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

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.

The title compound, C 13 H 15 ClN 2 O 6 , was synthesized by hypochlorous acid-mediated chlorination of N-acetyl-3-nitrol-tyrosine ethyl ester. The OH group forms an intramolecular O-HÁ Á ÁO hydrogen bond to the nitro group and the N-H group forms an intermolecular N-HÁ Á ÁO 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.

Related literature
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. (2007Janik et al. ( , 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 (Bruker, 2006); data reduction: SAINT; 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 (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-NO 2 Tyr) (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-NO 2 Tyr 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.
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.
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 1 H-NMR spectrum, both NANTEE and NACNTEE were dissolved in CD 3 OD and analyzed on a Bruker AV-400-liquid spectrometer. The 1 H-NMR data are reported in ppm downfield from TMS as an internal standard.  (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
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. U iso for H were assigned as 1.2 times U eq of the attached atoms (1.5 for methyl and OH). 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).  Ellipsoids at the 50% level, with H atoms having arbitrary radius.

Special details
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).