Reactivity of Tyrosyl–Proline toward Benzoylation in Aqueous 1,4-Dioxane

Article abstract of DOI:10.1134/S1070428020060111

Abstract: The kinetics of the reaction of L-tyrosyl-L-proline (Tyr–Pro) with di- andtrinitrophenyl benzoates in aqueous 1,4-dioxane (40 wt percent of water) were studiedin the temperature range 298–313 K. The reaction rate constant k₂₉₈ was fou

Full text of DOI:10.1134/S1070428020060111

ISSN 1070-4280, Russian Journal of Organic Chemistry, 2020, Vol. 56, No. 6, pp. 1034–1040. © Pleiades Publishing, Ltd., 2020.
Russian Text © The Author(s), 2020, published in Zhurnal Organicheskoi Khimii, 2020, Vol. 56, No. 6, pp. 933–940.
Reactivity of Tyrosyl–Proline toward Benzoylation
in Aqueous 1,4-Dioxane
T. P. Kustovaa,*, I. I. Loktevaa, L. B. Kochetovaa, and D. S. Khachatryanb
a Ivanovo State University, Ivanovo, 153025 Russia
b Institute of Chemical Reagents and Highly Pure Chemical Substances, “Kurchatov Institute” National Research Center,
Moscow, 107076 Russia
*e-mail: kustova_t@mail.ru
Received January 9, 2020; revised February 7, 2020; accepted February 11, 2020
Abstract—The kinetics of the reaction of L-tyrosyl-L-proline (Tyr–Pro) with di- and trinitrophenyl benzoates in
aqueous 1,4-dioxane (40 wt % of water) were studied in the temperature range 298–313 K. The reaction rate
constant k298 was found to vary in the range from 0.035 to 0.564 L·mol–1·s–1, and the activation barrier changed
from 39 to 51 kJ/mol. The neutral and anionic forms of the dipeptide were simulated by the DFT/B3LYP/
cc-pVTZ method. Natural bond orbital analysis of the electron density distribution in these forms showed that
the preferred nucleophilic center in the dipeptide molecule is the nitrogen atom of the primary amino group
rather than the oxygen atom of the phenolic hydroxy group.
Keywords: dipeptide, tyrosylproline, benzoylation, kinetics, 1,4-dioxane, 2,4- and 2,6-dinitrophenyl benzoates,
picryl benzoate
DOI: 10.1134/S1070428020060111
Up-to-date systematic data on the reactivity of
dipeptides in acyl transfer reactions are confined to our
previous data for the reactions of glycyl–glycine and
alanyl–alanine with a number of esters and for the reac-
tions of dipeptides with benzoyl chloride and sulfonyl
chlorides in aqueous 1,4-dioxane [1–4]. Meanwhile, in
recent publications, much attention has been given to
biological activity of both natural oligopeptides and
their functional derivatives, as well as to metabolism of
these compounds. Guzevatykh et al. [5, 6] analyzed
amino acid sequences of 130 opioid peptides belonging
to 30 families and revealed a high occurrence rate of
the Tyr–Pro dipeptide fragment; the authors also found
experimentally that Tyr–Pro is the shortest among
known amino acid sequences exhibiting analgesic
activity in thermal, mechanical, and chemical pain
stimulation in rats. It was found that Tyr–Pro analogs
obtained by modification at the proline carboxy group,
Tyr–Pro–X (where X = NH2, OMe, OEt), showed
a high activity. However, synthesis of Tyr–Pro deriva-
tives at the tyrosine α-amino group is restrained due
to the lack of data on the reactivity of this nucleo-
philic center.
centers in the Tyr–Pro molecule, nitrogen atom of the
primary amino group and oxygen atom of the phenolic
hydroxy group, by quantum chemical calculations of
structural, energetic, and electronic parameters of the
neutral and anionic forms of the dipeptide, as well as
by measuring the kinetics of its acylation.
The kinetics of the reactions of L-Tyr–L-Pro with
2,4- and 2,6-di- nitrophenyl and 2,4,6-trinitrophenyl
benzoates were studied in aqueous 1,4-dioxane in the
temperature range 298–313 K (Scheme 1). As we
showed previously [2], the only reactive species of
α-amino acids and dipeptides in reactions with esters at
pH 8.5–9 are the corresponding anions. The N-acyla-
tion in aqueous–organic medium can be accompanied
by hydrolysis of esters. We found in [2–4] that phenyl
benzoates are not hydrolyzed in water; therefore,
only alkaline hydrolysis of the ester (Scheme 2) was
taken into account in addition to the main reaction
(Scheme 1).
The reaction kinetics were measured by spectro-
photometry (λ 400 nm) using a large excess of the di-
peptide whose concentration was higher by two orders
of magnitude than the concentration of the acylating
agent (ester). The reaction rate was monitored by the
concentration of the products, di(tri)nitrophenoxide
The goal of the present work was to perform a com-
prehensive study of the reactivity of two nucleophilic
1034

REACTIVITY OF TYROSYL–PROLINE TOWARD BENZOYLATION
1035
Scheme 1.
O
O–
O
O
(NO2)n
+
N
Ph
O
NH2
HO
O–
N
O
O
(NO2)n
+
O
HO
HN
Ph
HO
n = 2, 3.
Scheme 2.
O
O
2OH–
+
(NO2)n
(NO2)n
+
Ph
O–
–O
Ph
O
n = 2, 3.
ions. The reaction rate with respect to the reagent (cac)
is given by Eq. (1):
unreactive zwitterionic (c±) and reactive anionic forms
(c) of an α-amino acid or dipeptide is higher than 4
(c±/c > 4), the rate of alkaline hydrolysis of the ester
can be neglected. In this case, the rate constant of the
reaction shown in Scheme 1 can be calculated by the
equation
∂cac
–
= [kh + (kα)c0]cac = kobscac,
(1)
∂τ
where α is the concentration ratio of the reactive
(anionic) form of Tyr–Pro and its overall concentration
in solution c0; k (L·mol–1·s–1) is the rate constant of
acylation of the reactive form; kh (s–1) is the rate
constant of hydrolysis of the acylating agent; and kobs
(s–1) is the observed rate constant:
k = kobs/c.
(5)
The contribution of hydrolysis of the acylating
agent to the overall reaction rate was estimated by
special kinetic experiment. For this purpose, the reac-
Transmission, %
kobs = kh + (kα)c0.
(2)
In order to maintain a definite concentration of
dipeptide anions, potassium hydroxide was added to
the reaction solution in an amount ensuring that a part
of the dipeptide remained in the zwitterionic form
which is inactive in the acylation. The concentration
of Tyr–Pro anions was equal to the concentration of
added alkali (c = cKOH), and its fraction in solution
α and the observed rate constant kobs were defined by
Eqs. (3) and (4):
25
20
15
10
0
α = cKOH/c0 = c/c0;
kobs = kh + k c.
(3)
(4)
0
200
400
600
800
1000
τ, s
Fig. 1. Transmission of the working solution versus time in
the reaction of Tyr–Pro with 2,4-dinitrophenyl benzoate in
aqueous 1,4-dioxane (40 wt % of water); temperature 313 K.
According to the results of our previous kinetic
studies [2–4], when the concentration ratio of the
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KUSTOVA et al.
1036
Table 1. Rate constats kobs and k for the reaction of L-proline with 4-nitrophenyl benzoate at different concentrations of the
amino acid anion c; X1 is the mole fraction of water in its mixture with 1,4-dioxane; temperature 298 K.
X1,
c, M
kobs×102, s–1
1.87±0.06
0.93±0.01
0.64±0.01
0.493±0.007
5.0±0.6
k, L·mol–1·s–1
1.10±0.04
1.10±0.02
1.13±0.02
1.16±0.02
2.6±0.3
0.0169
0.00846
0.00564
0.00423
0.0196
0.765
0.951
0.00980
0.00653
2.46±0.07
1.67±0.06
2.51±0.07
2.56±0.09
Table 2. Kinetic parameters of the reaction of Tyr–Pro with di(tri)nitropheyl benzoates in aqueous 1,4-dioxane (40 wt % of
water)
Temperature, K
c, M
kobs×104, s–1
k×102, L·mol–1·s–1
ΔH≠298, kJ/mol
–ΔS2≠98, J·mol–1·K–1
2,4-Dinitrophenyl benzoate
4.79±0.19
298
303
308
313
4.19±0.17
5.21±0.16
6.49±0.43
9.43±0.57
5.94±0.18
0.00876
37±2
145±7
7.40±0.49
10.8±0.65
2,6-Dinitrophenyl benzoate
3.45±0.25
298
0.00876
0.00876
3.02±0.22
–
–
2,4,6-Trinitrophenyl benzoate
298
303
308
25.3±0.3
28.9±0.3
42.6±0.6
56.4±0.1
37.3±0.5
54.8±0.5
49±2
91±6
tion of L-proline with 4-nitrophenyl benzoate was run
several times in a solvent with the same composition at
the same temperature with variation of the initial
amino acid concentration. The fact that the k values
remained constant within the examined temperature
range (Table 1) indicated insignificant contribution of
hydrolysis of the ester to the overall reaction rate.
The experimental kinetic parameters of the acyla-
tion process are presented in Table 2. It is seen that the
rate constants for the reactions of Tyr–Pro with
di(tri)nitrophenyl benzoates increase in the series
2,6-DNPB < 2,4-DNPB < 2,4,6-TNPB. The same ester
reactivity order was observed previously in the reac-
tions with Gly–Gly and Ala–Ala [2, 4]; obviously, it is
related to increase of the electrophilicity of these esters
in the same series. In addition, similarity of the activa-
tion parameters for the reactions of 2,4-DNPB with
Tyr–Pro (Table 2) and glycine should be noted
(ΔH≠298 = 31 kJ·mol–1, ΔS2≠98 = –134 J·mol–1·K–1) [4].
Figure 1 shows one of the experimental kinetic
curves for the benzoylation of Tyr–Pro. The shape of
the kinetic curve suggests the absence of catalytic or
autocatalytic processes in the system. It also implies
that the attack of the acylating agent is directed at only
one nucleophilic center of the dipeptide molecule,
nitrogen atom of the primary amino group. As will be
shown below, just that group exhibits strongly pro-
nounced electron-donor properties in comparison to
the alternative nucleophilic center, oxygen atom of the
phenolic hydroxy group.
Comparison of the kinetic parameters for the ben-
zoylation of α-amino acids and dipeptides in aqueous
1,4-dioxane (Table 3) indicates the determining effect
of the basicity of amino groups subjected to acylation
on the reaction rate. In going from Gly to Gly–Gly, the
pK value decreases by 1.55 log unit, and the acylation
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REACTIVITY OF TYROSYL–PROLINE TOWARD BENZOYLATION
1037
Table 3. Rate constants of N-acylation of α-amino acids and dipeptides in aqueous 1,4-dioxane (40 wt % of water),
temperature 298 K
Substrate
pKa
Ester
k,b L·mol–1·s–1
2.12
Substrate
pKa
Ester
k,b L·mol–1·s–1
2,4-DNPB
2,5-DNPB
2,6-DNPB
2,4,6-TNPB
2,4-DNPB
2,5-DNPB
2,6-DNPB
2,4,6-TNPB
2,4-DNPB
2,5-DNPB
2,6-DNPB
2,4,6-TNPB
2,4,6-TNPB
2,4,6-TNPB
2,4,6-TNPB
2,4-DNPB
11.1
7.9
0.42
Gly
9.78
L-Pro
10.64
0.30
3.0
11.2
36.5
0.75
1.67
3.1
0.54
L-Asn
8.80
9.15
8.23
8.14
0.09
DL-Ser
DL-Val
9.72
0.06
Gly–Gly
L-Ala–L-Ala
5.2
0.35c
a The values refer to protonation of the primary amino group in water [7].
b The error in the determination of rate constants did not exceed 5%.
c In aqueous 1,4-dioxane containing 60 wt % of water.
rate constant decreases by a factor of 3.6. According
to the data of [7], Tyr–Pro is characterized by a low
basicity in aqueous solution [pK(Tyr–Pro) 7.81, cf.
pK(Tyr) 9.11], which is likely to be responsible for the
lower (by an order of magnitude) rate constant k of
benzoylation of Tyr–Pro (Table 2) than of Gly–Gly.
Elongation of the peptide chain up to seven amino acid
residues leads to an insignificant decrease of pK
ΔE = 0 kcal/mol (conformer 1)
ΔE = 0.64 kcal/mol (conformer 2)
ΔE = 2.96 kcal/mol (conformer 3)
Fig. 2. Calculated strutures of the most stable conformers of the neutral form of Tyr–Pro and their relative energies. There is
intramolecular hydrogen bond in the carboxy group of each conformer.
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KUSTOVA et al.
1038
Geometric parameters of both forms were completely
optimized, and the energy minima on the potential
energy surface were identified by the absence of
imaginary frequencies in the corresponding Hessian
matrices. The anionic form of the dipeptide was gen-
erated by deprotonation of the proline carboxy group,
and its charge was assumed to be equal to –1, and
multiplicity, to 1.
Figure 2 shows three most stable structures of the
neutral form of Tyr–Pro, which were selected among
the possible conformers, and the most stable structure
of the deprotonated dipeptide is presented in Fig. 3.
It is seen that the geometric parameters of the neutral
form (conformer 1) significantly change in going to
the anionic form. In particular, the torsion angle
N1C5C6C7 and the distance between the cyclic frag-
ments of the molecule increase, whereas the torsion
angle N1C5C6N2 decreases due to formation of intra-
molecular hydrogen bond between the oxygen atom of
the carboxylate group and hydrogen atom of the pri-
mary amino group. As a result, the N–H bond becomes
longer (from 1.015 to 1.021 Å), which facilitates its
dissociation in the course of acylation. Furthermore,
the dipole moment of the molecule changes both its
direction and magnitude (μ = 4.38 and 11.23 D for the
neutral and anionic forms, respectively).
Fig. 3. Calculated structure of the anionic form of Tyr–Pro;
localization of LP(N2) and LP2(O2) on the nucleophilic
centers is shown.
(pK 7.31 and 7.34 for Tyr–Pro–Phe–Pro–Gly–Pro–Ile
and Tyr–Pro–Phe–Val–Glu–Pro–Ile, respectively) [7].
The linear correlation between logk for the reactions of
amino acids and dipeptides with 2,4,6-TNPB (Table 3)
and pK values of their amino groups was used to
predict the rate constants for the reactions of hepta-
peptides with 2,4,6-TNPB. Extrapolation of this
dependence gave k values at a level of 0.080–
0.084 L·mol–1·s–1.
Taking into account that the Tyr–Pro molecule pos-
sesses two nucleophilic centers capable of undergoing
electrophilic attack by benzoyl group, namely the
primary amino group and phenolic hydroxy group of
the tyrosine moiety, it seemed reasonable to compare
electron-donor properties of these groups on the basis
of quantum chemical calculations. For this purpose, the
neutral and anionic forms of Tyr–Pro were simulated
by DFT calculations using B3LYP functional and
cc-pVTZ basis set (Gaussian 03 software package [8]).
The electronic parameters of the two possible nu-
cleophilic centers, nitrogen atom of the primary amino
group (N2) and oxygen atom of the phenolic hydroxy
group (O2) were compared on the basis of NBO
analysis [9, 10] of electron density distribution in the
neutral and anionic forms. The results showed that
the oxygen atom has two unshared electron pairs,
LP1(sp1,2) with a lower energy and LP2(pπ) with
Table 4. Electronic characteristics of the two potential nucleophilic centers in the neutral and anionic forms of the dipeptide
Tyr–Pro
Neutral form
Anionic form
O2
Characteristic
Charge, a.u.
N2
O2
N2
–0.826
–0.629
–0.849
–0.782
–16.55 (LP1)
–8.68 (LP2)
–14.54 (LP1)
–6.52 (LP2)
Energy of lone electron pair, eV
Population of LP orbital
–8.51
1.95
–5.15
1.94
1.98 (LP1)
1.88 (LP2)
1.98 (LP1)
1.89 (LP2)
Interacting orbitals (donor →
acceptor)
LP →
LP1→
LP2→
LP →
LP1→
LP2→
σ*(C5–C6)
σ*(C12–C13) π*(C12–C13)
σ*(C5–C6)
σ*(C12–C13) π*(C12–C13)
Energy of donor–acceptor
interaction, kcal/mol
8.45
6.70
25.23
9.44
7.13
27.88
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REACTIVITY OF TYROSYL–PROLINE TOWARD BENZOYLATION
1039
a higher energy (Fig. 3; Table 4). However, the LP2
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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than the energy of LP2 on the oxygen atom. The energy
of LP(N2) increases in going from the neutral form of
the dipeptide to the anion, which enhances its electron-
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EXPERIMENTAL
The dipeptide L-Tyr–L-Pro as hydrochloride with
a purity of 99.95% was synthesized at the Institute of
Chemical Reagents and Highly Pure Chemical
Substances (“Kurchatov Institute” National Research
Center). 2,4-, and 2,6-Dinitro- and 2,4,6-trinitrophenyl
benzoates were prepared by acylation of the corre-
sponding nitrophenols with benzoyl chloride. All
reagents and solvents were purified until their physical
properties (melting or boiling point and refractive
index) completely coincided with reference data.
1,4-Dioxane of chemically pure grade was kept over
potassium hydroxide over 7 days and was then distilled
under atmospheric pressure over metallic sodium to
remove organic peroxide impurities. The binary solvent
was prepared from deionized water which was obtained
using a DV-1 deionizer. Working solutions of the di-
peptide and KOH in aqueous 1,4-dioxane and of di(tri)-
nitrophenyl benzoate in 1,4-dioxane were prepared
from accurately weighted amounts of the reagents and
were kept at a constant temperature over a period of
30 min before a kinetic run. The initial reactant con-
centrations were 10–2 and 10–4 M, respectively. The
optical densities of solutions were measured with
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