino acids and dipeptides with picryl benzoate in the water–
2-propanol solvent at 298 Ka
Parameter
obs×102, s–1
k–, l mol–1 s–1
Gly
2.19±0.04
15.1±0.3
α-Ala
1.69±0.05
4.7±0.1
Gly-Gly
1.07±0.09
3.0±0.2
α-Ala-α-Ala
0.366±0.007
1.01±0.02
β-Ala-β-Ala
1.85±0.03
5.10±0.08
k
α-Ala-α-Ala
β-Ala-β-Ala
k
–318, l mol–1 s–1
2.57
37
129
k
–318, l mol–1 s–1
13.1
37
116
Е–, kJ mol–1
Е–, kJ mol–1
–∆S≠, J mol–1 K–1
–∆S≠, J mol–1 K–1
a ctotal = 0.022 M, c– = 0.00362 mol l–1, cpicryl benzoate = 2×10–5 M, the mole fraction of water in solvent X(H2O) = 0.690.
and deprotonation of functional groups most strongly
affect the reactivity. It is known [1–9] that the
protonated amino group of α-amino acids, as well as
other aliphatic and aromatic amines, is inert in the N-
acylation, therefore in reactions with acylating agents
only two of the four forms of α-amino acids in the
solution can be involved: uncharged and anionic.
Zwitterions and cations are not active in these
reactions. It was also established that in aqueous-
organic media the rate of reaction of acylating agents
with anions of α-amino acids is significantly higher
than with non-ionized molecules.
divided by the total concentration of amino acid
(dipeptide) [Eq. (5)]:
kef = (c–/ctotal)k– = α–k–.
(5)
Here α = c–/ctotal is the fraction of anions in the total
concentration of amino acid (dipeptide) in solution.
According to [3], at the change of pH in the range
of 4–12 the fraction of the neutral molecules and
cations of α-amino acid in water–isopropanol solvent
containing more than 10 wt % of water is negligible,
so we may assume that c0 + c+ = 0, then ctotal = c± + c–.
It can be shown [2–5] that under these conditions the
kef is related to pH in accordance with Eq. (6):
The main factor determining the concentrations of
various ionic forms of amino acids and dipeptides in
solution is the acidity. By varying the pH, the
concentration of reactive species that determine the
effective rate of acylation [3, 5, 10] can be changed by
several orders of magnitude, and therefore it is of
interest to establish the dependence of the effective
kinetic parameters of the interaction of amino acids
and dipeptides with picryl benzoate on the pH of the
solution.
kef = k–Ka/(Ka + cH+),
(6)
where Ka is the acid dissociation constant of the
protonated amino acids (dipeptide).
While accounting for Eq. (6) and using Eqs. (7),
(8), we can calculate the effective values of the energy
and changes in the activation entropy, Eef and ΔSef≠ for
different pH:
Еef = E– + ΔHacH+/(Ka + cH+),
∆S≠ef = Rln (kefh/kBT) + Еef/T.
(7)
(8)
The equation of reaction rate constant [Eq. (1)] can
be written as [Eq. (3)]:
Here ΔHa is the enthalpy of dissociation of the
protonated amine, h is the Planck constant, kB is the
Boltzmann constant.
–dc/dτ = kefctotal
c
picryl benzoate = kobscpicryl benzoate
,
(3)
where ctotal is the total concentration of all forms of
amino acid (dipeptide) in solution, ctotal = c± + c0 + c– +
c+; c±, c0, c– and c+ are the concentrations of a zwitter-
ionic, uncharged, anionic, and cationic forms, respec-
Analysis of Eq. (6) shows that the dependence of
log kef on pH is different for different intervals of
acidity:
tively; cpicryl
is the concentration of picryl
benzoate
+
benzoate; kef is the effective rate constant.
(a) at pH = 4–8, where Ka << cH , the relationship is
linear with the slope equal to 1:
It follows from Eqs. (2) and (3) that the relationship
between kef and k– is defined by the Eq. (4):
log kef = log (k– Ka) + pH;
(9)
k
obs = k– c– = kefctotal
,
(4)
+
(b) at pH = 8–11, the values Ka and cH are
comparable, and Eq. (6) has the form:
which shows that the kef is the observed rate constant
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1812
KURITSYN et al.
Table 2. Effective kinetic parameters of the reactions of dipeptides with picryl benzoate in the water (40 wt %)–2-propanol
solvent at 298 K
рН
4
5
6
7
8
9
10
11
α-Ala-α-Ala
–log kef
Еef
4.45
84
3.45
84
2.45
84
1.46
82
0.578
71
0.104
47
0.008
38
0.003
37
–∆S≠ef
48
30
11
–2.9
16
89
118
121
β-Ala-β-Ala
–log kef
Еef
–∆S≠ef
5.15
74.5
94
4.15
74.5
74
3.15
2.15
74.5
36
1.16
74.0
19
0.209
69.9
14
–0.471
52.5
60
–0.677
39.1
74.5
55
101
Table 3.The ratio of kef of dipeptides and the corresponding amino acids
рН
γdip/am-acid
γk
γt
4
5
6
7
8
9
10
11
12
γII/I
9.8
24
9.8
24
9.5
24
9.2, 8.8а 6.6, 1.7b 1.8
23, 16а
18 5.4
0.39
0.86
0.22
0.28
0.20 0.195, 0.176а, 0.063b
0.22
0.22, 0.03а
50, 50а, 27b
110, 16а
γIIа/Iа
a γ, γk and γt were calculated from data in [3, 6]. b For the calculation the data of [11] were used.
log kef = log (k–Ka) – log (Ka + cH+);
(c) at pH = 11–13, Ka >> cH+, thus:
log kef = log k–.
(10)
Table 3 shows the values of γ for the reaction of I,
II, Ia, and IIa with picryl benzoate at different pH
calculated according to Eq. (12). As seen, the value of
γ depends on the acidity: in acidic and nearly neutral
media γ > 1, that is, dipeptides are more reactive; in
the alkaline medium the amino acids are more active in
the acylation (γ < 1). The pH value at which the
inversion of this ratio occurs (pHinv) is given by Eq. (13):
(11)
The results of calculation of the effective values of
the rate constants kef, the activation energy Eef, and
effective changes in the entropy of activation ΔS≠ef for
the reactions of picryl benzoate with IIa and IIb by
Eqs. (6)–(8) are presented in Table 2. These data show
that at pH < pKa the kef grows with increasing pH
value, and in an alkaline medium at pH > pKa it tends
to become constant. The values of Eef decrease with
increasing pH, and in the region close to the pKa a
sharp decrease occurs in Eef , from ~ 80 kJ mol–1 to
~40 kJ mol–1, ΔS≠ef dependence on pH is rather
complex, and passes through a maximum.
рНinv = рKa dip – log (γ – 1)
(13)
At pH = 4–8, in accordance with Eqs. (9), (12), the
parameter γ can be represented as follows:
γ = (k–dip/k–am-acid)·(Ka dip/Ka am-acid) = γkγt.
(14)
The value of γk in the expression (14) characterizes
the contribution of kinetic factors in the reactivity of
the amino acids and dipeptides, and the parameter γt
can be regarded as the thermodynamic factor. The
values of γk and γt for the reactions of I, II, Ia, and IIa
with picryl benzoate, as well as with benzoyl chloride
in aqueous dioxane [6] and N-acetyloxysuccinimide in
water [11] are presented in Table 3. Analysis of the
data in Table 3 shows that the kinetic factor is less than
unity in all the reactions, while the thermodynamic
factor contributes significantly more to the effective
rate of acylation. We can assume that γt is much larger
than γk also in the reactions of amino acids and
It must be borne in mind that all these changes of
effective kinetic parameters are associated not with the
changes in the acylation mechanism but rather with the
acid–base equilibria in the solutions of dipeptides
determining the concentration of their reactive forms.
The ratio of effective rate constants of acylation of
dipeptides kef dip and the corresponding amino acids
k
ef am-acid can be described using a coefficient γ:
γ = kef dip/kef am-acid
.
(12)
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 82 No. 11 2012

EFFECT OF pH ON THE REACTIVITY OF DIPEPTIDES
1813
Table 4. Electronic and energy characteristics of the amino
acids and oligopeptides anions calculated by the UHF/6-
31++G** method
oligopeptides with other acylating agents, which under
certain conditions may cause an increase in the
effective rate of acylation with increasing length of the
peptide chain. The kef may growth due to the growth of
γt which occurs due to the decrease in pKa of terminal
amino groups. In particular, in the series (15) the pKa
values decrease as follows: 9.6, 8.13, 7.91, and 7.75,
respectively [12–14], which leads to an increase in γt.
Therewith the values of k– and γk should decrease due
to diminishing basicity, but, owing to the
predominance of the thermodynamic factor over the
kinetic, the effective rate constant kef may increase.
Similar considerations are applicable in the case of α-
alanine and tri- and tetrapeptides.
Compound
–qN, au –q(NН2), au –EHOMO, eV ЕLUMO, eV
0.735
0.707
0.696
0.690
0.685
0.646
0.631
0.624
0.153
0.107
0.090
0.082
0.102
0.046
0.026
0.017
–
5.034
6.013
6.231
6.340
5.034
6.068
6.313
6.286
0.354
5.524
4.435
3.782
3.401
5.279
4.490
3.837
3.428
0.271
I
II
III
IV
Ia
IIa
IIIa
IVa
Gly, Gly–Gly, Gly–Gly–Gly (III),
Picryl benzoate –0.785a
Gly–Gly–Gly–Gly (IV).
(15)
a The charge on the carbonyl C atom.
We performed quantum-chemical simulation of the
anionic forms of compounds I–IV, Ia, IIa, as well as
L-α-Ala-L-α-Ala-L-α-Ala (IIIa) and L-α-Ala-L-α-Ala-
L-α-Ala-L-α-Ala (VIa).The calculations were carried
out with the software package Firefly 7.1.G [15], by
the method of UHF/6-31++G** with full geometry
optimization of the system. Some simulation results
are shown in Table 4.
decrease in k– in going from the amino acids to the
corresponding dipeptides (Table 1).
In this regard, we can assume that the kinetic
component of the effective rate of acylation of
dipeptides γk characterizes both the interaction of
molecular orbitals and the charge on the amino group.
In this case, as suggested above, the values of k– will
decrease in going from I to IV and from Ia to IVa due
to the decrease in EHOMO and also as a result of
reducing negative charge on the amino group (Table 4).
Comparison of the results for I–IV and Ia–IVa
with published data on the dissociation constants of
protonated amino groups of these compounds in water
[12–14] indicates the existence of linear dependence
between the values of pKa and charges on the amino
groups and q(NH2) (Fig. 1) . The charges on nitrogen
atoms qN of terminal amino groups in I–IV, Ia–IVa
also correlate with the pKa values. The dependence
shows that the thermodynamic component of the
effective rate of acylation γt is related to the charge
characteristics of amino groups.
The results of our study give some idea concerning
the acylation rate. The rate constant k– of the acylation
рKа
Therewith, the effect of the charge characteristics
on the rate of reaction (1) cannot be completely
excluded, as seen from the influence of basicity on the
k–. However, in several studies [7–9] it has been shown
that the reactions of α-amino acids with esters [3]
proceed predominantly under the orbital control. Table
4 shows calculated energies of frontier orbitals of the
nucleophiles I–IV, Ia–IVa and the acylating agent
picryl benzoate. The comparison of these values
suggests a possibility of orbital control also in the
reactions of dipeptides with picryl benzoate. A
decrease in the energy of HOMO of the dipeptides II
and IIa compared with the EHOMO of amino acids I and
Ia is consistent with the experimentally established
–q(NH2)
The plots of pKa of the protonated amino groups of amino
acids and peptides [12–14] vs. the charge on the amino
group q(NH2): (1) I, (2) II, (3) III, (4) IV, r = 0.999, (1a)
Ia, (2a) IIa, (3a) IIIa, (4a) IVa, r = 0.985.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 82 No. 11 2012

1814
KURITSYN et al.
2. Khripkova, L.N., Kuritsyn, L.V., and Kalinina, N.V.,
of reactive forms of amino acid with esters is much
higher than the k– of the corresponding dipeptides,
which is consistent with the higher basicity of amino
acids and charge characteristics of their amino groups.
The decrease in the basicity of the peptides in the
series (15) due to reduced negative charge on the
amino group and a decrease in EHOMO suggests that the
rate constant k– of the acylation of peptide anionic
forms II–IV, IIa–IVa should decrease with increasing
length of the peptide.
Zh. Obshch. Khim., 2004, vol. 74, no. 10, p. 1665.
3. Kuritsyn, L.V., Kustova, T.P., Sadovnikov, A.I.,
Kalinina, N.V., and Klyuev, M.V., Kinetika reaktsii
atsil’nogo perenosa (Kinetics of Anion Transfer
Reactions), Ivanovo: Ivanovo State Univ., 2006.
4. Kuritsyn, L.V., Lebedukho, A.Yu., Sadovnikov, A.I.,
Zh. Obshch. Khim., 2003, vol. 73, no. 4, p. 600.
5. Kuritsyn, L.V., Sadovnikov, A.I., Khripkova, L.N.,
Kalinina, N.V., and Shcherbakova, Yu.S., Zh. Obshch.
Khim., 2006, vol. 75, no. 1, p. 49.
Effective kinetic parameters of reactions of the
amide (peptide) bond are defined by the medium
acidity: increase in pH in the range 4–9 reduces by 4–5
orders of magnitude the effective rate constants kef of
dipeptide acylation. The ratio of effective rate
constants of amino acids acylation and of the
corresponding dipeptides also is pH-dependent: in
acidic, neutral and slightly alkaline media the kef of
dipeptides are higher compared with amino acids,
while in strong alkaline media the situation is reversed.
6. Kuritsyn, L.V. and Kalinina, N.V., Zh. Org. Khim.,
1994, vol. 30, no. 5, p. 723.
7. Ishkulova, N.R., Oparina, L.E. Kochetova, L.B., Kus-
tova, T.P., Kalinina, N.V., and Kuritsyn, L.V., Zh.
Obshch. Khim., 2010, vol. 80, no. 5, p. 794.
8. Kustova, T.P., Kochetova, L.B., and Kalinina, N.V., Zh.
Obshch. Khim., 2009, vol. 79, no. 5, p. 713.
9. Kochetova, L.B., Kalinina, N.V., and Kustova, T.P., Izv.
Akad. Nauk, Ser. Khim., 2009, no. 4, p. 725.
10. Kuritsyn, L.V. and Kalinina, N.V., Zh. Fiz. Khim., 1996,
Note that all the above mentioned changes in the
effective kinetic parameters of acylation are due to the
changes in the concentration of reactive forms of
amino acids and dipeptides in the system at varying the
pH of the medium.
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11. Hirata, H. and Nakasato, S., J. Jpn. Oil Chem. Soc.,
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12. Dawson, R.M.C., Elliott, D.C., and Elliott, W.H., Data
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ACKNOWLEDGMENTS
13. Edsall, J.T., Biophysical Chemistry, New York:
This work was supported by the Ministry of
Education and Science (project RNP.2.2.1.1.2820).
Academic Press, 1958.
14. Ellenbogen, E.J., J. Am. Chem. Soc., 1952, vol. 74,
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