Antioxidative properties of nitroxyl radicals and hydroxyamines in reactions with triplet and deaminated kynurenine

Article abstract of DOI:10.1007/s11172-010-0046-y

The reactions of triplet kynurenine and 4-(2-aminophenyl)-4-oxocrotonic acid, formed upon the thermal decomposition of kynurenine, with nitroxyl radicals and cyclic N-hydroxylamines were studied. Nitroxyl radicals were found to quench efficiently the trip

Full text of DOI:10.1007/s11172-010-0046-y

Russian Chemical Bulletin, International Edition, Vol. 59, No. 1, pp. 66—74, January, 2010
66
Antioxidative properties of nitroxyl radicals and hydroxyamines
in reactions with triplet and deaminated kynurenine
V. V. Yan´shole,a I. A. Kirilyuk,b I. A. Grigor´ev,b,c S. V. Morozov,b and Yu. P. Tsentalovicha
aInstitute "International Tomography Center," Siberian Branch of the Russian Academy of Sciences,
3a ul. Institutskaya, 630090 Novosibirsk, Russian Federation.
Fax: +7 (383) 333 1399. Eꢀmail: yura@tomo.nsc.ru
bN. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences,
9 prosp. Akad. Lavrent´eva, 630090 Novosibirsk, Russian Federation.
Fax: +7 (383) 330 9752
cNovosibirsk State University,
2 ul. Pirogova, 630090 Novosibirsk, Russian Federation.
Fax: +7 (383) 339 7101
The reactions of triplet kynurenine and 4ꢀ(2ꢀaminophenyl)ꢀ4ꢀoxocrotonic acid, formed
upon the thermal decomposition of kynurenine, with nitroxyl radicals and cyclic Nꢀhydrꢀ
oxylamines were studied. Nitroxyl radicals were found to quench efficiently the triplet state
of kynurenine (rate constant 3—6•108 L mol–1 s–1). The quenching proceeds via the spinꢀ
exchange mechanism and affords no new products. Neither nitroxyl radicals, nor hydroxyꢀ
lamines react with 4ꢀ(2ꢀaminophenyl)ꢀ4ꢀoxocrotonic acid under conditions similar to phyꢀ
siological.
Key words: kynurenine, 4ꢀ(2ꢀaminophenyl)ꢀ4ꢀoxocrotonic acid, triplet excited state,
quenching reactions, laser flash photolysis, nitroxyl radicals, hydroxylamines.
Cataract is progressing lenticular opacity resulting in
At the same time, it is assumed that the covalent addiꢀ
tion of these substances to lens proteins can modify
the proteins, namely, their coloration, crossꢀlinking,
a decrease in the solubility, and as a consequence, the
development of cataract.11—21 The lens proteins can be
modified by UV filters due to both thermal and photoꢀ
chemical reactions. We have earlier found22—24 that in
photochemical reactions the key intermediate is kynureꢀ
nine in the triplet state (T1) capable of reacting with amiꢀ
no acid residues of proteins. It was shown23,25 that kynureꢀ
nine and its derivatives are thermally unstable under physꢀ
iological conditions and undergo spontaneous deaminaꢀ
tion to form highly reactive compounds: 4ꢀ(2ꢀaminopheꢀ
nyl)ꢀ4ꢀoxocrotonic acid (2), being carboxyketoalkene, and
its derivatives. Carboxyketoalkenes can add to nucleophilic
amino acid residues of proteins (cysteine, histidine, and
lysine)15,16,26—28 distorting functionality of the proteins
and enhancing their susceptibility to UV irradiation.
In the normal state the lens contains various substancꢀ
es that protect it from oxidation processes, including gluꢀ
tathione, glutathione peroxidase, NADPH, catalase,
ascorbic acid, and superoxide dismutase. It has previously
been found that the lens proteins are protected from phoꢀ
toexcited UV filters by ascorbate capable of quenching
triplet molecules of kynurenine and its derivatives,24 while
worsening and, finally, in complete loss of vision. This
disease develops in more than 50% population of the Earth
who reached 65 years.1 At present cataract can be cured
only by surgery: the natural lens, which underwent opaciꢀ
ty, is removed and an intraocular lens is implanted. Eviꢀ
dently, the properties of the implant are far from natural
and, hence, vision cannot be reduced to a full extent. There
is no drug capable of restoring lens transparency. Thus,
struggling against this disease the main attention should
be given to prophylactics remedies.
It is known that the main reason for the development
of the ageꢀrelated cataract is an oxidative stress in the lens
nucleus leading to numerous postꢀtranslational modificaꢀ
tions of lens proteins. However, the mechanism of these
modifications remained unknown until recently. The works
have appeared indicating that a series of lowꢀmolecularꢀ
weight compounds that act in the lens as ultraviolet filters
play an important role in these processes.2—10 These comꢀ
pounds, viz., Lꢀkynurenine (1) and its derivatives, are metꢀ
abolic products of tryptophan; they absorb the major part
of the UV radiation in the range 300—400 nm penetrating
through the cornea, thus protecting the retina and the lens
itself from photoinduced damages and improving visual
ability due to reducing chromatic aberrations.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 67—75, January, 2010.
1066ꢀ5285/10/5901ꢀ0066 © 2010 Springer Science+Business Media, Inc.

Antioxidative properties of nitroxyl radicals
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
67
the protection from carboxyketoalkenes is performed by
reduced glutathione, which can reduce the double bond in
the side chain of acid 2 to form the adducts.28—30 The
concentration of these substances in human organism deꢀ
creases with aging, which decreases the efficiency of the
protective system. Therefore, the creation of new efficient
drugs corresponding to modern requirements and capable
of protecting the lens tissues from photochemical and therꢀ
mal reactions involving UV filters is a topical problem.
Exogenous antioxidants based on nitroxyl radicals ofꢀ
ten protect cells and living tissues from oxidation processꢀ
es induced by the UV irradiation more efficiently that
natural antioxidants.31 The nitroxyl radical 2,2,6,6ꢀtetꢀ
ramethylꢀ4ꢀhydroxypiperidineꢀ1ꢀoxyl (TEMPOL) has
earlier been used successfully for the protection of epitheꢀ
lial cells of the lens.32 It was also shown that the applicaꢀ
tion of the reduced form of this radical, viz., cyclic
Nꢀhydroxylamine (TEMPOLꢀN), results in cataractogeꢀ
nesis inhibition in vivo.33 The purpose of this work is to
evaluate the ability of nitroxyl radicals 2—16 and hydroxylꢀ
amines 17—21 of different structure to inhibit the key
stages of thermal and photoinduced transformations of
kynurenine resulting in the modification of lens proteins.
In particular, the ability of nitroxyl radicals (RR´NO•)
and their reduced forms, viz., hydroxylamines (RR´NOH),
to quench the triplet state of kynurenine 1 and reduce the
double bond in the side chain of carboxyketoalkene 2 has
been studied.
Experimental
D,LꢀKynurenine (Fluka, Germany) and organic solvents for
highꢀperformance liquid chromatography (Kriokhrom, Russia)
were used without additional purification. Doubleꢀdistilled waꢀ
ter and sodium phosphate (reagent grade) were used for the
preparation of buffer solutions. Nitroxyl radicals 3—6 (see Ref. 34),
7, 8 (see Ref. 35), and 9—13 (see Refs 36—40) were synthesized
by procedures described in the indicated works.
2,2,6,6ꢀTetramethylꢀ4ꢀtrimethylammoniopiperidineꢀ1ꢀoxyl
methylsulfate (14). Dimethyl sulfate (2.8 mL, 30 mol) and
Na2CO3 (5 g, 0.047 mmol) were added to a solution of 4ꢀaminoꢀ
2,2,6,6ꢀtetramethylpiperidineꢀ1ꢀoxyl (4ꢀaminoꢀTEMPO, ACROS
Organics) (1 g, 5.8 mmol) in acetone (20 mL). The suspension

68
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Yan´shole et al.
was vigorously stirred for 48 h. The precipitate was filtered off
and washed with acetone, and the resulting mixture of salts and
compound 14 was refluxed with MeOH, periodically separating
the colored solution by decantation until the extraction of
the radical ceased. The joined solutions were evaporated to dryꢀ
ness under reduced pressure. The residue was refluxed with
a MeOH—CHCl3 (1 : 3) mixture, and the obtained solution was
filtered and again evaporated to dryness under reduced pressure.
The residue was recrystallized from ethanol. The yield was 1.3 g
(70%), m.p. 244—249 °C. Found (%): C, 47.67; H, 8.80; N, 8.29;
S, 10.01. C13H29N2O5S. Calculated (%): C, 47.98; H, 8.98;
N, 8.61; S, 9.85. IR (KBr), ν/cm–1: 3047, 2980, 2945, 1502,
1487, 1469, 1228, 1183, 1061, 1016, 900, 747, 611, 579, 553.
3R(S),4S(R)ꢀ3ꢀCarbamoylꢀ4ꢀmethoxycarbonylꢀ2,2,5,5ꢀtetꢀ
ramethylpyrrolidineꢀ1ꢀoxyl (15) was isolated by chromatography
on silica gel (eluent CHCl3) as a byꢀproduct formed upon the
treatment of dicarboxylic acid 13 containing the products of
partial hydrolysis of transꢀ3,4ꢀdicyanoꢀ2,2,5,5ꢀtetramethylpyrꢀ
rolidineꢀ1ꢀoxyl40 with diazomethane in diethyl ether. M.p.
154—156 °C (from a hexane—ethyl acetate (1 : 1) mixture).
Found (%): C, 54.40; H, 7.87; N, 11.63. C11H19N2O4. Calcuꢀ
(СН, ОMe); 1732 (С=О); 1641 (С=N); 1440, 1376, 1319, 1281,
1198, 1179, 1151, 1084, 1055, 985, 883, 807.
Stage 2. 2,2ꢀBis(2ꢀmethoxycarbonylethyl)ꢀ5,5ꢀdiethylꢀ3,4ꢀ
dimethylimidazolidineꢀ1ꢀoxyl (24). Dimethyl sulfate (0.2 mL,
2.1 mmol) was added to a solution of 2,2ꢀbis(2ꢀmethoxycarbonylꢀ
ethyl)ꢀ5,5ꢀdiethylꢀ4ꢀmethylꢀ2,5ꢀdihydroimidazoleꢀ1ꢀoxyl (23)
(0.33 g, 1 mmol) in anhydrous diethyl ether (5 mL). Diethyl
ether was distilled off under reduced pressure, and the residue
was stirred for 1 h at 40—50 °C under reduced pressure and then
triturated with anhydrous diethyl ether. The residue that formed
was washed with diethyl ether and dissolved in EtOH, and
NaBH4 (40 mg, 1.06 mmol) was added by portions to the obꢀ
tained solution with stirring for 30 min. The reaction course was
monitored by TLC (Sorbfil, nꢀC6H14—CHCl3, 2 : 1). After the
reaction ceased, EtOH was distilled off under reduced pressure,
and the residue was diluted with water (10 mL) and extracted
with diethyl ether. The extract was dried with Na2SO4, and the
solvent was distilled off under reduced pressure. The residue was
chromatographed on a column packed with silica gel using an
nꢀC6H14—CHCl3 (2 : 1) mixture as eluent. The yield was 0.25 g
(75%), an orange oil. Found (%): С, 59.38; Н, 9.31; N, 7.90.
С17Н31N2O5. Calculated (%): C, 59.45; H, 9.10; N, 8.16. IR
(in thin layer), ν/cm–1: 2973, 2882, 2800 (СН, NСН3); 1736 (C=O);
1437, 1375, 1308, 1256, 1168, 1127, 987, 923, 888, 841.
lated (%): C, 54.31; H, 7.87; N, 11.51. IR (KBr), ν/cm–1
:
3396, 3319, 3215, 2984, 2941, 1735, 1673, 1630, 1462, 1439,
1416, 1369, 1295, 1265, 1245, 1207, 1181, 1020, 998, 967,
784, 740, 637.
Stage 3. 2,2ꢀBis(2ꢀcarboxyethyl)ꢀ5,5ꢀdiethylꢀ3,4ꢀdimethylꢀ
imidazolidineꢀ1ꢀoxyl disodium salt (16). A. A solution of NaOH
(80 mg, 2 mmol) in water (1 mL) was added to a solution of
2,2ꢀbis(2ꢀmethoxycarbonylethyl)ꢀ5,5ꢀdiethylꢀ3,4ꢀdimethylimidꢀ
azolidineꢀ1ꢀoxyl (24) (165 mg, 0.5 mmol) in EtOH (3 mL). The
reaction mixture was left to stay for 14 h at ~20 °C, then NaHCO3
(200 mg) was added, and the suspension was stirred for 4 h. The
residue was filtered off, the solution was evaporated to dryness
under reduced pressure, the residue was dissolved in isopropyl
alcohol and filtered, and nitroxyl radical 16 was precipitated
with diethyl ether. The yield was 110 mg (60%). Found (%):
С, 44.49; Н, 7.59; N, 6.77; Na, 11.38. С15Н25N2Na2O5•2.5H2O.
Calculated (%): C, 44.55; H, 7.48; N, 6.93; Na, 11.37. IR (KBr),
ν/cm–1: 2965, 2925, 2855, 2800 (СН, NMe); 1558 (br, C=O);
1423, 1357, 1337, 1277, 1242, 1233, 1193, 1086, 932, 872, 872.
B. The aqueousꢀalcohol solution of radical 16 obtained after
hydrolysis was mixed with NaHCO3 to remove alkali excess, the
hydrocarbonate precipitate was filtered off, the solution was
evaporated under reduced pressure, and the fineꢀcrystalline residue
that formed was triturated with an isopropyl alcohol—diethyl ether
(1 : 2) mixture. Yellow crystals were obtained. Found (%): С, 38.44;
Н, 5.87; N, 5.44; Na, 17.81. С15Н25N2Na2O5•2H2O•Na2CO3.
Calculated (%): C, 38.33; H, 5.83; N, 5.59; Na, 18.34. These
samples were used in the present study.
Hydroxylamines 17—21 (general procedure). Compounds
17—21 were synthesized according to an earlier described proꢀ
cedure43 by the hydrogenation of the corresponding nitroxyl radꢀ
icals with hydrogen under atmospheric pressure on the Pd/C
catalyst. A solution or a suspension of the nitroxyl radical
(10 mmol) in MeOH (50 mL) was placed in a conic flask equipped
with an attachment for gas supply with a valve at the outlet, and
the wet catalyst (Pd/C, 1%, 200 mg) was added. The flask was
purged with nitrogen and then with hydrogen and connected to
a gas meter. After the end of gas absorption (usually the amount
of the gas somewhat exceeded the calculated value), the hydroꢀ
genation was stopped. The hydroxylamines formed are active
reducing agents and can easily be oxidized in air to the starting
2,2ꢀBis(2ꢀcarboxyethyl)ꢀ5,5ꢀdiethylꢀ3,4ꢀdimethylimidazoliꢀ
dineꢀ1ꢀoxyl disodium salt (16) was synthesized in three stages
from 3ꢀhydroxyaminoꢀ3ꢀethylpentanꢀ2ꢀone hydrochloride (22),
which was prepared by an earlier described41 procedure.
Stage 1. 2,2ꢀBis(2ꢀmethoxycarbonylethyl)ꢀ5,5ꢀdiethylꢀ4ꢀmeꢀ
thylꢀ2,5ꢀdihydroimidazolꢀ1ꢀoxyl (23). 4ꢀKetoheptadioic (4ꢀoxoꢀ
heptanedicarboxylic) acid (4 g, 23 mmol) was dissolved in 25%
aqueous ammonia (30 mL), and the resulting solution was evaꢀ
porated to dryness under reduced pressure. Compound 22 (2 g,
11 mmol), ammonium acetate (4.5 g, 58 mmol), and MeOH
(20 mL) were added to the solid crystalline substance that formed.
The suspension was purged with argon for 30 min to remove
oxygen, argon was passed through the reaction vessel, which was
sealed, and the suspension was magnetically stirred for 6 h at
40—50 °C. The reaction mixture was stored for 60 days at this
temperature, 25% aqueous ammonia (20 mL) was added, and
the solution formed was purged with air for 72 h. The resulting
orange solution was evaporated under reduced pressure, CHCl3
(20 mL) and MeOH (20 mL) were added, the colorless precipiꢀ
tate formed was filtered off, and the solution was evaporated
under reduced pressure. A 10% solution of sodium carbonate
(20 mL) was added to the residue, which was extracted with
CHCl3. Diethyl ether (20 mL) was added to the remained aqueꢀ
ous solution, and the solution was carefully acidified with 10%
HCl to pH 2—3, which resulted in the orange coloration of the
organic layer. The ethereal solution was separated, the aqueous
solution was extracted with ether, the joined ethereal extracts
were dried with Na2SO4, and an excess of an ethereal solution of
diazomethane, prepared according to an earlier described proꢀ
cedure,42 until nitrogen evolution stopped. After 12 h, the reꢀ
sulting solution was evaporated under reduced pressure, and the
residue was chromatographed on a column with silica gel using
an nꢀC6H14—CHCl3 (2 : 1) mixture as eluent. The yield was 0.33 g
(9%), m.p. 94—96 °C (nꢀC6H14—C6H6, 2 : 1). Found (%):
С, 58.74; Н, 8.07; N, 8.47. С16Н27N2O5. Calculated (%):
C, 58.70; H, 8.31; N, 8.56. IR (KBr), ν/cm–1: 2958, 2926, 2850

Antioxidative properties of nitroxyl radicals
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
69
nitroxyl radicals and, therefore, after hydrogenation the reacꢀ
tion mixtures were acidified with HCl to pH 1—3. The catalyst
was filtered off, the solvent was distilled off under reduced presꢀ
sure, and the residue was recrystallized. The salts obtained are
stable upon prolong storage in air.
4ꢀ(2ꢀAminophenyl)ꢀ4ꢀoxocrotonic acid (2) was synthesized
by an earlier described procedure.28 An aqueous solution of
kynurenine 1 (5 mmol L–1, 20 mL) was alkalized to pH 8.3 by
the addition of NaOH monitoring with an Orion Research
pH meter (USA). Argon was bubbled through the resulting soluꢀ
tion for 10 min, after which the solution in the sealed ampule
was incubated in a thermostat for 24 h at 70 °C.
1ꢀHydroxyꢀ2,2,5,5ꢀtetramethylpyrrolidineꢀ3ꢀcarboxylic acid
hydrochloride (17). This compound was incorrectly described
previously,44 and for the characteristics of the zwitterion, see
Ref. 45. The yield was 70%, colorless crystals, m.p. 190—192 °C
(decomp.) (from concentrated HCl). Found (%): С, 48.17;
Н, 8.12; N, 6.26; Cl, 15.78. C9H18NClO3. Calculated (%):
C, 48.32; H, 8.11; N, 6.26; Cl, 15.85. IR (KBr), ν/cm–1: 1724,
1500, 1466, 1423, 1406, 1391, 1371, 1295, 1201, 1173, 1142,
853, 763, 721. 1H NMR (200 MHz, D2O), δ: 1.46 (br.s, 9 H,
3 Me); 1.56 (s, 3 Н, Me); 2.26 (br.m, 2 Н, СН2); 3.31 (br.m,
1 Н, СН); (200 MHz, D2O + N2D4), δ: 1.18 (s, 3 Н, Me); 1.22
(s, 3 H, Me); 1.34 (s, 3 Н, Me); 1.42 (s, 3 Н, Me); 2.08 (m, 2 Н,
СН2); 2.83 (m, 1 Н, СН). 13C NMR (100 MHz, СD3OD + N2D4),
δ: 20.32, 27.46, 28.41, 28.85 (Me); 40.81 (CH2); 53.34 (CH);
62.74, 66.91 (C(2), C(5)); 181.01 (СOOH).
The products formed were separated by chromatography
on a ZORBAX Eclipse XBDꢀC18 semipreparative column
9.4×250 mm using an acetonitrile—aqueous solution of trifluoꢀ
roacetic acid (0.05 wt.%) gradient. The content of acetonitrile in
a gradient solution was 0—30% (0—2 min), 30—55% (2—32 min),
55—100% (32—34 min), and 100% (34—40 min). The flow rate
was 0.9 mL min–1, and the detection was performed at five waveꢀ
lengths: λ = 254, 290, 315, 360, and 410 nm. The needed fracꢀ
tions were collected as the products were eluted. The isolated
solution of acid 2 was neutralized to pH 7.2 and stored at –18 °С.
Laser flash photolysis (LFP). Rate constants of quenching of
the kynurenine triplet state were measured by laser flash photolꢀ
ysis. The setup was described in detail in previous works.47,48
A solution was placed in a rectangular quartz cell and irradiated
with a QuantaꢀRay LABꢀ130ꢀ10 Nd:YAG laser (USA) at 355 nm
(pulse energy up to 150 mJ, pulse duration 8 ns). The detecting
system consisted of a DKsShꢀ150 arc xenon lamp operating in
the pulse mode with a pulse duration of 2 ms, a monochromator,
an Electron Tubes Ltd 9794B photomultiplier (Great Britain),
a digital twoꢀchannel oscillograph with a LeCroy 9310A 11ꢀbit
analogꢀtoꢀdigital converter (Switzerland) (400 MHz, time resoꢀ
lution 10 ns), and a system of filters and shutters. Since the
detecting light concentrated in a rectangular of 3×1 mm passes
through the cell along the front edge of laser radiation, in all
experiments the optical excitation and detection path lengths
were 1 mm and 0.8 cm, respectively; optical absorbance values
of the intermediates are given for l = 0.8 cm. Argon was bubbled
through the solution for 10 min before measurements and furꢀ
ther during the whole experiment to remove oxygen from the
system. The energy of the incident laser radiation was deterꢀ
mined by a Coherent FM power meter (USA).
1ꢀHydroxyꢀ3ꢀcarbomoylꢀ2,2,5,5ꢀtetramethylpyrrolidine hydroꢀ
chloride (18). The free base43 and hydrobromide46 were described
earlier. The yield was 70%, m.p. 216—220 °C (from isopropyl
alcohol). Found (%): С, 48.43; Н, 8.66; N, 12.37; Cl, 15.99.
C9H19N2ClO2. Calculated (%) C, 48.54; H, 8.60; N, 12.58;
Cl, 15.92. IR (KBr), ν/cm–1: 3308 br, 3138 br, 3018 br, 2813 br,
1659, 1622, 1500, 1482, 1435, 1387, 1372, 1307, 1158, 1054,
1
797, 718. H NMR (400 MHz, СD3OD—D2O (10 : 1)), δ: 1.44
(br.s, 3 Н, Me); 1.53 (s, 3 H, Me); 1.58 (s, 3 Н, Me); 1.60 (s, 3 Н,
Me); 2.36 (br.m, 2 Н, СН2); 3.19 (br.m, 1 Н, СН).
3ꢀAminomethylꢀ1ꢀhydroxyꢀ2,2,5,5ꢀtetramethylpyrrolidine
dihydrohloride (19). The yield was 70%, m.p. 198—203 °C (from
isopropyl alcohol). Found (%): С, 43.86; Н, 9.16; N, 11.11;
Cl, 28.42. C9H22N2Cl2O. Calculated (%): C, 44.09; H, 9.04;
N, 11.43; Cl, 28.92. IR (KBr) ν/cm–1: 3424 br, 2902 br, 2580 br,
2043 br, 1606, 1517, 1464, 1395, 1176, 1118, 979, 752. 1H NMR
(300 MHz; СD3OD—D2O (10 : 1)), δ: 1.32 (br.s, 3 Н, Me); 1.51
(br.s, 3 H, Me); 1.58 (br.s, 6 Н, 2 Me); 2.12 (br.m, 1 Н, Н(4));
2.35 (br.m, 1 Н, Н(4)); 2.53 (br.m, 1 Н, С(3)Н); 3.00 (1 Н,
ABd system, СН2N, JAB = 12.8 Hz, Jd = 10.3 Hz); 3.26 (1 Н,
ABd system, СН2N, JAB = 12.8 Hz, Jd = 3.3 Hz).
Absorption spectra of solutions were recorded on a Hewlett
Packard Agilent 8453 spectrophotometer (Germany).
Reactions of 2ꢀaminophenylꢀ4ꢀoxocrotonic acid (2) with nitrꢀ
oxyl radicals and hydroxylamines (general procedure). Solutions
of carboxyketoalkene 2 (3—4 mmol L–1) and compounds 3—21
(1—2•10–2 mol L–1) were prepared in a 0.1 М phosphate buffer
(pH 7.2). The prepared solutions were placed in 0.5ꢀmL ampules
and purged with argon for 10 min. The ampules were sealed and inꢀ
cubated at 37 °С. An individual ampule was used for each expeꢀ
rimental point. All ampules were simultaneously placed in a therꢀ
mostat and taken from it after different time intervals. The samples
taken from the thermostat were stored at –18 °C for several days.
Concentrations of acid 2 in incubated solutions were meaꢀ
sured by reverseꢀphase HPLC on an Agilent LC 1200 chromatoꢀ
graph equipped with a gradient pump and a multiwave optical
detector. Substances were separated on a ZORBAX Eclipse
XBDꢀC18 analytical column 4.6×150 mm using an acetoniꢀ
trile—aqueous solution of trifluoroacetic acid (0.05 wt.%) gradiꢀ
ent. The acetonitrile content in the gradient solution was 0%
(0—5 min), 0—50% (5—25 min), 50—100% (25—30 min),
100—0% (30—30.01 min), and 0% (30.01—35 min). The flow
rate was 0.2 mL min–1, and detection was carried out simultaꢀ
neously at five wavelengths: λ = 254, 290, 315, 360, and 410 nm.
1ꢀHydroxyꢀ2,2,5,5ꢀtetramethylpyrrolidineꢀ3R(S),4S(R)ꢀdiꢀ
carboxylic acid hydrochloride (20). The yield 70%, m.p. 260 °C
(decomp.) (from concentrated HCl). Found (%): С, 44.65;
Н, 6.84; N, 5.38; Cl, 13.12. C10H18NСlO5. Calculated (%):
C, 44.87; H, 6.78; N, 5.23; Cl, 13.24. IR (KBr), ν/cm–1: 3059 br,
2942 br, 1721, 1498, 1458, 1426, 1404, 1385, 1274, 1195, 1166,
1
1133, 1053, 857, 785, 723. H NMR (400 MHz, (CD3)2SO), δ:
1.21 (s, 6 H, 2 Me); 1.46 (s, 6 H, 2 Me); 3.19 (s, 2 H, 2 CH).
13C NMR (100 MHz, (CD3)2SO), δ: 20.30, 24.93 (Me); 51.35
(CH); 70.45 (CMe2); 170.51 (COOH).
1ꢀHydroxyꢀ2,2,6,6ꢀtetramethylꢀ4ꢀtrimethylammoniopiperiꢀ
dine hydrosulfate hydrochloride (21). The yield was 15%, m.p.
220—228 °C (from isopropyl alcohol). Found (%): С, 41.06;
Н, 8.79; N, 8.11; Cl 9.70; S, 9.32. C12H29N2ClO5S. Calculatꢀ
ed (%): C, 41.31; H, 8.38; N, 8.03; Cl, 10.16; S, 9.19. IR (KBr)
ν/cm–1: 1483, 1419, 1532, 1288, 1260, 1226, 1176, 1147, 1037,
958, 907, 874, 743. 1H NMR (300 MHz, СD3OD), δ: 1.56 (s, 6 Н,
2 Me); 1.59 (s, 6 H, 2 Me); 2.25 (2 H, ABd system, JAB = 14 Hz,
Jd = 12 Hz); 2.64 (br.s, 2 H, AB system, 2 CH2, J = 14 Hz); 3.94
(m, 1 Н, СН).

70
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Yan´shole et al.
The Agilent ChemStation program for Windows was used
ΔD
for recording chromatograms and integrating peak areas. The
change in the concentration of acid 2 was determined by a change
in the peak area at λ = 410 nm (absorption maximum in the
spectrum of compound 2).28
Results and Discussion
3
0.01
1
2
Quenching of triplet kynurenine. The photolysis of
kynurenine (1) in an aqueous solution results in the forꢀ
mation of the triplet state22 in a ∼2% quantum yield
0
,
where S1* is the excited singlet state, T1 is the triplet state,
and ISC stands for the intersystem crossing. Triplet
kynurenine formed upon intense laser irradiation can abꢀ
sorb the second photon and undergo photoionization to
form the radical cation and solvated electron.49 In turn,
the solvated electron can add to molecule 1 in the ground
state49,50 yielding an electron adduct. To minimize twoꢀ
quantum photoionization effects, all photochemical exꢀ
periments were carried out using minimum acceptable enꢀ
ergies of laser radiation (∼60 mJ pulse–1). In addition,
acetone (10 mmol L–1) was added to the solutions, beꢀ
cause its molecules are efficient traps for solvated elecꢀ
trons. Solvated electrons formed upon the photolysis of
compound 1 disappear in the reaction with acetone51
(ke = 6•109 L mol–1 s–1) with the formation of the elecꢀ
tron adduct Me2CO•–, which is rapidly protonated in
a neutral medium. The protonated radical of acetone is
a weak chromophore and does not contribute to the obꢀ
served transient spectra.
The transient absorption spectrum observed 280 ns afꢀ
ter the laser flash photolysis of an aqueous solution of
kynurenine 1 (1.6•10–4 mol L–1, buffer solution, pH 7.2)
is shown in Fig. 1. Similar spectra were obtained upon the
irradiation of solutions of compound 1 in the presence of
the nitroxyl radicals and their reduced forms. The negative
absorption with a maximum at λ = 360 nm corresponds to
the pulse decrease in the concentration of kynurenine in
the ground state, and the positive absorption is attributed
to triplet kynurenine, whose optical spectrum22,49 has maxꢀ
ima at λ = 280, 340, and 430 nm.
300
400
500
λ/nm
Fig. 1. Transient absorption spectra during the photolysis of an
aqueous solution of compound 1 (1.6•10–4 mol L–1) in the presꢀ
ence of radical 14 (1.3 mmol L–1): 1, 280 ns after the laser pulse;
2, 8 μs after the laser pulse. Solid line 3 is the absorption specꢀ
trum of the initial compound 1.
quenching rate constant, [T1] is the concentration of tripꢀ
let kynurenine, and [RR´NO•] is the radical concentraꢀ
tion. The values of the observed rate constant kobs
= k0 + kq[RR´NO•] obtained in the experiment deꢀ
pend linearly on the concentration of nitroxyl radicals
[RR´NO•] in the range from 10–3 to 10–2 mol L–1. The
=
kobs•106/s–1
1.5
ΔD
1.0
0.03
0.5
0
1
2
3
4
[RR´NO•]//mmol L–1
1
0.02
0.01
0
The decay kinetics of triplet kynurenine T1 was detectꢀ
ed in its absorption maximum at λ = 430 nm. The decay of
the signal of the triplet kynurenine in the absence of addiꢀ
tives and in the presence of nitroxyl radical 14 in different
concentrations is shown in Fig. 2. In the presence of radiꢀ
cal 14, the signal decay is well described by the exponenꢀ
tial dependence. Thus, the decay kinetics of the kynureꢀ
nine triplet state T1 can be described by the equation
2
3
4
0
2
4
6
Time/μs
Fig. 2. Kinetic curves of the decay of the signal of the triplet state
T1 in the presence of radical 14. [14]/mmol L–1 = 0 (1), 0.53 (2),
1.6 (3), and 4.8 (4). Solid lines are calculated curves. Inset: deꢀ
pendences of the observed rate constant of T1 decay on the nitrꢀ
oxyl radical concentration.
[T1] = [T1]0•exp{–(k0 + kq[RR´NO•])},
where k0 is the rate constant for the decay of the kynureꢀ
nine triplet state in the absence of quenchers, kq is the

Antioxidative properties of nitroxyl radicals
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
71
logkq
21.4
values of the quenching rate constants kq for different radꢀ
icals determined from the slope of the dependence of the
observed rate constant of triplet kynurenine decay on the
concentration of the added radical are listed in Table 1.
The temperature dependence of the quenching rate
constant of triplet kynurenine was measured for radical 3
(Fig. 3) and was used to determine the Arrhenius parameꢀ
ters for this constant: preꢀexponential factor logA =
21.2
21.0
= 12.0 0.7 and activation energy Еа = 18 3 kJ mol–1
The obtained Еа value is very close to the activation energy
for water viscosity (19 kJ mol–1). This indicates that the
quenching reaction is diffusionally controlled.
.
20.8
20.6
20.4
20.2
The transient absorption spectrum detected 8 μs after
the laser irradiation of a solution of compound 1
(1.6•10–4 mol L–1) in the presence of radical 14
(3.1 mmol L–1) is also shown in Fig. 1. It is seen that the
quenching of triplet kynurenine T1 results in the restoraꢀ
tion of the starting compound in the ground state, but no
new radicals or diamagnetic reaction products are formed.
An insignificant residual absorption observed at λ < 300 nm
increases with the laser energy increase quadratically and,
hence, can be assigned to the kynurenine radical cation
formed due to twoꢀquantum photoionization.49
0.0028
0.0030
0.0032
1/T
Fig. 3. Temperature dependence of the quenching rate conꢀ
stant of the triplet state T1 by nitroxyl radical 3 (circles).
Solid line is the calculation with the Arrhenius parameters
A = 9.2•1011 L mol–1 s–1 and Еа = 18.0 kJ mol–1
.
The channels of triplet kynurenine decay in the
absence of quenchers are mainly conversion to the
ground state and tripletꢀtriplet annihilation occurring with
the rate constant22,24 kTꢀT = 4.1•109 L mol–1 s–1. Under
physiological conditions (i.e., inside the lens), amino acid
residues of proteins, antioxidants, and oxygen act as naꢀ
tural quenchers of triplet kynurenine. We have earliꢀ
er shown24 that among amino acids the most efficient
Oxygen also quenches triplet kynurenine with a high rate
(kq = (2.1 0.5)•109 L mol–1 s–1). However, since the free
oxygen concentration in the eye lens is very low,52 this
reaction does not play a noticeable role in the evolution of
UV filters. Triplet kynurenine is quenched by amino acids
and antioxidants via the electron transfer mechanism to
form the kynurenine radical anion and the radical cation
of amino acid or antioxidant.24
The question about the mechanism of quenching of
singlet and triplet states of organic molecules (M*) by
radicals was repeatedly discussed in literature. In the genꢀ
eral case,53 three mechanisms of quenching of photoexꢀ
cited molecules by nitroxyl radicals RR´NO• are possible:
(1) energy transfer54,55 with the formation of the excited
state of a doublet RR´NO•*, (2) charge transfer,56,57 and
(3) electron spin exchange.54—58 The first mechanism asꢀ
sumes that the nitroxyl radical has an absorption band
with a lower energy than the energy of the excited state of
molecule M*. The quenching rate constant should depend
on both the energy of the excited state of molecule M* and
the position of the absorption band of the nitroxyl radical,
and the excited radical RR´NO•* should be the reaction
product. In the case of charge transfer, the quenching
reaction affords the radical cation (radical anion) of the
initial molecule. Finally, the quenching via the mechaꢀ
nism of electron spin exchange does not result in the apꢀ
pearance of new products.
quenchers of triplet kynurenine are tryptophan (kq
= (2.6 0.7)•108 L mol–1 s–1) and tyrosine (kq
=
=
= (6.7 1.5)•107 L mol–1 s–1), whereas among antioxiꢀ
dants this is ascorbate (kq = (8.5 1.2)•108 L mol–1 s–1).
Table 1. Quenching rate constants of triplet kynurenine by
nitroxyl radicals
Radical
Concentration of radical,
kq•108
/L mol–1 s–1
c•10–3/mol L–1
3
4
5
6
7
8
9
10
11
12
13
14
15
16
1.8—10.8
0.9—8.2
1.4—8.5
1.2—10.8
1.5—9.3
1.1—6.8
1.8—11.0
1.6—11.0
1.0—6.7
1.1—9.8
0.5—4.8
0.5—4.5
1.1—7.8
0.7—1.7
4.3 1.1
4.0 1.0
4.8 1.3
5.3 1.3
4.5 1.3
3.9 1.2
4.4 1.2
4.5 1.4
3.7 0.9
3.7 1.1
5.7 1.4
3.5 1.0
4.2 1.0
3.2 0.8
Figure 1 shows that no new paramagnetic or diamagꢀ
netic products are formed in solution upon the quenching
of triplet kynurenine by nitroxyl radicals (the spectrum

72
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Yan´shole et al.
[2]/mmol L–1
observed 8 μs after the laser pulse). Note that the molar
absorption coefficients of the kynurenine radicals are
close to those of the triplet and the lifetimes of these radiꢀ
cals are tens of microseconds.49 If the quenching proꢀ
ceeded via the charge transfer mechanism, noticeable
absorption signals of these radicals would be observed.
Therefore, we concluded that triplet kynurenine is quenꢀ
ched by nitroxyl radicals via the electron spin exchange
mechanism
1
2
4
3
2
T
1(↑↑) + RR´NO•(↓)
1(↑↓) + RR´NO•(↑).
Thus, nitroxyl radicals can protect the lens tissues from
reactions with photoexcited UV filters even better than
natural antioxidant ascorbate: the quenching of triplet
kynurenine by ascorbate produces free radicals that are
dangerous for biological tissues. It was shown that the
products of ascorbate photooxidation results in the glycoꢀ
sylation of lens proteins.59 In the case of nitroxyl radicals,
the quenching proceeds via the physical mechanism withꢀ
out the formation of chemical reaction products.
0
50
100
150
Time/h
Fig. 4. Kinetic curves of the decay of carboxyketoalkene 2 durꢀ
ing incubation at 37 °C: 1, without additives; 2, in the presence
of hydroxylamine 17 (1.5•10–2 mol L–1).
For all nitroxyl radicals studied in this work, the rate
constants are within 3—6•108 L mol–1 s–1. The lowest
quenching rate constant is detected for radical 16
containing the bulky substituents in the region of the radiꢀ
cal center. Probably, this indicates that the value of
the quenching rate constant is primarily determined by
the diffusion coefficient and steric factors, whereas the
donorꢀacceptor properties exert no noticeable effect on
the quenching rate. This additionally confirms that the
quenching proceeds via the physical mechanism of elecꢀ
tron spin exchange.
We also studied the ability of hydroxylamines 17—21
to quench the triplet state of kynurenine T1. It turned out
that even at the highest concentrations of hydroxylamines
(up to 10–2 mol L–1) the decay kinetics of triplet kynureꢀ
nine remains unchanged. This means that, unlike the
nitroxyl radicals themselves, their reduced forms do not
react with the triplet state of kynurenine.
also indicate that the nitroxyl radicals and hydroxylamines
exert no effect of the decay rate of molecules 2.
The results on the incubation of carboxyketoalkene 2
in the presence of nitroxyl radicals and hydroxylamines
show that the rate constants of the reactions of acid 2 with
these compounds are low compared to those for the reacꢀ
tions of this acid with nucleophilic amino acids and antiꢀ
oxidants. The rate constants for the addition of carboxꢀ
yketoalkene 2 to lysine and histidine at 37 °C (physiologiꢀ
cal temperature) lie in a range of 1—2•10–4 L mol–1 s–1
,
whereas for thiols (glutathione and cysteine) these conꢀ
stants are 2.1 and 36 L mol–1 s–1, respectively.28 Acꢀ
cording to our measurements, the rate constants of the
reaction of acid 2 with the nitroxyl radicals and hydrꢀ
oxylamines do not exceed 10–4 L mol–1 s–1. Thus, these
compounds cannot serve as an efficient antioxidant proꢀ
tection of lens proteins from the addition of deaminated
kynurenines.
Incubation with carboxyketoalkene 2
Thus, we established that the nitroxyl radicals and their
reduced forms are inert toward reactive molecules of
carboxyalkenes 2 formed due to the deamination of naꢀ
tural UV filters. At the same time, the nitroxyl radicals are
efficient quenchers of triplet states of kynurenine 1, and
the quenching proceeds via the electron spin exchange
mechanism that affords no additional products. Due
to this property and the well known ability of nitrꢀ
oxides to bind free radicals, there are wide prospects to
use these compounds in the development of solarꢀprotecꢀ
tive drugs.
To elucidate the influence of nitroxyl radicals RR´NO•
and hydroxylamines RR´NOH on the reduction of the
side chain of carboxyketoalkene 2, we incubated aqueous
solutions of acid 2 at 37 °C (pH 7.2) in the presence of
nitroxyl radicals and their reduced forms and measured
the change in the concentration of compound 2 during
incubation by HPLC. The kinetics of thermal decomposiꢀ
tion of acid 2 in the absence of additives and in the presꢀ
ence of hydroxylamine 17 (1.5•10–2 mol L–1) is illustratꢀ
ed in Fig. 4. No formation of any additional products was
observed in the chromatograms obtained after incubation
with the addition of RR´NO• or RR´NOH to the soluꢀ
tion. The kinetic curves for decay of acid 2 shown in Fig. 4
This work was financially supported by the Federal
Agency on Science and Innovations (State Contract
No. 02.512.11.2278), the Russian Foundation for Basic

Antioxidative properties of nitroxyl radicals
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
73
25. L. M. Taylor, J. A. Aquilina, J. F. Jamie, R. J. W. Truscott,
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Research (Project Nos 08ꢀ03ꢀ00539, 08ꢀ03ꢀ00432, and
07ꢀ03ꢀ00253), the Division of Chemistry and Materials
Science of the Russian Academy of Sciences (Project
No. 5.1.1), the Council on Grants at the President of the
Russian Federation (State Support Program for Leading
Scientific Schools, Grant NShꢀ3604.2008.3), the Russian
Academy of Sciences (Program "Fundamental Sciences
to Medicine," Project No. 22), and the Government of
Moscow (Program "New Methods and Technologies of
Early Diagnostics and Treatment of Oncological and
Other Dangerous Diseases").
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Received February 13, 2009;
in revised form April 13, 2009