Reactivity of α-Amino Acids in the Reaction with Esters in Aqueous–1,4-Dioxane Media

Article abstract of DOI:10.1134/S1070363218010127

The kinetics of the reaction of a series of α-amino acids with 4-nitrophenyl acetate, 4-nitrophenyl benzoate, and 2,4,6-trinitrophenyl benzoate in aqueous 1,4-dioxane medium has been studied. Kinetics of the reactions involving 4-nitrophenyl acetate and 2,4,6-trinitrophenyl benzoate has complied with the Br?nsted dependence and revealed linear correlation between rate constant logarithm and the energy difference of the frontier orbitals of α-amino acids anions.

Full text of DOI:10.1134/S1070363218010127

ISSN 1070-3632, Russian Journal of General Chemistry, 2018, Vol. 88, No. 1, pp. 80–85. © Pleiades Publishing, Ltd., 2018.
Original Russian Text © L.B. Kochetova, T.P. Kustova, L.V. Kuritsyn, 2018, published in Zhurnal Obshchei Khimii, 2018, Vol. 88, No. 1, pp. 84–89.
Reactivity of α-Amino Acids in the Reaction
with Esters in Aqueous–1,4-Dioxane Media
L. B. Kochetova*, T. P. Kustova, and L. V. Kuritsyn
Ivanovo State University, ul. Ermaka 39, Ivanovo, 153025 Russia
*e-mail: kochetova_lb@mail.ru
Received July 31, 2017
Abstract―The kinetics of the reaction of a series of α-amino acids with 4-nitrophenyl acetate, 4-nitrophenyl
benzoate, and 2,4,6-trinitrophenyl benzoate in aqueous 1,4-dioxane medium has been studied. Kinetics of the
reactions involving 4-nitrophenyl acetate and 2,4,6-trinitrophenyl benzoate has complied with the Brønsted
dependence and revealed linear correlation between rate constant logarithm and the energy difference of the
frontier orbitals of α-amino acids anions.
Keywords: α-amino acids, esters, N-acylation, water–1,4-dioxane
DOI: 10.1134/S1070363218010127
Acylation of α-amino acids is a point of interest due
to their crucial functions in biological objects, for
example, in biosynthesis of proteins, their post-
translational modifications, and metabolic processes [1–7].
This study aimed to investigate the kinetics of the
reactions of glycine (Gly), DL-valine (DL-Val), L-
proline (L-Pro), DL-α-alanine (DL-α-Ala), DL-leucine
(DL-Leu), DL-serine (DL-Ser), DL-threonine (DL-
Thr), DL-methionine (DL-Met), L-tryptophan (L-Trp),
and L-asparagine (L-Asn) with benzoic and acetic
acids esters substituted at the phenoxide unit: 4-nitro-
phenyl benzoate 1, 2,4,6-trinitrophenyl benzoate 2, and
4-nitrophenyl acetate 3 in a water (40 wt %)–1,4-
dioxane medium and to elucidate the main factors
determining the reactivity of substrates.
Acylation of amino acids is used in chemical syn-
thesis of polypeptides for building up the polypeptide
chain and protection of terminal and side functional
groups of α-amino acids. The acylation products (N-
acylamino acids) exhibit pharmacological activity:
vasodilatory, neuroprotective, and analgesic actions
[3, 4], inducing or inhibition of the proliferation of
T lymphocytes in vitro [4], suppression of proliferation
of cell line of rectal carcinoma [5], induction of insulin
secretion [6], and prevention of skin aging [7]. Acyl-
amino acids are low-toxic and do not cause undesirable
side effects [8, 9].
It is known that amino acids can exist in four forms
in a liquid phase: cationic, anionic, zwitter-ionic, and
molecular, depending on the medium acidity. Under
the experimental conditions used in this study, only the
anionic form is reactive. The reaction of an α-amino
acid anion with esters occurs as shown in Scheme 1.
N-Acylamino acids serve as constituents of hygienic
and cosmetic products as well as herbicides and
fungicides which can be easily degraded by environ-
ment microorganisms. At the same time, N-acylamino
acids may be low-toxic for warm-blooded animals, non-
phytotoxic, and may accelerate plants growth [10].
No catalytic and autocatalytic reactions were ob-
served in the experiment.
The esters hydrolysis can accompany the N-acyla-
tion in aqueous-organic media. It has been shown in
some reports [12, 13] that phenyl benzoates are not
hydrolyzed in water; therefore, only the target reaction
and alkaline hydrolysis were accounted for in the
calculation of the rate constants (Scheme 2).
Kinetic data on acylation of α-amino acids in dif-
ferent aqueous-organic media which are optimal for
industrial synthesis [10, 11] are necessary for elabora-
tion of large-scale methods of synthesis of acylamino
acids. However, the relevant data have been limited.
The specific pH value (8.5–9) was created to
minimize the contribution of the hydrolysis into the
80

REACTIVITY OF α-AMINO ACIDS IN THE REACTION WITH ESTERS
81
Scheme 1.
k
NH2CHCOO_ + R2COO
R1
R2CONHCHCOO_ + HO
R1
(NO2)n
(NO2)n
(CH ) SCH
1
R = H (Gly), CH3 (Ala), CH(CH3)2 (Val), CH2CH(CH3)CH3 (Leu), CH2OH (Ser), CH(CH3)OH (Thr),
2 2
3
(Met), CH2CONH2 (Asn),
(Trp),
(Pro); R2 = CH , Ar; n = 1, 3.
CH2
COOH
3
N
H
N
H
Scheme 2.
kh
RCOO
+ OH_
RCOO_ + HO
(NO2)n
(NO2)n
process kinetics. The alkali was added to a solution of
the amino acid, and the latter existed as a mixture of
zwitter-ion form and the form containing non-
protonated amino group capable of the acylation.
Under such conditions, concentration of the molecular
form of amino acids and its contribution to the reaction
rate are negligibly low compared with the anionic form
[14]. The earlier kinetic study has shown that if the
concentrations ratio of the unreactive zwitter-ionic
form (с±) and the reactive anionic form (с) exceeds
four (с±/c > 4), the rate of alkali hydrolysis can be
neglected compared with the rate of the N-acylation [12].
Hence, in the absence of hydrolysis, the rate constant
of N-acylation of the amino acid reactive form can be
calculated using Eq. (5).
k = ko/с.
(5)
The observed rate constants ko and the rate constants
k of the reactions of α-amino acids anions with com-
pounds 1–3 in aqueous 1,4-dioxane calculated using
Eq. (5) are given in Tables 1 and 2. The independence
of the acylation rate constant k of the concentration of
amino acids anions indicated the insignificant
contribution of ester hydrolysis to the observed rate of
the reaction.
The kinetics was investigated in large excess of the
amino acid (102–103-fold) with respect to the acylation
agent, and the change in the acylation agent concentra-
tion (сac) could be expressed by Eq. (1).
Comparison of the data in Tables 1 and 2 showed
that the rate constants of the reactions of the studied α-
amino acids with compound 3 were significantly
higher than those for compound 1. The decrease in the
k values in the case of acylation with ester 1 was
caused by the electronic effect of the aromatic ring
reducing its electrophilicity due to the conjugation
with the carbonyl group, as compared with compound
3 (no conjugation). At the same time, the k values of
acylation of α-amino acids with ester 2 were signi-
ficantly higher than those for ester 3. The reactivity of
ester 2 was caused by two factors: the electronic
influence of the aromatic π-system of the acyl part of
the ester and the action of three nitro groups on the
carbonyl reactive site. The second factor was prevail-
ing, as confirmed by quantum-chemical simulations:
the positive charge q(C) at the carbonyl carbon atom of
ester 2 was slightly higher (0.81 a.u.) than that for ester
3 (0.775 a.u.). Furthermore, the LUMO energy of
molecule of compound 2 was significantly lower
–dcac/dτ = [kh + (kα)c0]cac = koсac.
(1)
Here α is the fraction of anionic form of the α-amino
acid with respect to its total concentration in the solu-
tion 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 acylation agent; ko (s–1) is the
observed rate constant [Eq. (2)].
ko = kh + (kα)с0.
(2)
Concentration of the anionic form equals concentra-
tion of the added alkali (с = сNaOH); its fraction in the
solution α and the observed rate constant ko are
determined by Eqs. (3), (4).
α = cNaOH/c0 = c/c0,
ko = kh + kс.
(3)
(4)
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 1 2018

82
KOCHETOVA et al.
Table 1. Rate constants of acylation ko and k of α-amino acids with esters 1–3 in water (40 wt%)–1,4-dioxane medium at 298 Ka
Amino acid
pKII [17]
с×103, mol/L
ko×103, s–1
k, L mol–1 s–1
1
2
DL-Ala
DL-Leu
L-Asn
3.40
3.40
7.21
0.111±0.002
0.652±0.008
0.645±0.010
0.0331±0.003
0.0190±0.003
0.0089±0.002
L-Asn
7.21
3.40
7.21
7.21
7.21
5.41±0.02
5.67±0.08
27.1±0.6
22.2±0.4
19.2±0.1
0.750±0.003
1.67±0.02
3.80±0.08
3.03±0.06
2.67±0.02
DL-Ser
DL-Thr
DL-Met
L-Trp
3
Gly
9.78
9.72
10.64
9.87
9.64
8.80
9.15
9.25
9.21
9.38
3.55
3.40
4.26
3.40
3.40
3.40
3.40
7.21
7.21
7.21
3.73±0.030
2.50±0.050
19.9±0.500
1.380±0.080
0.890±0.009
0.153±0.007
0.299±0.006
0.822±0.008
0.728±0.004
1.280±0.020
1.05±0.01
0.73±0.02
DL-Val
L-Pro
4.67±0.01
DL-Ala
DL-Leu
L-Asn
DL-Ser
DL-Thr
DL-Met
L-Trp
0.41±0.02
0.261±0.003
0.045±0.002
0.088±0.002
0.114±0.001
0.101±0.001
0.178±0.003
а (с) Initial concentration of amino acid anions.
(0.32 eV) than ЕLUMO of ester 3 (1.64 eV). The nitro
groups of compound 2 in positions 2 and 6 of the
phenoxide ring do not create steric hindrance to the
nucleophile attack, because the phenoxide ring was
rotated by 71.1° with respect to the plane of the acyl
part of molecule of ester 2. The combination of these
factors explains higher reactivity of ester 2 in the
reactions with α-amino acids as compared with ester 3.
The compliance with the Brønsted dependence was
observed for the reactions of α-amino acids with esters
2 and 3 [Eq. (6)].
log k = а + βR1pKII.
Here а is a constant parameter for the reaction series,
R1 is the sensibility to the basicity of nucleophile, and
(6)
β
KII is the second acid dissociation constant of the
amino acid. The described dependence is described by
Eqs. (7), (8) for compounds 2 and 3, respectively.
Rate constants of the reactions of α-amino acids
with ester 2 (Tables 1 and 2) were 3–4 orders of
magnitude lower than the k values for the reactions of
the same α-amino acids with benzoyl chloride 4 [15].
The difference was due to lower nucleophilicity and
higher elimination ability of the leaving group of acid
chlorides compared to their esters.
log k = (–7.5±0.9) + (0.85±0.09)pKII,
n = 8, r = 0.97;
(7)
log k = (–11.1±0.8) + (1.10±0.08)pKII,
n = 14, r = 0.97.
(8)
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 1 2018

REACTIVITY OF α-AMINO ACIDS IN THE REACTION WITH ESTERS
83
Table 2. Rate constants of acylation of Gly, L-Pro, and L-Val with esters 1 and 2 in water (40 wt%)–1,4-dioxane mediuma
с×103,
mol/L
с×103,
mol/L
Reaction Т, K
Gly + 1 298
ko×103, s–1
k, L mol–1 s–1 Reaction Т, K
ko×103, s–1
k, L mol–1 s–1
5.77
8.66
17.3
16.9
4.23
16.9
8.46
4.23
8.46
16.9
1.73
1.23
1.08
0.250
0.350
0.438
0.670
0.250
0.350
0.438
0.670
0.523± 0.005 0.0907±0.0009 L-Pro + 1 298
16.9
8.46
5.64
4.23
0.668
0.634
0.596
12.7
15.5
19.9
8.63
5.76
4.32
18.7±0.6
9.31±0.01
6.39±0.02
4.79±0.05
24.4±0.7
24.2±0.6
21.5±0.7
1.11±0.04
1.10±0.01
1.13±0.01
1.13±0.01
37±1
0.870±0.009 0.101±0.001
1.67±0.02
0.096±0.001
1.65±0.01 0.0974±0.0004
0.598±0.006 0.140±0.001
303
308
L-Pro + 2 298
L-Val + 1 298
L-Val + 2 298
2.36±0.02
1.17±0.01
0.140±0.001
0.139±0.001
38±1
36±1
0.846±0.006 0.200±0.001
0.142±0.007 0.0112±0.0006
0.170±0.004 0.0110±0.0003
0.231±0.002 0.0116±0.0002
1.61±0.01
3.30±0.02
19.0±0.7
13.7±0.7
11.9±0.2
3.20±0.03
4.65±0.06
5.83±0.09
8.71±0.07
3.73±0.04
4.93±0.02
6.22±0.05
9.98±0.09
0.190±0.001
0.195±0.001
11.0±0.04
11.1±0.08
11.0±0.02
12.8±0.1
Gly + 2 298
45±6
30±4
22±4
5.2±0.7
5.1±0.7
5.2±0.9
303
13.3±0.2
13.3±0.2
13.8±0.1
308
14.9±0.2
14.5±0.1
14.4±0.1
14.9±0.3
а (с) Initial concentration of amino acid anions.
Besides the data in Table 1, the rate constants of the
reactions of ester 3 with diamino- and dicarboxylic
acids [16] were included in Eq. (8). The рKII values in
water [17] were used for derivation of the correlations,
in view of the absence of available KII values for the
aqueous-dioxane solvent for some of the studied amino
acids.
influence of the amino group basicity on the rate of the
α-amino acids acylation. That fact was consistent with
other data on the reactions of acyl transfer [14].
We determined the values of the activation
parameters of glycine reactions with esters 1 and 2.
The accessible temperature range was limited by the
decrease in the solubility of the reactants and products
on cooling and acceleration of the acylation agents
hydrolysis on heating.
In should be noted that the correlation between
logarithm of the rate constant k of the α-amino acids
reactions with the corresponding acetates and benzoates
was close to linear, as well as that between log k of
α-amino acids acylation with the esters and compound
4 [15], the correlation coefficients being at least 0.93.
The marked linear relationship and the holding the
Brønsted dependence was due to the determinative
The activation energy of glycine acylation with
ester 1 equaled 52±3 kJ/mol, significantly higher than
for the reaction with ester 2 (22±3 kJ/mol). That fact
was in good agreement with the increase in the rate
constants with the increase in the number of nitro
groups in the ester molecule. The determined values of
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 1 2018

84
KOCHETOVA et al.
Table 3. Quantum-chemical parameters of α-amino acids anions simulated via DFT//B3LYP/6-31+G(d,p) method
Amino acid r(N–H1), Å
r(N–H2), Å
q(N), a.u.
q(NH2), a.u.
ЕHOMO, eV
ЕLUMO, eV
∠H1NH2, deg
Gly
Val
Ser
1.020
1.018
1.015
1.018
1.023
1.017
1.019
1.020
1.027
1.019
1.029
1.023
1.018
1.021
99.89
103.61
103.34
108.68
107.43
107.57
105.75
–0.462
–0.454
–0.451
–0.477
–0.428
–0.457
–0.451
–0.180
–0.143
–0.144
–0.162
–0.129
–0.148
–0.159
–0.57
–0.98
–0.84
–1.05
–1.30
–1.34
–0.98
3.70
2.95
3.18
2.61
2.50
2.18
2.29
Thr
Met
Trp
Asn
activation entropy (–98±9 and –160±9 J mol–1 K–1 for
compounds 1 and 2, respectively) indicated that the
transition state structure was more ordered in the case
of 2,4,6-trinitrophenyl benzoate as compared to 4-nitro-
phenyl benzoate.
determining role of basicity of amino groups in the
acylation kinetics. The simulation results showed that
the orbital parameters of the reagents could be used to
describe the reactivity of α-amino acids anions and
esters in acylation.
We simulated the α-amino acids anions using the
DFT//B3LYP/6-31+G(d,p) method; selected results are
given in Table 3. The presented data showed that the
structure of amino group was not significantly changed
in the series of the anions: the variation of the N–H
bond length and the bond angle between the N–H
bonds were as low as 0.014 Å and 8.79°, respectively.
EXPERIMENTAL
α-Amino acids [Gly (“pure” grade), DL-α-Ala
(“analytical pure” grade), DL-Val (“pure” grade), DL-
Leu (“pure” grade), L-Pro (“analytical pure grade),
DL-Met (“analytical pure” grade), DL-Thr (“analytical
pure grade), DL-Ser (“analytical pure” grade), L-Trp
(“analytical pure” grade), L-Asn (“analytical pure”
grade)] were dried at 373 K for 1 h and then were used
without further purification. Esters 1 and 2 were
obtained via acylation the corresponding nitro deriva-
tives of phenol with benzoyl chloride. 4-Nitrophenol
and acetic anhydride were used for preparation of ester
3. Sodium hydroxide (“analytical pure” grade) was
used as received. 1,4-Dioxane (“pure” grade) was kept
over KОН for several days and distilled over metal
sodium. Bidistilled water for the preparation of the
mixed water–dioxane solvent was obtained using a
DV-1 deionized water generator. Physical properties of
the reagents (melting point, boiling point, refraction
index, etc.) coincided with the reference data [19–22]
after the purification.
The results of simulation of α-amino acids anions
were compared with the kinetic data for their reactions
with esters 2 and 3 in aqueous 1,4-dioxane (Table 1). It
was found that the rate constants of amino acids
acylation k were not related to the values of the charge
on the nitrogen atoms q(N) and α-amino groups
q(NH2) of the anions. At the same time, linear
correlation between log k values and the energy gap
between the frontier orbitals (ΔЕ) of α-amino acids
anions [Eq. (9)] was observed for both reaction series.
ΔЕ = –(ЕHOMO – ЕLUMO).
The derived equations were as follows [Eqs. (10),
(11)].
(9)
log k = –(5.6±0.6) + (1.58±0.18)ΔЕ,
n = 7, r = 0.96;
(10)
Kinetics of α-amino acids reactions with the esters
was investigated using indicator spectrophotometric
method, the reaction products (mono- and trinitro-
phenols) serving as the indicators being transformed
into the corresponding yellow-colored anions under the
experimental conditions. The reaction progress was
monitored by measuring the increasing absorbance of
the solution at λ 400 nm using a SF-26 spectrophoto-
meter equipped with a Scsh300 digital voltmeter and a
log k = (0.32±0.12) – (0.339±0.034)ΔЕ,
n = 7, r = 0.98.
(11)
In summary, rate constants of the reactions (10) of
α-amino acids with substituted phenyl esters of
benzoic and acetic acids were obtained in this study.
The observed Brønsted dependence indicated the
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 1 2018

REACTIVITY OF α-AMINO ACIDS IN THE REACTION WITH ESTERS
85
9. Merck Index, Budavari, S., Ed., New York: Merck&Co.,
temperature-controlled cell holder as described in
detail elsewhere [12].
1989, p. 1400.
10. Mikhalkin, A.P., Russ. Chem. Rev., 1995, vol. 64, no. 3,
Quantum-chemical simulation of amino acids
anions and ester molecules was performed using the
DFT//B3LYP/6-31+G(d,p) and HF/6-31G(d) methods,
respectively, implemented in the Firefly 7.1.g software
package [18]. The correspondence of the structures to
true minimums in the potential energy surface during
the geometry optimization was confirmed by the absence
of imaginary frequencies in the vibration spectrum.
p. 259. doi 10.1070/RC1995v064n03ABEH000149
11. Gershkovich, A.A. and Kibirev, V.K., Khimicheskii
sintez peptidov (Chemical Synthesis of Peptides), Kiev:
Naukova Dumka, 1992.
12. Khripkova, L.N., Candidate Sci. (Chem.) Dissertation,
Ivanovo, 2003.
13. Khripkova, L.N., Kuritsyn, L.V., Kalinina, N.V., and
Sadovnikov, A.I., Russ. J. Gen. Chem., 2004, vol. 74,
no. 10, p. 1543. doi 10.1007/s11176-005-0052-1
REFERENCES
14. Kuritsyn, L.V., Kustova, T.P., Sadovnikov, A.I.,
Kalinina, N.V., and Klyuev, M.V., Kinetika reaktsii
atsil’nogo perenosa (Kinetics of Acyl Transfer Reac-
tions), Ivanovo: Ivanovsk. Gos. Univ., 2006.
1. Waluk, D.P., Doctoral Thesis, Stockholm, 2012.
2. Schachter, D. and Taggart, J.V., J. Biol. Chem., 1954,
vol. 208, no. 1, p. 263.
15. Kuritsyn, L.V. and Kalinina, N.V., Zh. Ogr. Khim.,
3. Hanuš, L., Shohami, E., Bab, I., and Mechoulam, R.,
Biofactors, 2014, vol. 40, no. 4, p. 381. doi 10.1002/
biof.1166.
4. Burstein, S.H., Rossetti, R.G., Yagen, B., and Zurier, R.B.,
Prostaglandins Other Lipid Mediat., 2000, vol. 61,
nos. 1–2, p. 29.
5. Gustafsson, S.B., Lindgren, T., Jonsson, M., and
Jacobsson, S.O.P., Cancer Chemother. Pharmacol.,
2009, vol. 63, no. 4, p. 691. doi 10.1007/s00280-008-0788-5
6. Ikeda, Y., Iguchi, H., Nakata, M., Ioka, R.X., Tanaka, T.,
Iwasak, S., Magoori, K., Takayasu, S., Yamamoto, T.T.,
Kodama, T., Yada, T., Sakurai, T., Yanagisawa, M., and
Sakai, J., Biochem. Biophys. Res. Commun., 2005,
vol. 333, no. 3, p. 778. doi 10.1016/j.bbrc.2005.06.005
1994, vol. 30, no. 5, p. 723.
16. Kochetova, L.B., Kalinina, N.V., Solov’eva, D.S.,
Ditsina, O.Yu., Kuritsyn L.V., and Kustova, T.P.,
Butlerovsk. Soobshch., 2016, vol. 45, no. 1, p. 145.
17. IUPAC Stability Constants Database SCUERY © 2005,
IUPAC and Academic Software SCQUERY, Version 5.20.
18. Granovsky, A.A., Firefly 7.1.G. http://www.classic.
chem.msu. su/gran/firefly/index.html.
19. Svoistva organicheskikh soyedinenii (Properties of Organic
Compounds), Potekhin, A.A., Ed., Leningrad: Khimiya,
1984.
20. Karyakin, Yu.V. and Angelov, I.I., Chistye khimicheskie
veshchestva (Pure Chemical Substances), Moscow:
Khimiya, 1974.
21. Spravochnik khimika (Chemist’s Handbook), Nikol’skii, B.P.,
Ed., Moscow: Khimiya, 1964, vol. 3.
22. Gordon, A.J. and Ford, R.A., The Chemist’s Com-
panion. A Handbook of Practical Data, Techniques and
References, New York: Wiley, 1972.
7. Dumont, S., Cattuzzato, L., Trouve, G., Chevrot, N.,
and Stolz, C., Int. J. Cosmet. Sci., 2010, vol. 32, no. 1,
p. 9. doi 10.1111/j.1468-2494.2009.00525.x
8. Mashkovskii, М.D., Lekarstvennye sredstva (Drugs),
Мoscow: Novaya Volna, 2006.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 1 2018