ISSN 1070-4272, Russian Journal of Applied Chemistry, 2013, Vol. 86, No. 7, pp. 986−991. © Pleiades Publishing, Ltd.

, 2013.
Original Russian Text © N.V. Dolgopyatova, V.Yu. Novikov, I.N. Konovalova, N.M. Putintsev, 2013, published in Zhurnal Prikladnoi Khimii, 2013, Vol. 86,
No. 7, pp. 1052−1058.
ORGANIC AND PETROCHEMICAL
PROCESSES

Mechanism of Acid Hydrolysis of N-Acetyl-D-glucosamine

N. V. Dolgopyatovaa, V. Yu. Novikovb, I. N. Konovalovaa, and N. M. Putintseva

a Murmansk State Technical University, Murmansk, Russia
b Knipovich Polar Research Institute of Marine Fishery and Oceanography, Murmansk, Russia
e-mail: nowitaly@yandex.ru

Received May 16, 2013

Abstract—The hydrolysis kinetics of the chitin monomer, N-acetyl-D-glucosamine, in HCl, HClO4, and H3PO4
was studied in relation to the acid concentration. The rate constants of N-acetyl-D-glucosamine deacetylation and
D(+)-glucosamine formation in HClO4 and H3PO4 were determined for the first time. The rate of the acetamide
bond hydrolysis in N-acetyl-D-glucosamine depends on the concentrations of hydrogen ions and water. The nu-
cleophilicity of the acid residues does not affect the rate of N-acetyl-D-glucosamine hydrolysis.
DOI: 10.1134/S1070427213070070

Amino saccharides D(+)-glucosamine and N-acetyl- N-Acetyl-D-glucosamine can be prepared by

D-glucosamine widely occur in the nature, play an
important role in biology, and are used in medicine,
organic and bioorganic chemistry, in cosmetic and food
industry, and in other branches [1]. The use of these
amino saccharides as antiarthritics is of most practical
interest [2, 3].
D(+)-Glucosamine is prepared by exhaustive
depolymerization and deacetylation of natural
polysaccharide chitin under the action of concentrated
HCl on heating [4].
Also, an enzymatic procedure for preparing
glucosamine from chitin [5] and enzymatic procedures
for chitin depolymerization under the action of various
chitinolytic enzymes [6] have been suggested.
N-Acetyl-D-glucosamine can be prepared from
chitin by mild acid hydrolysis [5, 6]. Partial hydrolysis
is usually performed with hydrochloric acid, and the
hydrolysis products contain, along with the monomer,
also small oligomers (chitooligosaccharides). Acid
hydrolysis of chitin and chitosan leads to cleavage of
both glycoside (degradation, depolymerization) and
acetamide (deacetylation) bonds. Therefore, products
of partial acid hydrolysis contain acetylated and
deacetylated compounds.
acetylation of D(+)-glucosamine. A simple procedure for
D(+)-glucosamine acetylation with various acetylating
agents such as acetic anhydride [7] or acetyl chloride
[10] is the most widely used.
As found in [11], deacetylation of chitin and N-acetyl-
D-glucosamine in hydrochloric acid is described by a
first-order rate equation, and the dependence of the
rate of this reaction on the acid concentration passes
through a maximum at the HCl concentration of 29.8%.
Gizatulina et al. [12] studied the influence exerted on the
chitin degradation kinetics by the degree of ionization
and nucleophilicity of different acids.
Studies of the acid hydrolysis of chitin showed
that the glycoside bonds are cleaved first, and the
deacetylation mainly occurs at the step of N-acetyl-D-
glucosamine [7, 8]. The practical use of N-acetyl-D-
glucosamine requires either preservation of the acetyl
group or its removal by deacetylation.
We found no data in the literature on how the
hydrolysis kinetics of N-acetyl-D-glucosamine is
influenced by the degree of ionization and by the
nucleophilicity of various acids.
To refine the mechanism of acid deacetylation and
find the optimal conditions for practical use of chitin
monomers, we studied the specific features of N-acetyl-

986
 MECHANISM OF ACID HYDROLYSIS OF N-ACETYL-D-GLUCOSAMINE
D-glucosamine hydrolysis in relation to the kind and
concentration of the acid and evaluated the rate constants
of this reaction.
Materials and methods. D(+)-Glucosamine was
prepared by hydrolysis of chitin, which was isolated
from red king crab shells by the known procedure [13].
The chitin hydrolysis to glucosamine was performed in
concentrated (12.2 M) HCl with continuous stirring at
98°C for 2.5 h. The chitin to acid ratio was 1 : 20.
D(+)-Glucosamine (GlA) (C6H13NO5, 2-amino-2-
deoxy-D-glucose) is an amino saccharide. It is a strong
base, exhibits the properties of amines, and readily
forms stable salts.
N-Acetyl-D(+)-glucosamine (AcGIA) was prepared
in 75.5% yield by acetylation of glucosamine with
acetic anhydride by the procedure described in [9], in
accordance with the equation
C6H11NO5 + (CH3CO)2O
→ C8H15NO6 + CH3COOH. (1)
Hydrolysis of N-acetyl-D-glucosamine was
performed in various acids (HCl, HClO4, H3PO4) with
equivalent concentrations from 1 to 12.2 M at 80°C.
The resulting solutions were neutralized with aqueous
NaOH, filtered, and analyzed to determine the content
of N-acetyl-D-glucosamine and D(+)-glucosamine
(GlA).
The content of GlA in hydrolysis products was
determined spectrophotometrically using the reaction
with p-dimethylaminobenzaldehyde [14]. The total
content of reducing saccharides (AcGlA + GlA) was
estimated spectrophotometrically using the reaction
with K3[Fe(CN)6] [15]. The concentration of AcGlA
was calculated from the difference between the total
amount of AcGlA + GlA and the amount of GlA.
The rate of deacetylation of chitin and N-acetyl-D-
glucosamine, like the rate of any chemical reaction,
depends on the reactant concentrations and reaction
conditions. As shown in [11], the rate of deacetylation
of chitin and N-acetyl-D-glucosamine depends on the
HCl concentration, passing through a maximum at
~8 M HCl.
In this work we studied the kinetics of N-acetyl-
D-glucosamine hydrolysis and D(+)-glucosamine
formation in solutions of various acids differing in
concentration, degree of ionization, and nucleophilicity
of the acid residues: perchloric, hydrochloric, and
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 86 No. 7 2013
987
phosphoric acids.
The hydrolysis of N-acetyl-D-glucosamine consists
in deacetylation and is accompanied by the formation of
D(+)-glucosamine (formed as a salt under the conditions
of acid hydrolysis) and acetic acid, which are major
reaction products:
C8H15NO6 + H2O → C6H11NO5 + CH3COOH. (2)
Figures 1a and 1b show as example the kinetic
curves of N-acetyl-D-glucosamine hydrolysis and D(+)-
glucosamine formation in HCl, HClO4, and H3PO4
solutions at equivalent acid concentration of 6 M and
temperature of 80°C (for other acid concentrations
studied, the trends were similar).
Analysis of the kinetic curves of N-acetyl-D-
glucosamine hydrolysis and D(+)-glucosamine
formation at a constant acid concentration (Figs. 1a,
1b) shows that the reactions can be described by first-
order (pseudo-first-order) rate equations. To ensure
the pseudo-first-order conditions, one of the reactants
(aqueous acid solution) is taken in excess, and its
concentration remains virtually constant in the course
of the reaction. Therefore, for calculating the rate
constants of N-acetyl-D-glucosamine hydrolysis and
D(+)-glucosamine formation, we used the formula for
the kinetics of first-order reactions [16].
Figure 2 shows how the rate constants of N-acetyl-
D-glucosamine deacetylation, kA (Fig. 2a), and of
D(+)-glucosamine formation, k (Fig. 2b), depend on
the equivalent molar concentration of HCl, HClO4,
and H3PO4. As can be seen, the rate of N-acetyl-D-
glucosamine hydrolysis passes through a maximum and
depends on the kind of the acid. In the case of HCl, the
rate constant passes through a maximum at 7.8 M HCl,
after which it somewhat decreases and reaches constant
values at 10 M HCl.
In HClO4, the reaction rate constant is maximal at a
concentration of ~6 M, and with a further increase in the
concentration kA decreases by a factor of 5.
In phosphoric acid, the rate constant of hydrolysis
of acetamide bonds in N-acetyl-D-glucosamine is
considerably lower than in HCl and HClO4. As in HClO4,
a local maximum is observed at an acid concentration
of ~6 M. Appreciable increase in the hydrolysis rate is
observed at the equivalent acid concentration of 20–
22 M.
Comparison of the rate constants of N-acetyl-D-
988 DOLGOPYATOVA et al.
(a)
сAcGlA, mM
τ, min
(b)
сGlA, mM
τ, min
Fig. 1. Kinetic curves of N-acetyl-D-glucosamine hydrolysis
and D(+)-glucosamine accumulation in (1) HCl, (2) HClO4,
and (3) H3PO4 solutions (equivalent acid concentration 6 M).
(сAcGlA, сGlA) N-Acetyl-D-glucosamine and D(+)-glucosamine
concentrations.
glucosamine hydrolysis, kA, and of D(+)-glucosamine
formation, k, in the acids studied (Fig. 2) shows that, at
all the examined HCl concentrations, the values of kA
and k in HCl virtually coincide.
In hydrolysis of N-acetyl-D-glucosamine in
oxidizing acids (perchloric, phosphoric), the D(+)-
glucosamine formation rate is lower than the N-acetyl-
D-glucosamine decomposition rate. For example, in
HClO4 at сН = 5 M the hydrolysis rate constant is kA =
4.3 × 10–4 min–1, whereas the rate constant of D(+)-
glucosamine formation is k = 2.2 × 10–4 min–1.
Appreciably lower rate of D(+)-glucosamine
formation compared to N-acetyl-D-glucosamine
hydrolysis may be due to the fact that the hydrolysis
in perchloric and phosphoric acids is accompanied by
oxidative degradation of the carbohydrates, competing
with AcGlA deacetylation and causing the loss of
GlA, which leads to the observed decrease in the GlA
formation rate.
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 86 No. 7 2013
(a)
kA × 104 s–1
kA × 104 s–1
(b)
kA × 104 s–1
kA × 104 s–1
c, M
Fig. 2. Rate constants of (a) N-acetyl-D-glucosamine
hydrolysis (kA) and (b) D(+)-glucosamine accumulation (k)
as functions of the acid concentration c. Acid: (1) HCl, (2)
HClO4, and (3) H3PO4; 80°C.
Apparently, the rate of hydrolysis of acetamide bonds
in N-acetyl-D-glucosamine primarily depends on the
concentration of hydrogen ions, i.e., is determined by
the degree of the acid ionization. In accordance with pKa
(–8.95 for HClO4, –6.7 for HCl, 2.12 for H3PO4), the
hydrolysis rate decreases in the order HClO4 > HCl >
H3PO4.
The relationships obtained can be rationalized
by considering the mechanism of hydrolysis of the
acetamide bond.
As discussed in the literature, the hydrolysis of the
O
||
amide bond in alkylamides (R—C—NH—R') starts
with protonation of the carbonyl oxygen atom [17] or
amide nitrogen atom [18]. N-Acetyl-D-glucosamine
differs from alkylamides in that it is a bioorganic
compound containing a carbohydrate residue occurring
the hemiacetal and hydroxy carbonyl tautomeric forms.
 MECHANISM OF ACID HYDROLYSIS OF N-ACETYL-D-GLUCOSAMINE 989

_
O :O :
+

:
H3C C N C6H11O5 H3C C N C6H11O5 (3)

H H

Probably, in N-acetyl-D-glucosamine, protonation of charge, which is energetically favorable. In the process,

the carbonyl oxygen atom is more probable because of
the mesomeric interaction with the lone electron pair of
the nitrogen atom:
In this case, the partial positive charge arising on
the nitrogen atom will decrease its basicity, making the
protonation of the carbonyl oxygen atom in N-acetyl-D-
glucosamine more probable.
Protonation of the oxygen atom of the acetamide
group yields a carbocation with delocalized positive
a mesomeric structure is formed in which the positive
charge is delocalized over C, O, and N atoms.
Thus, hydrolysis of the acetamide bond in N-acetyl-
D-glucosamine starts with the protonation of the carbonyl
oxygen atom to form a carbocation which subsequently
undergoes nucleophilic attack of a water molecule. The
oxonium structure formed in the process undergoes a
series of transformations to form the final products: D(+)-

O OH
k1
H3C C NH C6H11O5 + H+ H3C C+ (4)

NH C6H11O5

OH OH
k2
C+ (5)
: :

H3C + HOH H3C C NH C6H11O5

NH C6H11O5 O
H + H

OH OH
+ (6)
H3C C NH C6H11O5 H3C C NH C6H11O5
:

O OH H
H + H

OH O
+ (7)
H3C C NH C6H11O5 H3C C OH + NH2C6H11O5
_ +
H
OH H

glucosamine and acetic acid. These transformations can
be presented as the following steps (k1 and k2 are the rate
constants of the corresponding reactions):
To calculate the reaction rate, we can use the method
of steady-state concentrations, because, in accordance
with the mechanism of the acetamide bond hydrolysis,
the first step of the reaction, protonation of the carbonyl
group, is reversible with rapid attainment of the
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 86 No. 7 2013
equilibrium [16]. Probably, the hydrolysis of N-acetyl-
D-glucosamine is an acid-catalyzed reaction [16]. The
rate of the N-acetyl-D-glucosamine consumption is
equal to the rate of the D(+)-glucosamine formation
(as demonstrated by the experimental data presented in
Fig. 2 for the hydrolysis of N-acetyl-D-glucosamine in
HCl as example).
Initially, N-acetyl-D-glucosamine is in a large excess,
990
and the hydrolysis can be considered to be the only
occurring process. The limiting step of the hydrolysis
of the acetamide bond is the hydrolysis of protonated
N-acetyl-D-glucosamine [reaction (5)]; therefore, the
overall reaction rate is (8)
Under the conditions of N-acetyl-D-glucosamine
hydrolysis, the water concentration is much higher than
the concentrations of the other reaction participants,
and its variation with time can be neglected. Strong
acids HCl and HClO4 and moderately strong acid
H3PO4 as sources of Н+ ions are also taken in a large
excess. Because of high concentration of these acids,
the dissociation of weak acetic acid (reaction product)
is suppressed. Therefore, the concentration of Н+ ions
in the course of the reaction also remains constant. The
intermediates formed in accordance with the reaction
mechanism are unstable, and their concentration can be
neglected. Using the steady-state approximation [16],
we can express the rate of the N-acetyl-D-glucosamine
hydrolysis as follows:
d[CH3CONHC6H11O5]
– ————————— = keff [CH3CONHC6H11O5], (9)
dt
where keff = k2k1[Н+][H2O] (10); keff is the effective
rate constant of N-acetyl-D-glucosamine hydrolysis,
and specifically this quantity is determined from the
experimental data.
сAcGlA, mM
τ, min
Fig. 3. Acid hydrolysis of N-acetyl-D-glucosamine (1) without
LiNO3 added and (2) with the addition of LiNO3. сHCl = 25%,
T = 80°C. (τ) Time.
DOLGOPYATOVA et al.
Thus, the calculated effective rate constants of the
N-acetyl-D-glucosamine hydrolysis depend both on the
concentration of hydrogen ions (i.e., on the degree of
acid ionization) and on the water concentration.
The maximum in the dependence of the hydrolysis
rate constant on the acid concentration (Fig. 2) can be
primarily attributed to the dependence of keff not only on
the concentration of hydrogen ions, but also on the water
concentration. Probably, with an increase in the acid
concentration, the concentration of water as the main
hydrolyzing agent decreases, which leads to a decrease
in the reaction rate. Furthermore, at, e.g., an HCl
concentration сН = 6–9 M, the acid activity coefficient,
according to [17], is in the range 4.3–9.4. Hence, the
activity of hydrogen ions considerably exceeds their
molar concentration. Apparently, this fact should also
be taken into account when analyzing the influence of
the concentration of strong acids on the hydrolysis rate.
This assumption is confirmed by the results shown
in Fig. 3. In this case, we added to the reaction mixture
a water-binding agent, anhydrous lithium nitrate. As
follows from Fig. 3, in the presence of lithium nitrate
the rate of the N-acetyl-D-glucosamine hydrolysis
somewhat decreases.
For strong acids such as HCl and HClO4, we can also
attempt to account for the maximum in the dependence
of the hydrolysis rate constant on the acid concentration
(Fig. 2) from the viewpoint of the Debye–Hückel theory
of strong electrolytes.
A decrease in the hydrolysis rate after reaching the
maximal values of kA may be due to the formation of
ion pairs (+) (–) in a strong electrolyte solution in this
concentration range [18]. This may lead to a decrease in
the total activity of hydrogen ions in solution and hence
to a decrease in the hydrolysis rate.
A drastic decrease in the hydrolysis rate at HClO4
concentrations exceeding the values corresponding to
the maximum of kA may also be due to the fact that
HClO4, being a strong oxidant, is consumed for the
monosaccharide oxidation.
CONCLUSIONS
(1) The rate of hydrolysis of the acetamide bond in
the chitin monomer, N-acetyl-D-glucosamine, depends
on the concentrations (activities) of hydrogen ions and
water; the nucleophilicity of the acid residues does not
affect the hydrolysis rate.
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 86 No. 7 2013
 MECHANISM OF ACID HYDROLYSIS OF N-ACETYL-D-GLUCOSAMINE
(2) Deacetylation of N-acetyl-D-glucosamine under
homogeneous conditions is an acid-catalyzed reaction
and can be described by a pseudo-first-order rate
equation. The rate of the D(+)-glucosamine formation is
influenced by the redox properties of the acid in which
the hydrolysis was performed.
(3) The results obtained expand the views on the
mechanisms of elementary reactions of the hydrolysis of
chitin and its monomers and can be used in improvement
of the technology of N-acetyl-D-glucosamine and D(+)-
glucosamine production.
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