supplementary materials


hb6933 scheme

Acta Cryst. (2012). E68, o2810-o2811    [ doi:10.1107/S1600536812036380 ]

N-Acetyl-5-chloro-3-nitro-L-tyrosine ethyl ester

T. T. Mutahi, B. J. Edagwa, F. R. Fronczek and R. M. Uppu

Abstract top

The title compound, C13H15ClN2O6, was synthesized by hypochlorous acid-mediated chlorination of N-acetyl-3-nitro-L-tyrosine ethyl ester. The OH group forms an intramolecular O-H...O hydrogen bond to the nitro group and the N-H group forms an intermolecular N-H...O hydrogen bonds to an amide O atom, linking the molecules into chains along [100]. The crystal studied was a non-merohedral twin, with a 0.907 (4):0.093 (4) domain ratio.

Comment top

Peroxynitrite (PN), an oxidant formed during the down-regulation of nitric oxide (.NO) (Uppu & Pryor, 1999), is known to cause oxidation of both free- and protein-bound amino acids (AAs) (Alverez et al., 1999; Beckman 2009; Uppu et al., 1996). The reactivity of PN towards AAs in proteins can be accounted for by the side chains of constituent AAs in particular those present in cysteine, methionine, tyrosine, tryptophan, and histidine. Among the various AAs with reactive side chains, the oxidation of Tyr by PN results in the formation of a characteristic nitro product, 3-nitroTyr (3-NO2Tyr) (Beckman, 2009; Ceriello, 2002; Crow, 1999; Darwish et al., 2007) which is often used as a marker of PN formation in vivo. Hypochlorous acid (HOCl) is another oxidant that can also be formed at sites of inflammation, catalyzed by the enzyme myeloperoxidase. Like PN, HOCl is mostly reactive towards the side chains of cysteine, methionine, tyrosine, tryptophan, and histidine and cause posttranslational modifications of proteins resulting in chlorinated products. 3-Chloro-L-tyrosine is one the products that has been well characterized and used as a biomarker of HOCl formation in vivo (Crow, 1999; Pitt & Spickett, 2008; Winterbourn, 2002). Now, a question that follows naturally but never addressed in detail is what happens when HOCl and PN are produced in the same biological milieu and react with AA side chains in proteins. The significance of these combined oxidations on the issue of biomarker validation could be truly overwhelming given the report by Whiteman and Halliwell (1999) wherein it was shown that the 3-NO2Tyr was in fact lost to some unknown product(s) following oxidation with HOCl. Another important consequence could be that we need additional biomarkers and their validation.

Herein, we report the synthesis and characterization of the oxidation product of HOCl reaction with N-acetyl-3-nitro-L-tyrosine ethyl ester (NANTEE), a model for protein-bound 3-NO2Tyr. When HOCl was a limiting reagent (hypochlorite/HOCl < NANTEE), the major product was found to be N-acetyl-5-chloro-3-nitro-L-tyrosine ethyl ester (NACNTEE). This product was purified by reversed phase (RP) high-performance liquid chromatography (HPLC). Its identification was based on single-crystal X-ray crystallographic analysis (Fig. 1) and 1H-NMR assignments (Figs. 2–4).

The structure is shown in Fig. 1. The absolute configuration at the asymmetric center C8 is S, in agreement with the known configuration of the starting material. Molecular geometry is normal, except for the nitro group, which has slightly long C3—N1 distance, 1.473 (4) Å and asymmetric N—O distances, N1—O2 1.249 (3) and N1—O3 1.169 (3) Å. The shape of the N1 ellipsoid is somewhat peculiar, while ellipsoids for other atoms in the molecule appear normal. The two C—C—N angles at the nitro-substituted C atom C3 also differ by 3.5 (3)°. These features suggest the possibility of a slight disorder involving rotation of the phenyl group, such that the Cl atom nearly superimposes upon N1 a small fraction of the time. This would lead to a slightly misplaced refined N1 position and account for the observed irregularities.

The nitro group lies nearly in the phenyl plane, with O2—N1—C3—C2 torsion angle 2.6 (3)°, and it accepts an intramolecular hydrogen bond from the OH group, having O1···O2 distance 2.570 (3) Å. The tyrosine N-acetyl NH group donates an intermolecular hydrogen bond to O6 (at x + 1, y, z), forming chains in the [1 0 0] direction.

Related literature top

For background to peroxynitrite and its reactions with amino acids, see: Alvarez et al. (1999); Beckman (2009); Ceriello (2002); Crow (1999); Dahaoui et al. (1999); Darwish et al. 2007; Janik et al. (2007, 2008); Koszelak & van der Helm (1981); Pieret et al. (1972); Pitt & Spickett (2008); Soriano-García (1993); Stout et al. (2000); Uppu & Pryor (1999); Uppu et al. (1996); Whiteman & Halliwell (1999); Winterbourn (2002).

Experimental top

Chemicals and solvents used in the preparation and recrystallization of NACNTEE were obtained as follows: NANTEE, potassium phosphate monobasic, sodium phosphate dibasic, sodium hydroxide, sodium hypochlorite (chlorine content: ca. 5%), CD3OD from Sigma (St. Louis, MO); formic acid (88%) from Fishers chemicals (Fair Lawn, NJ); ammonium hydroxide (28–30%)from VWR (Goshen Parkway, PA); HPLC grade methanol from EMD Chemicals (Gibbstown, NJ). Water with resistance of 18 megaohms/cm or higher was used.

Oxidation of NANTEE was performed by reacting equimollar concentrations of NANTEE with hypochlorite/HOCl. Briefly, NANTEE (8.5 mg) was dissolved in 2.8 mL of 0.2 M phosphate buffer, pH 7.0 to make a 10 mM NANTEE solution. A solution of 56 µL of hypochlorite (stock solution) was added drop-wise to the 10 mM NANTEE solution while stirring. Aliquots (200 µL each) of the reaction mixture were analyzed by reversed phase HPLC using Supleco LC18 column (150 x 4.6 mm, particle size: 5µ) and an isocratic mobile phase consisting of 0.05M ammonium formate buffer solution (50%) and methanol (50%) at pH of 3.93 and a flow rate of 1 mL/min. The absorbance was set at 410 nm. The HPLC system used in this research was a Lab Alliance series II/III liquid chromatography equipped with Lab Alliance model 500 UV-Vis detector and Peak Simple 329 chromatography data system. The peaks corresponding to pure NANTEE and the product were collected and concentrated. The amorphous powder was recrystallized from methanol to give yellow needles of NACNTEE. For 1H-NMR spectrum, both NANTEE and NACNTEE were dissolved in CD3OD and analyzed on a Bruker AV-400-liquid spectrometer. The 1H-NMR data are reported in ppm downfield from TMS as an internal standard.

N-acetyl-3-nitro-L-tyrosine ethyl ester (Fig. 3): 1H-NMR (400 MHz, CD3OD): δ 1.23 (t, J = 7.1 Hz, 3H), 1.91 (s, 3H), 2.95 (dd, J = 14.0, 8.7 Hz, 1H), 3.14 (dd, J = 14.0, 5.8 Hz, 1H), 4.15 (q, J = 7.1 Hz, 2H), 4.63 (dd, J = 8.8, 5.8 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H), 7.47 (dd, J = 8.6, 2.2 Hz, 1H), 7.92 (d, J = 2.1 Hz, 1H), 8.52 (s, 1H).

N-acetyl-5-chloro-3-nitro-L-tyrosine ethyl ester (Fig. 4): 1H-NMR (400 MHz, CD3OD): δ 1.23 (t, J = 7.1 Hz, 3H), 1.92 (s, 3H), 2.92 (dd, J = 14.1, 8.7 Hz, 1H), 3.12 (dd, J = 14.1, 5.8 Hz, 1H), 4.16 (q, J = 7.0 Hz, 2H), 4.63 (dd, J = 8.6, 5.8 Hz, 1H), 7.60 (d, J = 2.1 Hz, 1H), 7.86 (d, J = 2.1 Hz, 1H), 8.43 (s, 1H) (Fig. 4). The chemical shifts derived from the proton NMR spectrum of the product are consistent with the structure of N-acetyl-5-chloro-3-nitro-L-tyrosine ethyl ester, specifically, the doublet at 7.08 ppm corresponding to the proton at the ortho position of the OH group on the aromatic ring in the starting material disappears in the product due to chloride substitution.

Refinement top

H atoms on C were placed in idealized positions, with C—H distances 0.95–1.00 Å. A torsional parameter was refined for each methyl group. N—H and hydroxy H atom positions were refined. Uiso for H were assigned as 1.2 times Ueq of the attached atoms (1.5 for methyl and OH).

Computing details top

Data collection: APEX2 (Bruker, 2006); cell refinement: SAINT (Bruker, 2006); data reduction: SAINT (Bruker, 2006); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: ORTEP-3 for Windows (Farrugia, 1997); software used to prepare material for publication: SHELXTL (Sheldrick, 2008).

Figures top
[Figure 1] Fig. 1. Ellipsoids at the 50% level, with H atoms having arbitrary radius.
[Figure 2] Fig. 2. Chlorination of N-acetyl-3-nitro-L-tyrosine ethyl ester by hypochlorite/hypochlorous acid
[Figure 3] Fig. 3. 1H-NMR spectrum of N-acetyl-3-nitro-L-tyrosine ethyl ester dissolved in CD3OD and analyzed on a Bruker AV-400-liquid spectrometer.
[Figure 4] Fig. 4. 1H-NMR spectrum of N-acetyl-5-chloro-3-nitro-L-tyrosine ethyl ester in CD3OD and analyzed on a Bruker AV-400-liquid spectrometer.
N-Acetyl-5-chloro-3-nitro-L-tyrosine ethyl ester top
Crystal data top
C13H15ClN2O6F(000) = 344
Mr = 330.72Dx = 1.506 Mg m3
Monoclinic, P21Cu Kα radiation, λ = 1.54184 Å
Hall symbol: P 2ybCell parameters from 1900 reflections
a = 5.1513 (4) Åθ = 7.9–67.6°
b = 10.6761 (9) ŵ = 2.63 mm1
c = 13.2849 (8) ÅT = 90 K
β = 93.689 (4)°Lath, yellow
V = 729.10 (9) Å30.34 × 0.11 × 0.03 mm
Z = 2
Data collection top
Bruker Kappa APEXII DUO area-detector
diffractometer
2307 independent reflections
Radiation source: IµS microfocus2299 reflections with I > 2σ(I)
QUAZAR multilayer optics monochromatorRint = 0.058
φ and ω scansθmax = 68.2°, θmin = 6.7°
Absorption correction: multi-scan
(TWINABS; Sheldrick, 2002)
h = 66
Tmin = 0.468, Tmax = 0.925k = 1212
7589 measured reflectionsl = 1515
Refinement top
Refinement on F2Secondary atom site location: difference Fourier map
Least-squares matrix: fullHydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.034H atoms treated by a mixture of independent and constrained refinement
wR(F2) = 0.091 w = 1/[σ2(Fo2) + (0.029P)2 + 0.4076P]
where P = (Fo2 + 2Fc2)/3
S = 1.07(Δ/σ)max < 0.001
2307 reflectionsΔρmax = 0.32 e Å3
208 parametersΔρmin = 0.20 e Å3
2 restraintsAbsolute structure: Flack (1983), 961 Friedel pairs
Primary atom site location: structure-invariant direct methodsFlack parameter: 0.078 (17)
Crystal data top
C13H15ClN2O6V = 729.10 (9) Å3
Mr = 330.72Z = 2
Monoclinic, P21Cu Kα radiation
a = 5.1513 (4) ŵ = 2.63 mm1
b = 10.6761 (9) ÅT = 90 K
c = 13.2849 (8) Å0.34 × 0.11 × 0.03 mm
β = 93.689 (4)°
Data collection top
Bruker Kappa APEXII DUO area-detector
diffractometer
2307 independent reflections
Absorption correction: multi-scan
(TWINABS; Sheldrick, 2002)
2299 reflections with I > 2σ(I)
Tmin = 0.468, Tmax = 0.925Rint = 0.058
7589 measured reflectionsθmax = 68.2°
Refinement top
R[F2 > 2σ(F2)] = 0.034H atoms treated by a mixture of independent and constrained refinement
wR(F2) = 0.091Δρmax = 0.32 e Å3
S = 1.07Δρmin = 0.20 e Å3
2307 reflectionsAbsolute structure: Flack (1983), 961 Friedel pairs
208 parametersFlack parameter: 0.078 (17)
2 restraints
Special details top

Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes.

Refinement. The crystal not single, and was treated as a nonmerohedral twin by rotation of 4.6 degrees about reciprocal axis 0.070 1.000 - 0.042 and real axis 0.300 1.000 - 0.019 The twin law is: (0.991, 0.000, -0.032, 0.006, 1.000, 0.014, 0.215, -0.021, 1.002)

The structure was refined versus. TWIN5 data, yielding BASF=0.093 (4).

Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) top
xyzUiso*/Ueq
Cl10.24004 (11)0.34934 (7)0.34487 (4)0.02854 (18)
O10.6456 (4)0.53157 (19)0.31400 (14)0.0267 (4)
H1O0.792 (8)0.585 (4)0.330 (3)0.040*
O21.0367 (4)0.66367 (19)0.38081 (15)0.0331 (5)
O31.1791 (4)0.65600 (18)0.53520 (16)0.0286 (5)
O40.2148 (4)0.23412 (19)0.84645 (13)0.0267 (4)
O50.4182 (4)0.37138 (19)0.95279 (13)0.0316 (5)
O60.0002 (3)0.58322 (18)0.81904 (14)0.0245 (4)
N11.0294 (4)0.6262 (2)0.46967 (16)0.0248 (5)
N20.4220 (4)0.5472 (2)0.79647 (15)0.0175 (4)
H2N0.568 (4)0.579 (3)0.797 (2)0.021*
C10.4584 (5)0.4103 (3)0.43817 (19)0.0220 (6)
C20.6440 (5)0.4968 (2)0.41110 (19)0.0215 (5)
C30.8183 (5)0.5385 (2)0.4902 (2)0.0216 (5)
C40.8039 (5)0.4991 (2)0.58960 (18)0.0175 (5)
H40.92140.53190.64100.021*
C50.6194 (4)0.4125 (2)0.61350 (18)0.0163 (5)
C60.4450 (5)0.3693 (2)0.53587 (18)0.0190 (5)
H60.31470.31050.55100.023*
C70.6044 (4)0.3609 (2)0.71852 (17)0.0175 (5)
H7A0.76950.37920.75810.021*
H7B0.58470.26880.71460.021*
C80.3772 (4)0.4158 (2)0.77388 (18)0.0166 (5)
H80.21490.40850.72870.020*
C90.3412 (4)0.3398 (3)0.86964 (18)0.0202 (5)
C100.2262 (5)0.6225 (2)0.81958 (18)0.0194 (5)
C110.2946 (6)0.7547 (3)0.8436 (2)0.0266 (6)
H11A0.24720.80760.78490.040*
H11B0.48220.76120.86050.040*
H11C0.19980.78270.90110.040*
C120.1647 (7)0.1488 (3)0.9296 (2)0.0381 (7)
H12A0.14470.19690.99230.046*
H12B0.31190.08990.94140.046*
C130.0777 (7)0.0789 (3)0.9011 (3)0.0415 (8)
H13A0.22260.13790.89080.062*
H13B0.11420.02020.95510.062*
H13C0.05650.03230.83860.062*
Atomic displacement parameters (Å2) top
U11U22U33U12U13U23
Cl10.0268 (3)0.0336 (3)0.0250 (3)0.0030 (3)0.0000 (2)0.0031 (3)
O10.0312 (11)0.0286 (10)0.0207 (9)0.0023 (9)0.0058 (8)0.0033 (7)
O20.0380 (12)0.0302 (11)0.0321 (11)0.0054 (9)0.0103 (9)0.0044 (8)
O30.0221 (10)0.0241 (10)0.0401 (12)0.0107 (8)0.0066 (9)0.0074 (8)
O40.0307 (10)0.0265 (10)0.0234 (9)0.0079 (8)0.0060 (7)0.0089 (8)
O50.0478 (12)0.0295 (11)0.0175 (9)0.0062 (10)0.0015 (8)0.0004 (8)
O60.0149 (9)0.0286 (10)0.0303 (10)0.0014 (8)0.0026 (7)0.0090 (8)
N10.0261 (12)0.0275 (12)0.0221 (12)0.0139 (10)0.0115 (10)0.0057 (9)
N20.0139 (10)0.0191 (10)0.0196 (10)0.0005 (8)0.0027 (8)0.0019 (8)
C10.0225 (12)0.0236 (13)0.0200 (12)0.0036 (11)0.0024 (10)0.0037 (10)
C20.0221 (12)0.0219 (12)0.0209 (13)0.0097 (10)0.0058 (9)0.0001 (10)
C30.0183 (13)0.0170 (12)0.0306 (14)0.0038 (10)0.0113 (10)0.0040 (10)
C40.0160 (12)0.0157 (11)0.0210 (12)0.0033 (9)0.0030 (9)0.0010 (9)
C50.0148 (11)0.0151 (11)0.0196 (12)0.0035 (10)0.0051 (9)0.0009 (9)
C60.0200 (11)0.0150 (12)0.0223 (11)0.0043 (10)0.0034 (8)0.0009 (9)
C70.0158 (10)0.0185 (12)0.0188 (11)0.0015 (10)0.0049 (8)0.0011 (10)
C80.0150 (11)0.0163 (11)0.0188 (12)0.0002 (10)0.0018 (9)0.0004 (9)
C90.0178 (11)0.0210 (12)0.0225 (12)0.0088 (11)0.0063 (9)0.0025 (11)
C100.0181 (13)0.0257 (13)0.0143 (11)0.0049 (10)0.0001 (9)0.0001 (9)
C110.0296 (14)0.0239 (14)0.0264 (13)0.0019 (12)0.0012 (10)0.0039 (11)
C120.0403 (18)0.0433 (18)0.0317 (16)0.0048 (15)0.0093 (13)0.0229 (14)
C130.050 (2)0.0364 (17)0.0398 (17)0.0137 (16)0.0147 (14)0.0072 (15)
Geometric parameters (Å, º) top
Cl1—C11.745 (3)C5—C61.402 (3)
O1—C21.343 (3)C5—C71.507 (3)
O1—H1O0.96 (4)C6—H60.9500
O2—N11.249 (3)C7—C81.538 (3)
O3—N11.169 (3)C7—H7A0.9900
O4—C91.329 (4)C7—H7B0.9900
O4—C121.467 (3)C8—C91.530 (3)
O5—C91.198 (3)C8—H81.0000
O6—C101.237 (3)C10—C111.485 (4)
N1—C31.473 (4)C11—H11A0.9800
N2—C101.341 (3)C11—H11B0.9800
N2—C81.449 (3)C11—H11C0.9800
N2—H2N0.823 (18)C12—C131.483 (5)
C1—C61.375 (4)C12—H12A0.9900
C1—C21.393 (4)C12—H12B0.9900
C2—C31.409 (4)C13—H13A0.9800
C3—C41.393 (4)C13—H13B0.9800
C4—C51.377 (4)C13—H13C0.9800
C4—H40.9500
C2—O1—H1O91 (2)H7A—C7—H7B107.8
C9—O4—C12117.4 (2)N2—C8—C9111.5 (2)
O3—N1—O2123.9 (2)N2—C8—C7110.6 (2)
O3—N1—C3119.6 (2)C9—C8—C7109.38 (19)
O2—N1—C3116.5 (2)N2—C8—H8108.4
C10—N2—C8121.0 (2)C9—C8—H8108.4
C10—N2—H2N117 (2)C7—C8—H8108.4
C8—N2—H2N122 (2)O5—C9—O4125.5 (2)
C6—C1—C2122.1 (2)O5—C9—C8124.5 (3)
C6—C1—Cl1118.9 (2)O4—C9—C8110.0 (2)
C2—C1—Cl1119.0 (2)O6—C10—N2121.1 (2)
O1—C2—C1118.5 (2)O6—C10—C11122.3 (2)
O1—C2—C3125.9 (2)N2—C10—C11116.6 (2)
C1—C2—C3115.6 (2)C10—C11—H11A109.5
C4—C3—C2122.8 (2)C10—C11—H11B109.5
C4—C3—N1116.9 (2)H11A—C11—H11B109.5
C2—C3—N1120.3 (2)C10—C11—H11C109.5
C5—C4—C3120.1 (2)H11A—C11—H11C109.5
C5—C4—H4120.0H11B—C11—H11C109.5
C3—C4—H4120.0O4—C12—C13107.8 (2)
C4—C5—C6118.1 (2)O4—C12—H12A110.1
C4—C5—C7122.4 (2)C13—C12—H12A110.1
C6—C5—C7119.5 (2)O4—C12—H12B110.1
C1—C6—C5121.4 (2)C13—C12—H12B110.1
C1—C6—H6119.3H12A—C12—H12B108.5
C5—C6—H6119.3C12—C13—H13A109.5
C5—C7—C8112.9 (2)C12—C13—H13B109.5
C5—C7—H7A109.0H13A—C13—H13B109.5
C8—C7—H7A109.0C12—C13—H13C109.5
C5—C7—H7B109.0H13A—C13—H13C109.5
C8—C7—H7B109.0H13B—C13—H13C109.5
C6—C1—C2—O1179.6 (2)C4—C5—C6—C10.9 (3)
Cl1—C1—C2—O10.9 (3)C7—C5—C6—C1177.4 (2)
C6—C1—C2—C30.6 (4)C4—C5—C7—C8105.2 (3)
Cl1—C1—C2—C3178.02 (18)C6—C5—C7—C876.6 (3)
O1—C2—C3—C4179.5 (2)C10—N2—C8—C975.9 (3)
C1—C2—C3—C41.6 (4)C10—N2—C8—C7162.1 (2)
O1—C2—C3—N11.3 (4)C5—C7—C8—N268.5 (3)
C1—C2—C3—N1177.5 (2)C5—C7—C8—C9168.3 (2)
O3—N1—C3—C42.3 (4)C12—O4—C9—O50.1 (4)
O2—N1—C3—C4178.1 (2)C12—O4—C9—C8179.6 (2)
O3—N1—C3—C2176.9 (2)N2—C8—C9—O522.1 (3)
O2—N1—C3—C22.6 (3)C7—C8—C9—O5100.6 (3)
C2—C3—C4—C52.3 (4)N2—C8—C9—O4158.4 (2)
N1—C3—C4—C5176.9 (2)C7—C8—C9—O478.9 (2)
C3—C4—C5—C61.9 (3)C8—N2—C10—O62.4 (4)
C3—C4—C5—C7176.4 (2)C8—N2—C10—C11178.7 (2)
C2—C1—C6—C50.3 (4)C9—O4—C12—C13150.8 (3)
Cl1—C1—C6—C5178.34 (19)
Hydrogen-bond geometry (Å, º) top
D—H···AD—HH···AD···AD—H···A
O1—H1O···O20.96 (4)1.63 (4)2.570 (3)168 (3)
N2—H2N···O6i0.82 (2)2.23 (2)2.999 (3)156 (3)
Symmetry code: (i) x+1, y, z.
Hydrogen-bond geometry (Å, º) top
D—H···AD—HH···AD···AD—H···A
O1—H1O···O20.96 (4)1.63 (4)2.570 (3)168 (3)
N2—H2N···O6i0.823 (18)2.23 (2)2.999 (3)156 (3)
Symmetry code: (i) x+1, y, z.
Acknowledgements top

Upgrade of the diffractometer was made possible by grant No. LEQSF (2011–12)-ENH-TR-01, administered by the Louisiana Board of Regents. This publication was made possible by National Institute of Health (NIH) grant No. P20RR16456 (the BRIN Program of the National Center for Research Resources), National Science Foundation (NSF) grant HRD-1043316 (the HBCU-UP ACE implementation program) and US Department of Education grant PO31B040030 (Title III, Part B - Strengthening Historically Black Graduate Institutions). The contents of this publication are solely the responsibility of authors and do not necessarily represent the official views of the NSF, NIH or US Department of Education.

references
References top

Alvarez, B., Ferrer-Sueta, G., Freeman, B. A. & Radi, R. (1999). J. Biol. Chem. 274, 842–848.

Beckman, J. S. (2009). Arch. Biochem. Biophys. 484, 114–116.

Bruker (2006). APEX2 and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA.

Ceriello, A. (2002). Int. J. Clin. Pract. Suppl. 129, 51–58.

Crow, J. P. (1999). Methods in Enzymology, Vol. 301, Nitric Oxide Part C: Biological and Antioxidant Activities, edited by L. Packer, pp. 151–160. New York: Academic Press.

Dahaoui, S., Jelsch, C., Howard, J. A. K. & Lecomte, C. (1999). Acta Cryst. B55, 226–230.

Darwish, R. S., Amiridze, N. & Aarabi, B. (2007). J. Trauma, 63, 439–442.

Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565.

Flack, H. D. (1983). Acta Cryst. A39, 876–881.

Janik, A., Chyra, A. & Stadnicka, K. (2007). Acta Cryst. C63, o572–o575.

Janik, A., Jarocha, M. & Stadnicka, K. (2008). Acta Cryst. B64, 223–229.

Koszelak, S. N. & van der Helm, D. (1981). Acta Cryst. B37, 1122–1124.

Pieret, A. F., Durant, F., Germain, G. & Koch, M. (1972). Cryst. Struct. Commun. 1, 75–77.

Pitt, A. R. & Spickett, C. M. (2008). Biochem. Soc. Trans. 36, 1077–1082.

Sheldrick, G. (2002). TWINABS. University of Göttingen, Germany.

Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.

Soriano-García, M. (1993). Acta Cryst. C49, 96–97.

Stout, K. L., Hallock, K. J., Kampf, J. W. & Ramamoorthy, A. (2000). Acta Cryst. C56, e100.

Uppu, R. M. & Pryor, W. A. (1999). J. Am. Chem. Soc. 121, 9738–9739.

Uppu, R. M., Squadrito, G. L. & Pryor, W. A. (1996). Arch. Biochem. Biophys. 327, 335-343.

Whiteman, M. & Halliwell, B. (1999). Biochem. Biophys. Res. Commun. 258, 168–172.

Winterbourn, C. C. (2002). Toxicology, 181–182, 223–227.