Stoichiometric C6-oxidation of hyaluronic acid by oxoammonium salt TEMPO+Cl- in an aqueous alkaline medium

Article abstract of DOI:10.1016/j.carbpol.2015.04.054

This paper reports the selective oxidation of hyaluronic acid (HA) by stoichiometric quantity of 2,2,6,6-tetramethylpiperidine-1-oxoammonium chloride (TEMPO⁺) in aqueous alkaline medium. High efficiency of the HA oxidation and quantitative yield of carboxy-HA per starting TEMPO⁺, as well as unusual behavior of the oxidation system generating an oxygen upon alkali-induced oxoammonium chloride decomposition are demonstrated. The scheme for HA oxidation involving both TEMPO⁺ and oxygen produced upon the TEMPO⁺Cl^- decomposition and/or air oxygen is proposed. For comparison, the data on stoichiometric oxidation of such substrates as dermatan sulfate, water-soluble potato starch, methyl 2-acetamido-2-deoxy-β-d-glucopyranoside and ethanol are presented.

Full text of DOI:10.1016/j.carbpol.2015.04.054

Carbohydrate Polymers 130 (2015) 69–76
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Stoichiometric C6-oxidation of hyaluronic acid by oxoammonium salt
+
−
TEMPO Cl in an aqueous alkaline medium
∗
Irina Yu Ponedel’kina , Elvira A. Khaibrakhmanova, Tatyana V. Tyumkina,
Irina V. Romadova, Victor N. Odinokov
Institute of Petrochemistry and Catalysis, Russian Academy of Sciences, Ufa 450075, Russian Federation
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper reports the selective oxidation of hyaluronic acid (HA) by stoichiometric quantity of 2,2,6,6-
Received 19 January 2015
Received in revised form 8 April 2015
Accepted 21 April 2015
+
tetramethylpiperidine-1-oxoammonium chloride (TEMPO ) in aqueous alkaline medium. High efficiency
+
of the HA oxidation and quantitative yield of carboxy-HA per starting TEMPO , as well as unusual
behavior of the oxidation system generating an oxygen upon alkali-induced oxoammonium chloride
Available online 1 May 2015
+
decomposition are demonstrated. The scheme for HA oxidation involving both TEMPO and oxygen pro-
+
−
duced upon the TEMPO Cl decomposition and/or air oxygen is proposed. For comparison, the data on
stoichiometric oxidation of such substrates as dermatan sulfate, water-soluble potato starch, methyl
Keywords:
Hyaluronic acid
Oxidation
2
-acetamido-2-deoxy-ˇ-d-glucopyranoside and ethanol are presented.
©
2015 Elsevier Ltd. All rights reserved.
2
,2,6,6-Tetramethylpiperidine-1-
oxoammonium
chloride
Hydroxylamine TEMPOH
Radical TEMPO
Chemical compounds studied in this article:
Hyaluronic acid (CID 3084049)
2
2
,2,6,6-Tetramethylpiperidine-1-oxyl (CID
724126)
1
. Introduction
Hyaluronic acid (HA) is a natural heteropolysaccharide of a
appreciably differs in properties from natural HA. Carboxy-HA
has a better solubility in water (Crescenzi, Francescangeli, Renier,
& Bellini, 2001) and an unusually high stability to the action of
testicular hyaluronuridase (Ponedel’kina et al., 2007), which is
important for the development of prolonged action drugs. Being
a promising biomaterial and a carrier for other biomolecules,
carboxy-HA is an attractive object for conjugation with physio-
logically active amines (Bellini et al., 2002; Ponedel’kina, Lukina,
Khaibrakhmanova, Sal’nikova, & Odinokov, 2011).
linear structure belonging to the class of glycosaminoglycans
and consisting of alternating d-glucuronic acid and N-acetyl-
d-glucosamine units. The biocompatibility, water-retaining and
reparative and regenerative properties, and the ability to form
high-viscosity aqueous solutions (hydrogels) are valuable proper-
ties of HA demanded in medicine and cosmetics. By derivatization
at the carboxyl, hydroxyl, or acetamido groups, it is possible to
modify the physicochemical and biological properties of HA and
to develop new products for biomedicine and cosmetics (Kogan,
The C6-oxidation of HA is usually performed with an oxidative
system comprising sodium hypochlorite as a stoichiometric oxi-
dant, the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) nitroxyl
radical as a catalyst, and sodium bromide as a co-catalyst
(Scheme 1). Higher conversion of the primary alcohol groups of
HA to carboxy groups (80–100%) is achieved by oxidation in an
ˇ
Soltés, Stern, & Gemeiner, 2007; Mero & Campisi, 2014; Prestwich,
011; Schanté, Zuber, Herlin, & Vandamme, 2011). For example,
2
oxidation of the C6 primary hydroxyl groups of the N-acetyl-d-
glucosamine unit of HA affords polyuronic acid, i.e. carboxy-HA
◦
alkaline medium (pH 10), at a temperature of ∼0 C, and a reac-
(
or percarboxylated HA) (Bellini, Crescenzi, & Francescangeli, 2002;
tion time of 70–80 min (Crescenzi et al., 2001; Jiang, Drouet, Milas,
& Rinaudo, 2000). On the other hand, upon oxidation of HA with
sodium hypochlorite in a concentrated aqueous solution of NaHCO3
or with trichlorocyanuric acid in aqueous DMF, aldehyde hydrate
groups (5–18%) can be introduced into the macromolecules (Buffa,
Kettou, Pospi sˇ ilová, Berková, & Velebn y´ , 2011). Aldehyde hydrates
Ponedel’kina, Odinokov, Saitgalina, & Dzhemilev, 2007), which
∗ Corresponding author. Tel.: +7 347 284 2750; fax: +7 347 284 2750.
E-mail address: ponedelkina@rambler.ru (I.Y. Ponedel’kina).
http://dx.doi.org/10.1016/j.carbpol.2015.04.054
0
144-8617/© 2015 Elsevier Ltd. All rights reserved.

7
0
I.Y. Ponedel’kina et al. / Carbohydrate Polymers 130 (2015) 69–76
-
4-acetylamino-2,2,6,6-tetramethyl-1-oxopiperidinium tetrafluo-
-
e
roborate (Bobbitt’s salt) in the presence of pyridine bases (in
CH Cl ), primary alcohols containing an oxygen atom (of the ether
+
N
N
2
2
O
O
group) in the ˇ-position to the alcohol group are over-oxidized to
dimeric esters, and their yield depends on the nature of the pyridine
base (Bobbitt et al., 2014; Merbouh, Bobbitt, & Brückner, 2004). The
mechanism of this over-oxidation of alcohols containing ˇ oxygen
has not yet been elucidated.
N-Acetyl-d-glucosamine unit in HA also contains an oxygen
atom (pyranosyl) in the ˇ-position to the hydroxymethyl group,
and under basic conditions, this can affect the selectivity of oxi-
dation and the structure of oxidized products. Meanwhile, the
known ability of cation TEMPO+ to decay in aqueous alkalis (Endo,
Miyazawa, Shiihashi, & Okawara, 1984; Golubev et al., 1965;
Golubev & Sen’, 2011) or to react with hydroxylamine TEMPOH (Eq.
.
TEMPO+
TEMPO
+
RCOOH + 2TEMPOH + 2H+
RCH OH + 2TEMPO + H O
2
2
-
+
+
-
TEMPOH + OBr + H
TEMPO + H O + Br
2
Br- + OCl-
-
-
OBr + Cl
Scheme 1. Oxidation of primary hydroxyl groups of polysaccharides by the
TEMPO/NaOCl/NaBr system in water.
and aldehydes are known to be intermediates in the TEMPO oxida-
tion of alcohols to carboxylic acids (Bragd, van Bekkum, & Besemer,
004; Ponedel’kina, Khaibrakhmanova, & Odinokov, 2010).
According to the generally accepted scheme of C6-oxidation
of polysaccharides with the NaOCl–NaBr–TEMPO system in aque-
ous alkali, the active oxidant is the TEMPO oxoammonium cation
formed in situ from TEMPO on treatment with hypohalite. As the
hydroxymethyl group is oxidized to the carboxy group, TEMPO
is reduced to the hydroxylamine TEMPOH. Then TEMPOH is oxi-
dized to TEMPO+ by NaOBr formed from NaBr under the action of
hypochlorite (Bragd et al., 2004; Jiang et al., 2000).
(1)) (Israeli et al., 2005) can also affect the efficiency of HA oxidation.
TEMPO + TEMPOH → 2TEMPO + H+
+
(1)
2
Therefore, we studied both the products of HA oxidation and the
+
+
transformations of the TEMPO cation. We believe this study could
be helpful for better understanding of the mechanism of both stoi-
chiometric and catalytic oxidation of alcohols with participation of
TEMPO derivatives.
+
2. Experimental
Oxidation of polysaccharides, including HA, by a stoichiometric
amount of oxoammonium salts, has not been performed previously.
Meanwhile, this method has long been used for selective oxida-
tion of low-molecular-weight primary and secondary alcohols to
aldehydes (or carboxylic acids) and ketones both in organic and
aqueous medium (Bobbitt, Bartelson, Bailey, Hamlin, & Kelly, 2014;
De Nooy, Besemer, & van Bekkum, 1996; Golubev, Rozantsev, &
Neiman, 1965; Luderer et al., 2011; Qiu, Pradhan, Blanck, Bobbitt,
2.1. Materials
In the oxidation experiments two samples of sodium
hyaluronate were used: HMW HA from bacterial source
6
(M = 1.5 × 10 , supplier Leko Style, St.-Petersburg) and LMW
4
HA from human umbilical cord (M = 4–6 × 10 ), which was iso-
lated as described elsewhere (Ponedel’kina et al., 2005). Other
polysaccharides and low-molecular-weight compounds used in
the oxidation were: dermatan sulfate (DS) from pig skin, which
was prepared as elsewhere (Ponedel’kina, Khaibrahmanova,
Odinokov, Khalilov, & Dzhemilev, 2010), starch (Sigma–Aldrich),
ethanol and methyl 2-acetamido-2-deoxy-ˇ-d-glucopyranoside
(MeNAG, for preparation see Section 2.3). TEMPO, 2-acetamido-2-
deoxy-d-glucopyranoside (d-GlcNAc), sodium dithionite Na2S2O4
and cation exchanger Dowex 50WX4 were purchased from Acros
Organics; NaOD and DCl were purchased from Sigma–Aldrich; D2O
was bought from Eurisotop. Molar extinctions of TEMPO in water
were determined as 1960 and 320 at 245 and 290 nm, respectively
(the ratio ε245/ε290 ∼ 6). Other chemicals were of analytical reagent
grade.
&
Bailey, 2011). This paper reports the first application of this
effective method for the oxidation of HA in order to prepare
carboxy-HA. As the stoichiometric oxidant, we have chosen 2,2,6,6-
+
−
tetramethylpiperidin-1-oxoammonium chloride (TEMPO Cl ) as it
is easy to prepare and readily soluble in water.
The mechanisms of both catalytic and stoichiometric oxidation
of alcohols with oxoammonium cation under basic conditions have
not been ultimately established. The mechanism considering the
formation of the intermediate complex of alkoxide anion and the
oxoammonium cation followed by intramolecular hydrogen atom
transfer resulting in the aldehyde (ketone) and TEMPOH as the
key step is regarded as most likely (Scheme 2) (Bailey, Bobbitt, &
Wiberg, 2007; Semmelhack, Schmid, & Cortés, 1986). The main rea-
sons in favor of this mechanism are the quantitative yields of target
products and the absence of by-products.
+
−
2.2. Preparation of TEMPO Cl and TEMPOH
However, it has been found not long ago that the selectiv-
ity of oxidation by oxoammonium salts and the composition
of products can depend substantially on the alcohol structure
and oxidation conditions. For instance, upon oxidation with
+
−
Oxoammonium salt TEMPO Cl was prepared by reacting of
TEMPO radical with Cl according to the procedure described in the
literature (Golubev, Zhdanov, & Rozantsev, 1970). TEMPO (200 mg,
2
-O
H
R
HO
H
R
+ B
BH+
-
R(H)
R(H)
R
+
N
-O
R
.. +
O
+
O
N
N
R(H)
R(H)
O
H
OH
Scheme 2. Mechanism of alcohol oxidation by oxoammonium cation in basic media.

I.Y. Ponedel’kina et al. / Carbohydrate Polymers 130 (2015) 69–76
71
Table 1
1
.28 mmol) was dissolved in 3 ml CCl4 after then 3 ml of 1.5%
+
Experimental conditions for HA oxidation by TEMPO .
Cl2 solution in CCl4 was added. The yellow precipitate was sepa-
a
rated, washed with CCl , dried (yield 100%) and stored not more
Entry
[HA], mmol/ml,
HA:TEMPO+
pH
DO, %
[TEMPO] /
4
+
−
[TEMPO+] , %
than 3 days. Molar extinctions of TEMPO Cl were determined as
ε245 = 1730, ε290 = 860 (the ratio ε245/ε290 ∼ 2), and ε470 = 19.
Hydroxylamine TEMPOH was synthesized by the TEMPO reduc-
tion with sodium dithionite according to literature (Ozinskas &
Bobst, 1980). Briefly, to a water–acetone (1:1) solution of TEMPO
0
HMW
LMW
HMW
LMW
b
1
2
3
4
5
6
7
0.0125, 1:1
0.0125, 1:1
0.0125, 1:1
0.0125, 1:2
0.0125, 1:0.5
0.0125, 1:0.25
0.0063, 1:1
0.0063, 1:1
0.0063, 1:2
0.0125, 1:1
8.5
42
43
60
50
72
100
–
46
30
33
67
20
35
27
18
42
56
29
22
19
41
–
b
10.2
11.5
10.2
10.2
10.2
10.2
10.2
10.2
10.2
45b
90
(
200 mg, mmol) excess Na S O was added at room tempera-
2 2 4
b
26
ture and under flowing Ar. The suspension was stirred ∼10 min
after then acetone was removed under vacuum. The product was
extracted from aqueous layer with Et O (3× 3 ml). After Et O evap-
8b
50
–
–
56
55
100
55
43
26
41
56
c
2
2
8
50
1
oration, 160 mg TEMPOH (80%) was obtained. H NMR (400 MHz;
D O) ı 1.58 (br. s, 6H), 1.14 (s, 12H); C NMR (125 MHz; D O) ı
9
100
13
10d
51b
2
2
6
3.9 (C-2,6), 38.0 (C-3,5), 29.4, 18.9 (CH ), 16.0 (C-4).
It should be noted, that under storing in air atmosphere the slow
a Quantity of TEMPO after oxidation was determined from UV spectra at 245 nm.
TEMPOH solutions at the wave length do not absorb.
3
b
Determined by IR.
Reactions were carried out in Ar.
Reactions were carried out at ∼20 C.
transformation of colorless TEMPOH crystals to red-orange TEMPO
was observed.
c
d
◦
2.3. Preparation of MeNAG
ꢀ
ꢀ
D O) ı 176.4 (C-6), 176.1 (C-6 ), 175.3 (CH CON), 104.2 (C-1), 101.7
2
3
MeNAG was synthesized from d-GlcNAc (Scheme 1S) accord-
ing to the work (Cai, Ling, & Bundle, 2005; Mack, Basabe, &
Brossmer, 1988). To a suspension of d-GlcNAc (2 g, 9 mmol) in
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(
(
C-1 ), 82.7 (C-3 ), 81.7 (C-4), 77.6 (C-5), 77.1 (C-5 ), 74.8 (C-3), 73.6
ꢀ
ꢀ
ꢀꢀ
C-2), 72.0 (C-4 ), 55.5 (C-2 ), 23.7 (CH CON).
3
For FTIR analysis, the samples were converted in their acidic
4
0 ml dry acetone BF ·Et O (4 g, 2.8 mmol) was added. The mix-
3
2
forms with a cation exchange resin Dowex 50WX4 in H+ form and
were prepared in the form of films.
ture was stirred and boiled under reflux for 12 min with the
exclusion of moisture, then cooled in ice, treated with 13 ml
triethylamine, and added dropwise to cold solution of sodium car-
bonate (14.5 g in 100 ml water). Acetone and triethylamine were
2.4.1. Oxidation in D O for the NMR analysis
2
◦
removed under diminished pressure at <30 C. The obtained mix-
Oxidation of LMW HA, DS, starch, ethanol and MeNAG
ture was multiple extracted with ether, and the combined extracts
were dried with MgSO4 and concentrated. The crude product was
dissolved in 80 ml dry methanol, and p-toluenesulfonic acid (PTSA)
(0.0125 mmol/3.3 ml D O) for the NMR analysis was carried out
2
analogously to the typical procedure (HA:TEMPO+ = 1:2, 2 ± 2 ◦C,
pH 10.2). To regulate the reaction pH, NaOD and DCl were used.
Reaction solution was placed onto NMR tube and NMR spectra were
monitored (Fig. 3 and Figs. 6S, 8S, 10S–13S).
For oxidized DS DOtotal was found from the ratio of intensi-
ties of signals at 5.03 ppm (H-4 in oxidized galactosamine) and
(
0.5 g, 3 mmol) was added. After 3 h the reaction mixture was
neutralized with triethylamine, and methanol was removed in vac-
uum. The residue was multiple washed with CH Cl in order to
2
2
remove PTSA salt and then was purified by column chromatogra-
phy (methanol:chloroform, 1:1) on silica gel to give MeNAG (1 g,
2.10 ppm (CH CON, as reference); the content of units with alde-
3
4
.5 mmol) as white powder. Yield 50%.
hyde and aldehyde hydrate groups was found using signals at
9.22 and 5.19 ppm respectively; carboxyl groups content was cal-
culated as the difference between DOtotal and content of units
with aldehyde + aldehyde hydrate groups (Fig. 6S) (Ponedel’kina,
Khaibrahmanova, Odinokov, et al., 2010). For oxidized starch
DOtotal was determined from the ratio of intensities of signals
at 4.09 ppm (H-5 in oxidized unit) and 5.43–5.62 ppm (anomeric
region, as reference); the content of aldehyde hydrate units
was found using signals at 5.31–5.38 ppm; the content of units
with carboxyl groups was calculated as the difference between
DOtotal and aldehyde hydrate content (Fig. 8S) (Ponedel’kina,
Araslanova, Tyumkina, Lukina, & Odinokov, 2014). For oxidized
MeNAG the DO, i.e. the content of methyl 2-acetamido-2-deoxy-
ˇ-d-glucopyranosyluronic acid, was found as 100%-unreacted
MeNAG. The last was calculated from the ratio of signals at 3.95 ppm
(H-6) and 4.45 ppm (H-1, as reference) (Figs. 10S and 11S). In the
case of oxidized ethanol, NMR signals for methyl protons at 2.26
(aldehyde), 1.93 (acid) and 1.20 ppm (unoxidized ethanol) were
used (Figs. 12S and 13S).
1
H NMR (400 MHz; D O) ı 4.45 (d, H-1, J = 8.4), 3.95 (d, H-
2
1,2
ꢀ
, J6,6 = 12.1), 3.44–3.78 (H-2–5, H-6 ), 3.52 (s, CH O), 2.05 (s,
ꢀ
3
6
CH CON), cf. Perkins, Johnson, Phillips, and Dwek (1977) (Fig. 9S).
3
2.4. Typical oxidation procedure
HA (50 mg, 0.125 mmol of primary hydroxyl groups) was dis-
solved in 10 or 20 ml of distilled water, then the temperature and
pH of HA solution were adjusted to required values (Table 1). When
an inert atmosphere was required argon was passed through the
solution for 15 min and above the solution for all reaction time. The
required quantity of TEMPO+ (0.24–48 mg, 0.00125–0.25 mmol,
Table 1) in 1 ml H O was added to HA solution under vigorous stir-
2
+
ring. After TEMPO addition, 0.4 M NaOH was immediately added
to neutralize the acidic reaction products and maintain the reaction
pH at the necessary level. Reaction was stopped at 15 min by adding
2
ml methanol, and then the solution was neutralized. Samples of
oxidized HMW HA were precipitated with three volumes of MeOH,
and LMW HA solutions were concentrated to 2–3 ml under vac-
◦
uum (temperature of the bath ∼60 C) and were also treated with
+
−
2
.5. Effect of basic media on the TEMPO Cl stability
MeOH. The resulting precipitates were centrifuged, washed with
◦
MeOH and diethyl ether and dried in vacuum at 60 C for 2 h. Oxi-
+
−
TEMPO Cl (0.24 or 2.4 mg) in 1 ml H O was added to water
dized samples (44–48 mg) were obtained as white water-soluble
powders or fibers.
2
◦
(
10 ml) cooled to 2 ± 2 C. The pH of solutions with finishing
+
−
1
ꢀꢀ
TEMPO Cl concentrations of 0.022 (0.00011) or 0.22 (0.0011) mg
(mmol)/ml was adjusted to 10.2, and UV spectra were recorded at
various timepoints between 1 and 15 min (Table 2).
Carboxy-HA. H NMR (500 MHz; D O) ı 4.55 (H-1 ), 4.48 (H-1),
2
ꢀꢀ ꢀꢀ ꢀꢀ
3
4
.87 (H-2 ), 3.78 (H-5 ), 3.77 (H-3 ), 3.70 (H-5), 3.70 (H-4), 3.61 (H-
ꢀ
ꢀ
13
), 3.59 (H-3), 3.34 (H-2), 2.03 (3H, CH CON); C NMR (125 MHz;
3

7
2
I.Y. Ponedel’kina et al. / Carbohydrate Polymers 130 (2015) 69–76
Table 2
aqueous solutions at various pH and temperatures, variable reac-
tant concentrations and ratio, either with free access of air oxygen
or under Ar (Table 1).
+
−
Absorption of TEMPO Cl solutions depending on the reaction time.
+
[TEMPO ], mg/ml
A245/A290
TEMPO+ was used in no more than twofold excess and
added to the HA solution as one portion for the following
0
mina
1 min
5 min
15 min
0
0
.22
.022
0.96/0.49b
0.11/0.06
0.56/0.11b
0.06/0.01
0.56/0.11b
0.06/0.01
0.57/0.11b
0.06/0.01
+
reasons. According to the equation RCH OH + 2TEMPO + H O→
2
2
+
RCOOH + 2TEMPOH + 2H (see Scheme 1), during oxidation of the
a
Measurements were made at neutral pH.
Absorptions are given for solutions diluted twofold.
alcohol to the carboxylic acid, the TEMPO+ cation is converted
b
to the hydroxylamine TEMPOH. In an alkaline medium, TEMPOH
and TEMPO+ can undergo comproportionation (Eq. (1)) to give the
+
−
4
−1 −1
2.5.1. Oxygen evolution measurement in the TEMPO Cl solution
TEMPO radical (k = 10 M s ) (Israeli et al., 2005).
◦
In addition, TEMPO+ is unstable to the action of aque-
ous alkalis, which was demonstrated previously in relation
Water (30 ml) was cooled to 2 ± 2 C and adjusted to pH 10.2,
after then dissolved oxygen was removed by passing Ar for 15 min
up to concentration of 0.00 mg/l, accordingly to oximeter read-
to
2,2,6,6-tetramethyl-4-hydroxypiperidin-1-oxo
chloride
+
−
+
−
ing. TEMPO Cl (72 mg, 0.375 mmol) was added to water under
Ar atmosphere and the concentration of the resulting oxygen was
monitored for 15 min. Then the TEMPO Cl solution was kept for
(4-hydroxy-TEMPO Cl ) (Golubev et al., 1965), 4-methoxy-2,2,6,6-
tetramethylpiperidin-1-oxo bromide (4-methoxy-TEMPO+Br−)
+
−
(Endo et al., 1984) and 2,2,6,6-tetramethyl-4-oxopiperidin-1-oxo
3
h under Ar for further oxygen evolution monitoring.
perchlorate (4-oxo-TEMPO+ClO4−) (Golubev & Sen’, 2011). The
major product formed upon decomposition of these salts is the
corresponding radical. Besides radical, other byproducts can be
formed at alkaline pH (see Footnote below). When TEMPO is
2
.5.2. TEMPOH stability under reaction conditions
TEMPOH (19 mg, 0.125 mmol) was added to an aerated solution
+
used in a more than twofold excess or when TEMPO+ is gradually
added to a solution with an alkaline pH, the probability of side
reactions can increase, while the degree of oxidation (DO) of HA
can hence decrease. To prevent this, the pH 10.2 was chosen. It is
also optimal for the catalytic oxidation (Jiang et al., 2000). The pH
8.5 was difficult to control due to rapid formation of acidic reaction
products, which moved the pH toward more acidic values. At pH 7
and lower, HA oxidation did not occur.
of HMW HA (50 mg/11 ml H O) prepared as for the oxidation pro-
2
◦
cedure (2 ± 2 C, pH 10.2) and mixture was stirred for 15 min. At
various timepoints between 1 and 15 min UV spectra were mon-
itored for the TEMPO detection. Under these conditions the UV
spectra of TEMPOH remained unchanged. The TEMPOH/HA solu-
tion was then kept for a week at room temperature but the TEMPO
formation was not detected.
+
2.6. Analyses
The stoichiometric HA oxidation with the TEMPO cation under
basic conditions proceeded very rapidly, and already after 1–2 min
the peak at 470 nm for TEMPO+ in the vision spectrum was com-
pletely disappeared indicating the end of the oxidation (Fig. 2).
As was shown earlier, catalytic TEMPO oxidation of HA was much
longer (Jiang et al., 2000).
One- ( H and 13C) and two-dimensional (HSQC) NMR spectra
1
were recorded on a Bruker Avance III 500 MHz (500.17 MHz for
H and 125.78 MHz for C) and on a Bruker Avance II 400 MHz
400.13 MHz for H and 100.62 MHz for C) spectrometers. Sam-
1
13
1
13
(
As expected, the HA oxidation with the TEMPO+ cation was
accompanied by the formation of the hydroxylamine TEMPOH
(Fig. 3, the reaction conditions as in entry 4). Its signals were
observed at 1.14 (s, 12H) and 1.58 (br. s, 6H) ppm in 1H NMR spec-
trum. However, the intensities ratio of these signals (>2) and the
presence of additional unknown signals in the near area of the spec-
trum gave evidence for the formation of some quantity of products
of the oxoammonium chloride decomposition. Apart from TEM-
POH, a considerable amount of the TEMPO radical was detected
(Fig. 2), although attempts were made to avoid its formation. The
yield of TEMPO reached a maximum also after 1–2 min, being equal
ples were analyzed as solutions in D O (15 mg/ml) at room
temperature using acetone as internal standard (ı 2.22 ppm for
2
1
13
H and 31.45 ppm for C). For HMW HA samples the pulse con-
ditions were as follows: for the 1H NMR spectrum, 90 pulse flip
angle, acquisition time (AQ) = 2.05 s, high-power pulse (P1) = 8.00 s,
spectral width (SW) = 4000 Hz, data points (TD) = 16 384, num-
◦
ber of scans (NS) = 32; for the 13C NMR spectrum, 30 pulse flip
◦
angle, P1 = 4.7 s, AQ = 0.9 s, dummy scans (DS) = 2, Relaxation Delay
(
RD) = 1.0 s, SW = 18 116 Hz, TD = 32 768; for the HSQC spectrum,
AQ = 0.32 s, RD = 0.84 s, SW(F1) = 7043.1 Hz, SW(F2) = 1600.5 Hz,
TD = 1024 × 128.
+
Dissolved oxygen concentration (mg/l) was measured with an
oximeter Mark 303 T. Absorbance spectra were obtained in 1 cm
cell using a Perkin Elmer Lambda 750 UV/VIS spectrometer. FTIR
spectra were recorded using a Bruker Vertex 70 spectrometer. The
reaction pH was controlled with pH meter HANNA 2210.
to 18–67% of the TEMPO cation taken in the reaction (Table 1), and
it was higher in an aerated solution (entry 7) than under Ar (entry
+
8). A decrease in the TEMPO concentration down to catalytic also
resulted in a higher yield of the radical. For HA to TEMPO+ ratios
of 1:0.1, 1:0.025, and 1:0.01 (other conditions as in entry 2), the
percentages of TEMPO were 27, 38, and 58%, respectively. It is clear
that the more TEMPO is formed upon HA oxidation, the lower the
3
. Results and discussions
+
yield of TEMPOH (approximately TEMPO –TEMPO); however, due
The oxidation of HMW and LMW HA with the oxoammonium
salt TEMPO Cl (below TEMPO ) (Scheme 3) was performed in
to side reactions, there was no sense to determine its yield more
accurately. For example, in entry 4 (DO of LWM HA 100%) the yield
+
−
+
OH
TEMPO+
COOH
O
COOH
COOH
O
O
O
HO
HO
O
O
O
O
HO
HO
NHAc
NHAc
OH
OH
carboxy-HA
HA
Scheme 3. Oxidation of hyaluronic acid by TEMPO+.

I.Y. Ponedel’kina et al. / Carbohydrate Polymers 130 (2015) 69–76
73
+
TEMPO+
of TEMPOH calculated as TEMPO –TEMPO (100–41) was 59%, while
RCH OH
2
H
O, OH-
2
1
the same found from H NMR spectrum (Fig. 3) was 48% per starting
+
TEMPO .
The 13C NMR spectra of all of the oxidized HA samples exhibited
O
2
RCHO
no signal for the aldehyde group. The minor signal at ∼5.20 ppm
H O
TEMPO
2
corresponding to the methine proton in the CH(OH) group (Bubb,
2
1
2
003), present in the H NMR spectra of some samples, was indica-
RCH(OH)2
RCOOH
tive of a slight content of aldehyde hydrate units; see Fig. S1
Supplementary Data). The hydroxamic test for ester groups was
(
TEMPO
+
O2
negative. Evidently because of aqueous alkaline conditions, the
pyranosyl oxygen located in the ˇ position to the alcohol group of
N-acetyl-d-glucosamine did not affect the selectivity of HA oxida-
tion and the oxidation of HA hydroxyl groups afforded exclusively
carboxyl groups. The content of these groups (i.e. DO) in HA was
found from the IR spectra (Fig. 1) according to reported method
TEMPOH
Scheme 4. Oxidation of hyaluronic acid.
In most cases, in both aerated solutions and an inert medium, the
DO of HA nearly corresponded to TEMPO consumed in the reaction
except for LWM HA samples oxidized at pH 8.5 (entry 1) and 11.5
entry 3). Their DO values were 60 and 72%, respectively, instead
of the expected 50%. In all cases, the DO of HA was higher than it
followed from the amount of hydroxylamine TEMPOH formed.
Most likely, the low yield of TEMPOH was caused by the redox
reactions typical of the TEMPO+ cation to give the TEMPO radical.
However, in these reactions, TEMPO is spent not for HA oxidation;
hence, the deficient TEMPO should be counterbalanced by another
oxidant. In aerated solutions, the role of the second oxidant can
be performed by air oxygen. In addition, O2 can be generated in
the reaction medium, possibly, upon decomposition of oxoamm-
onium chloride. Indeed, when we removed the dissolved oxygen
from water by Ar purging (pH 10.2) and then added the TEMPO+
salt in a concentration of 0.013 mmol/ml, then the formation of
O2 was detected by an oximeter. During 15 min, the oxygen con-
centration increased from 0 to 0.53 mg/l, and another 3 h later it
reached 4.5 mg/l. Sodium hydroxide was required to maintain the
+
(Jiang et al., 2000) and using 2D HSQC experiment (Fig. 4).
The DO of HA depended mainly on the reactant ratio. Under
(
identical oxidation conditions, the DO of almost all HWM HA sam-
ples was somewhat lower than in the case of LWM HA (Table 1).
Apparently, due to the high viscosity of HWM HA, the reactant mis-
+
cibility was insufficiently high and TEMPO was partially consumed
for side reactions rather than for HA oxidation. In less concentrated
solutions and at room temperature where the viscosity was lower,
the HWM HA oxidation was more effective (cf. entries 4 and 9, 2
and 10).
+
+
pH of this reaction at 10.2. Apparently, apart from TEMPO and O ,
2
1
decomposition of the salt afforded acidic products. At high con-
centration of the TEMPO+ salt (0.013 mmol/ml), it is difficult to
detect the formation of the TEMPO radical, especially in the first
+
minutes of the reaction. However, at lower TEMPO concentrations
(
0.0011 and 0.00011 mmol/ml), when the influence of the alkaline
+
medium increased, fast conversion of the TEMPO cation to the
TEMPO radical was observed (yield ∼50%) (Table 2).
+
Thus, the obtained data suggest that along with TEMPO , molec-
ular oxygen is involved in HA oxidation. The HA oxidation process
can be described by the following chart (Scheme 4). The alcohol
groups of HA are first oxidized to the aldehyde groups, this is
accompanied by consumption of an equimolar amount of TEMPO+
and gives an equivalent amount of TEMPOH. Some of the remaining
+
TEMPO undergoes comproportionation reaction with an equimo-
lar amount of TEMPOH to give the TEMPO radical (see Eq. (1)).
1
+
−
For the reaction and decomposition products of 4-hydroxy-TEМРO Cl
+
−
(
Golubev et al., 1965), 4-methoxy-TEМРO Br (Endo et al., 1984), and 4-oxo-
TEМРO ClO4 (Golubev & Sen’, 2011) under alkaline conditions, see the relevant
+
−
+
−
references. Only 4-oxo-TEМРO ClO4 was studied in detail. Among the nine iden-
tified products of decomposition, the yield of the 4-oxo-TEМРO radical was 60%,
and no oxygen was found. On the other hand, at рН 9–11 and room tempera-
+
−
ture, 4-methoxy-TEМРO Br was quantitatively converted to 4-methoxy-TEМРO,
+
−
the other product being Н2O2 (∼90%). If decomposition of TEМРO Cl also gives
Н
2O2, the formation of O2 that we detected can be due to its reaction with
the TEMPO cation: TEMPO + H2O2 → TEMPO + O2 + 2H (Sen, Golubev, Kulyk, &
+
+
+
Rozantsev, 1976). Hence, it should be noted that decomposition of oxoammonium
+
−
salts (including TEМРO Cl ) has not been systematically studied as a function of
substituent in the piperidinium ring, the counter-ion type, and the reaction con-
ditions. All the listed factors can have a very pronounced influence on the redox
properties of oxoammonium salts both in the decomposition and in oxidation of
+
Fig. 1. Fragments of FTIR spectra of (a) intact HMW HA and (b) partially oxidized
alcohols. For example, it was found that 4-methoxy-TEМРO Br is an order of mag-
HMW HA (DO 45%) in their H+ form. The degree of HA oxidation was calculated using
+
−
nitude less reactive in alcohol oxidation than 4-methoxy-TEМРO Cl (Miyazawa,
−
1
−1
the areas of bands at 1728 cm (CO2H) and at 1555 cm (NH, as the reference).
Endo, Shiihashi, & Okawara, 1985).

7
4
I.Y. Ponedel’kina et al. / Carbohydrate Polymers 130 (2015) 69–76
+
−
Fig. 2. UV spectra of 0.0125 M TEMPO Cl prior to reaction and TEMPO formed after 1–2 min oxidation (conditions as in entry 2).
Fig. 3. 1H (A) and 13C NMR (B) spectra of mixture of carboxy-HA and TEMPOH in reaction solution after LMW HA oxidation in D2O (15 mg/3.3 ml D2O, HA:TEMPO Cl = 1:2,
+
−
◦
2
± 2 C, pH 10.2). Unlabeled signals are attributed to carboxy-HA and given in experimental part.
The rest of TEMPO+ is spent for oxidation of some of the aldehyde
groups of HA (apparently, as aldehyde hydrate groups; Qiu et al.,
Apart from the above side reactions, the low yield of TEMPOH
and, hence, the high yield of the TEMPO radical in the oxidation of
HA may be due to the known re-oxidation reaction of the hydroxy-
lamine TEMPOH by air oxygen (Eq. (2)) (Ozinskas & Bobst, 1980):
2
011) to carboxyl groups being thus reduced to TEMPOH or it partly
decomposes under the action of alkali to give TEMPO, O and other
2
+
products. The air oxygen and/or oxygen generated upon TEMPO
TEMPOH + (1/2)O → TEMPO + (1/2)H O
(2)
2
2
decomposition oxidizes another part of the aldehyde groups of HA,
thus counterbalancing TEMPO+ spent for side reactions. It is diffi-
cult to estimate the contribution of each of the side reactions. It is
quite possible that decomposition in which TEMPO+ is consumed
However, TEMPOH that we synthesized proved to be stable and
did not react with oxygen under the process conditions. Therefore,
this pathway to the TEMPO radical was ruled out.
The proposed scheme of HA oxidation involving oxygen
accounts not only for the formation of TEMPO but also for the fact
that the DO of some LWM HA is higher than the expected DO (50%)
by 10–22% (entries 1 and 3).
for the formation of O prevails over comproportionation in which
2
TEMPO+ is consumed for the formation of TEMPO. Otherwise, the
DO of HA would be lower in an inert atmosphere than it is observed
in the experiment (entry 8).

I.Y. Ponedel’kina et al. / Carbohydrate Polymers 130 (2015) 69–76
75
Fig. 4. Fragment of HSQC spectrum of partially oxidized LMW HA sample. DO of HA hydroxymethyl groups was found as follows. Intensities of marked C4/H4 cross-peaks
for oxidized (72.0/3.61 м.д, Iox) and non-oxidized (69.9/3.51 м.д, Inon-ox) glucosamine units were determined by contour integration and DO was calculated by formula
DO = Iox/(Iox + Inon-ox).
High efficiency of stoichiometric oxidation and unusual behav-
ior of the oxidation system in whole are interesting in application
to other substrates with primary alcohol groups. Therefore, such
polysaccharides as glycosaminoglycan DS and water-soluble potato
starch were involved in oxidation. Ethanol was used as a simplest
low-weight-molecular alcohol, and MeNAG was chosen as nearest
oxidized substrate more than alkali pH (cf. Fig. 3 and Figs. 6S, 8S,
10S–13S).
As for the scheme proposed for HA oxidation (Scheme 4), the
question of its applicability to other substrates such as DS, starch,
ethanol or MeNAG remains open. The reaction ability of alcohols
strongly depends on their structure and, presumably, the HA behav-
ior in oxidation with TEMPO+ is unique.
analog of N-acetyl-ˇ-d-glucosamine unit (Table 3). The oxidation
+
conditions were as in entry 4 (Table 1): RCH OH:TEMPO = 1:2, pH
2
◦
1
0.2, 2 ± 2 C. In these conditions the reaction rate was extremely
+
rapid as in the case of HA, and cation TEMPO was consumed during
1
–2 min. However, in contrast to HA the DOtotal of all substrates was
Table 3
lower than 100%, and oxidized products, except oxidized MeNAG,
contained both acids and aldehydes (aldehyde hydrates). Their
composition depended on the structure of used alcohol but prac-
tically did not depend on atmosphere (air or Ar), in which the
+
−
Oxidation of DS, starch, MeNAG and ethanol by TEMPO Cl .
substrate DOtotal, %a Oxidized products, mol%
TEMPO+
consumed
[TEMPO]/
[TEMPO ]0, %
b
+
Aldehyde Aldehyde Acid
hydrate
+
oxidation was performed. The TEMPO consumed only in oxida-
tion and calculated based on the stoichiometry of the oxidation of
alcohol to acid and aldehyde was 31–56% (Table 3). Therefore, it is
not necessary to discuss the deficiency of oxidizer, as in the case of
HA oxidation. The remaining oxoammonium salt was consumed
in side reactions of decomposition and/or comproportionation.
Among products of these reactions, the radical TEMPO (Table 3)
and hydroxylamine TEMPOH prevailed (Figs. 6S, 8S, 10S–13S).
Other unknown products of TEMPO decomposition were more
diversified and were formed with higher yield than upon HA oxi-
dation; their quantity and composition depended on the nature of
DS
Starch
70
42
4
0
24
23
42
19
56
31
81
50
54
5
0
0
0
0
54
45
54
45
70
71
MeNAG
c
c
4
4
5
4
3
4
5
0
0
40
48
44
50
52
50
Ethanol
a
DO was found from 1H NMR (see Section 2 and Figs. 6S, 8S, 10S–13S).
TEMPO consumed was calculated based on the oxidation stoichiometry of alco-
+
b
+
hol to aldehyde/aldehyde hydrate and acid.
c Reactions were carried out in Ar.

7
6
I.Y. Ponedel’kina et al. / Carbohydrate Polymers 130 (2015) 69–76
Golubev, V. A., Zhdanov, R. I., & Rozantsev, É. G. (1970). Reaction of iminoxyl radicals
4
. Conclusion
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The oxidation of HA (HMW and LMW) by 0.25–2 equivalents
+
−
of oxoammonium salt TEMPO Cl occurs rapidly and selectively
in water–alkaline medium and leads to formation of carboxy-HA
with nearly quantitative yield of carboxyl groups per the start-
ing TEMPO . Among products of the cation TEMPO reduction,
apart from expected hydroxylamine TEMPOH the radical TEMPO
was found in the considerable amount (18–67% per the starting
09.037
+
+
Jiang, B., Drouet, E., Milas, M., & Rinaudo, M. (2000). Study on TEMPO-mediated
selective oxidation of hyaluronan and the effects of salt on the reaction kinet-
ics. Carbohydrate Research, 327(4), 455–461. http://dx.doi.org/10.1016/S0008-
6
215(00)00059-8
+
TEMPO ). We assumed that TEMPO is formed upon the interaction
Kogan, G., Sˇ oltés, L., Stern, R.,
& Gemeiner, P. (2007). Hyaluronic acid: A
+
of TEMPO with TEMPOH (i.e. comproportionation reaction) and/or
natural biopolymer with a broad range of biomedical and industrial appli-
cations. Biotechnology Letters, 29(1), 17–25. http://dx.doi.org/10.1007/s10529-
upon alkali-induced TEMPO+ decomposition. Dioxygen evolution
006-9219-z
was observed in the decomposition reaction. The side reactions
account for the deficiency of TEMPO+ as the oxidant and this defi-
ciency is counterbalanced by participation of air oxygen and/or
oxygen generated upon TEMPO+ decomposition. A new expanded
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scheme for HA oxidation with combined participation of both
TEMPO+ and molecular oxygen is proposed. Stoichiometric oxida-
0008-6215(88)84154-5
tion of other substrates such as DS, starch, MeNAG or ethanol was
less effective in compared with HA, and these results do not allow
us to make a conclusion about general character of the scheme
proposed for HA.
Merbouh, N., Bobbitt, J. M., & Brückner, C. (2004). Oxoammonium salts. 9. Oxidative
dimerization of polyfunctional primary alcohols to esters. An interesting ␤ oxy-
gen effect. Journal of Organic Chemistry, 69(15), 5116–5119. http://dx.doi.org/10.
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Miyazawa, T., Endo, T., Shiihashi, S., & Okawara, M. (1985). Selective oxidation of
Acknowledgments
+
−
alcohols by oxoaminium salts (R2N = O X ). Journal of Organic Chemistry, 50(8),
332–1334. http://dx.doi.org/10.1021/jo00208a047
1
Ozinskas, A. J., & Bobst, A. M. (1980). Formation of N-hydroxy-amines of spin labeled
This study was supported by the Russian Foundation for Basic
Research (“My first grant” number 14-03-31131).
1
nucleosides for H-NMR analysis. Helvetica Chimica Acta, 63(6), 1407–1411.
http://dx.doi.org/10.1002/hlca.19800630609
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1
13
H- and
C-N.M.R spectra of d-glucopyranose, 2-acetamido-2-deoxy-d-
Appendix A. Supplementary data
glucopyranose, and related compounds in aqueous media. Carbohydrate
Research, 59(1), 19–34. http://dx.doi.org/10.1016/S0008-6215(00)83289-9
Ponedel’kina, I. Y., Araslanova, D. I., Tyumkina, T. V., Lukina, E. S.,
& Odi-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carbpol.2015.04.
nokov, V. N. (2014). Partially oxidized potato starches from bromide-free
TEMPO-mediated reaction: Characterization of monosaccharide composition.
Starch/Stärke, 66(5–6), 444–449. http://dx.doi.org/10.1002/star.201300130
Ponedel’kina, I. Y., Khaibrakhmanova, E. A., & Odinokov, V. N. (2010). Nitroxide-
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